Neutralizing Antibodies Against HIV-1 BRU and SF2 Isolates Generated in Mice Immunized with Recombinant Vaccinia Virus Expressing HIV-1 (BRU) Envelope Glycoproteins and Boosted with Homologous gp160

Section: Immunizationmentioning

confidence: 99%

Polyvalent Envelope Glycoprotein Vaccine Elicits a Broader Neutralizing Antibody Response but Is Unable To Provide Sterilizing Protection against Heterologous Simian/Human Immunodeficiency Virus Infection in Pigtailed Macaques

Cho

Kim

Lee

et al. 2001

“…To neutralize HIV-1, an antibody must be able to bind to the native, trimeric virusassociated Env complex (11,12,63,64). Most Env-based vaccine candidates tested to date have been either monomeric gp120 subunits or various forms of the uncleaved gp160 or gp140 (gp120 plus gp41 ectodomain) precursor protein (4,25,26,37,47,68,80,(96)(97)(98). The use of uncleaved gp140 or gp160 protein has been considered necessary because the labile, noncovalent gp120-gp41 association in cleaved Env leads to the dissociation of gp120 from gp41 (30,49,54,74).…”

mentioning

confidence: 99%

Enhancing the Proteolytic Maturation of Human Immunodeficiency Virus Type 1 Envelope Glycoproteins

Binley

Sanders

Master

et al. 2002

Self Cite

142

165

In virus-infected cells, the envelope glycoprotein (Env) precursor, gp160, of human immunodeficiency virus type 1 is cleaved by cellular proteases into a fusion-competent gp120-gp41 heterodimer in which the two subunits are noncovalently associated. However, cleavage can be inefficient when recombinant Env is expressed at high levels, either as a full-length gp160 or as a soluble gp140 truncated immediately N-terminal to the transmembrane domain. We have explored several methods for obtaining fully cleaved Env for use as a vaccine antigen. We tested whether purified Env could be enzymatically digested with purified protease in vitro. Plasmin efficiently cleaved the Env precursor but also cut at a second site in gp120, most probably the V3 loop. In contrast, a soluble form of furin was specific for the gp120-gp41 cleavage site but cleaved inefficiently. Coexpression of Env with the full-length or soluble form of furin enhanced Env cleavage but also reduced Env expression. When the Env cleavage site (REKR) was mutated in order to see if its use by cellular proteases could be enhanced, several mutants were found to be processed more efficiently than the wild-type protein. The optimal cleavage site sequences were RRRRRR, RRRRKR, and RRRKKR. These mutations did not significantly alter the capacity of the Env protein to mediate fusion, so they have not radically perturbed Env structure. Furthermore, unlike that of wild-type Env, expression of the cleavage site mutants was not significantly reduced by furin coexpression. Coexpression of Env cleavage site mutants and furin is therefore a useful method for obtaining high-level expression of processed Env.The Env glycoprotein complex mediates receptor binding and membrane fusion during human immunodeficiency virus type 1 (HIV-1) infection of susceptible cells (66). It is synthesized as a polypeptide precursor (gp160) that oligomerizes to form a heavily glycosylated trimer (20,24). At a late stage of synthesis, most probably in the trans-Golgi network (TGN), gp160 is cleaved by furin (17,18,(55)(56)(57)(58) or other, related subtilisin-like proteases (17,18,28,38,58,90) into the surface (SU; gp120) and transmembrane (TM; gp41) subunits (34,43,(55)(56)(57)(58)82). Cleavage occurs at a motif at the gp120-gp41 juncture that contains a basic amino acid tetrad, R-X-(R/K)-R (where X is any amino acid). The gp120 and gp41 proteins then remain noncovalently associated, forming the functional, native gp120 3 -gp41 3 complex (20,24,66).During fusion, the gp120 protein interacts with the virus receptor and coreceptor on target cells. This triggers conformational changes that lead to the insertion of a hydrophobic fusion peptide, located at the N terminus of gp41, into the target cell membrane (66). Cleavage of gp160 is essential for fusion, since uncleaved gp160 is fusion incompetent (9,33,39,48). Generally, only cleaved Env is incorporated into virions (22), although uncleaved Env can be virion associated (39,48). By analogy with other enveloped viruses such as influenza A virus (5,...

