Immunostimulatory CpG motifs induce CTL responses to HIV type I oligomeric gp140 envelope protein

In the present study we investigated the immunomodulatory effects of two adjuvants, liposomal lipid A [L(LA)] and CpG-containing oligodeoxynucleotides (CpG ODN), to the HIV-1 ogp140 envelope protein. Administration of each of these adjuvants separately with unencapsulated ogp140 resulted in low antibody titres. Encapsulation of ogp140 in liposomes containing lipid A resulted in a sixfold increase in anti-ogp140 antibodies. The antibody titres were further enhanced threefold by the addition of CpG ODN. Priming and boosting BALB/c mice with unencapsulated ogp140 with L(LA) or encapsulation in liposomes containing lipid A induced a mixed Th1/Th2 type of immune response. In contrast, immunization with L(ogp140 + LA) plus CpG ODN switched the immune response to a Th-1 response with elevated anti-ogp140 IgG2a antibodies and IFN-gamma levels. Both adjuvants induced excellent ogp140-specific proliferative and CTL responses. Therefore, for the induction of high titre antibodies, but not for cellular responses, the antigen and lipid A have to be present in the same liposomes. These results can have significant implications in directing the Th1 or Th2 differentiation of antigen-specific immune responses in the context of vaccine development.     

Antibodies to Lipids and Liposomes: Immunology and Safety

Naturally occurring antibodies to phospholipids and cholesterol are widespread; they occur commonly during the course of acute infections; they are not causally related to the anti-phospholipid syndrome; they have been associated with other clinical entities only as an epiphenomenon; and they have not been implicated as causing any clinical syndrome or disease. There are theoretical and experimental reasons to believe that normal cells and tissues are protected from binding of antibodies to bilayer lipids by steric hindrance due to adjacent larger molecules, such as large or charged adjacent glycolipids or proteins on the cell surface. There are also reasons to believe that certain natural antibodies to lipids can even serve useful normal functions. Antibodies to liposomal lipids induced by liposomes containing lipid A appear to have characteristics that are similar or identical to naturally occurring antibodies to lipids, and it is therefore believed that such antibodies would not cause adverse clinical effects. Numerous Phase I and II human clinical trials of experimental vaccines containing liposomes and lipid A have shown a high level of safety.     

Memory B cell antibodies to HIV-1 gp140 cloned from individuals infected with clade A and B viruses

Understanding the antibody response to HIV-1 in humans that show broad neutralizing serologic activity is a crucial step in trying to reproduce such responses by vaccination. Investigating antibodies with cross clade reactivity is particularly important as these antibodies may target conserved epitopes on the HIV envelope gp160 protein. To this end we have used a clade B YU-2 gp140 trimeric antigen and single-cell antibody cloning methods to obtain 189 new anti-gp140 antibodies representing 51 independent B cell clones from the IgG memory B cells of 3 patients infected with HIV-1 clade A or B viruses and exhibiting broad neutralizing serologic activity. Our results support previous findings showing a diverse antibody response to HIV gp140 envelope protein, characterized by differentially expanded B-cell clones producing highly hypermutated antibodies with heterogenous gp140-specificity and neutralizing activity. In addition to their high-affinity binding to the HIV spike, the vast majority of the new anti-gp140 antibodies are also polyreactive. Although none of the new antibodies are as broad or potent as VRC01 or PG9, two clonally-related antibodies isolated from a clade A HIV-1 infected donor, directed against the gp120 variable loop 3, rank in the top