“…Additionally, monomeric gp120 and uncleaved gp160 molecules have been shown to be poor antigenic representations of virion-associated gp160. Evidence suggests that an immunogen capable of eliciting virus-neutralizing antibodies may be an important component of an effective human immunodeficiency virus type 1 (HIV-1) vaccine (19,32,42,69,87,99). Such an immunogen should faithfully represent the antigenic structure of the virion-associated envelope complex, since neutralizing capacity has been observed with antibodies directed against epitopes contained on the native Env trimer (10,12,68,73,82).…”

mentioning

confidence: 99%

Covalently Linked Human Immunodeficiency Virus Type 1 gp120/gp41 Is Stably Anchored in Rhabdovirus Particles and Exposes Critical Neutralizing Epitopes

McKenna

Pomerantz

Dietzschold

et al. 2003

Rabies virus (RV) vaccine strain-based vectors show significant promise as potential live-attenuated vaccines against human immunodeficiency virus type 1 (HIV-1). Here we describe a new RV construct that will also likely have applications as a live-attenuated or killed-particle immunogen. We have created a RV containing a chimeric HIV-1 Env protein, which contains introduced cysteine residues that give rise to an intermolecular disulfide bridge between gp120 and the ectodomain of gp41. This covalently linked gp140 (gp140 SOS) is fused in frame to the cytoplasmic domain of RV G glycoprotein and is efficiently incorporated into the RV virion. On the HIV-1 virion, the gp120 and gp41 moieties are noncovalently associated, which leads to extensive shedding of gp120 from virions and virus-infected cells. The ability to use HIV-1 particles as purified, inactivated immunogens has been confounded by the loss of gp120 during preparation. Additionally, monomeric gp120 and uncleaved gp160 molecules have been shown to be poor antigenic representations of virion-associated gp160. Evidence suggests that an immunogen capable of eliciting virus-neutralizing antibodies may be an important component of an effective human immunodeficiency virus type 1 (HIV-1) vaccine (19,32,42,69,87,99). Such an immunogen should faithfully represent the antigenic structure of the virion-associated envelope complex, since neutralizing capacity has been observed with antibodies directed against epitopes contained on the native Env trimer (10,12,68,73,82). However, formulating vaccines capable of eliciting neutralizing antibodies has been quite difficult because of the labile nature of gp120-gp41 interactions and the antigenic differences between virion-associated gp160 and monomeric or dissociated subunits (11,15,53,54,67).Following oligomerization in the endoplasmic reticulum, the gp160 precursor protein is cleaved by cellular proteases and is transported to the cell surface (28, 49). The mature, virionassociated form of the HIV-1 Env glycoprotein is a trimeric molecule composed of three gp120 and three gp41 subunits held together by weak noncovalent interactions (30,73,103). This structure is highly flexible and undergoes substantial conformational changes upon gp120 binding with CD4 and chemokine coreceptors, which leads to exposure of the fusion peptides of gp41 that insert into the target cell membrane and mediate viral entry (16,44,46,47,58,93,103). During the course of HIV-1 infection, multiple forms of gp120 and gp41 subunits are shed from virions and virus-infected cells due to the noncovalent interactions between gp120 and gp41 and between gp41 subunits (38,54,62,71). This "viral debris" is an attractive target for the humoral immune response; however, these dissociated, largely monomeric subunits are immunologically distinct from virion-associated gp160 multimers. It is widely held that this antigen shedding is an immune avoidance mechanism evolved by HIV-1 to elicit an inappropriate antibody response and thus draw the hosts' immune defens...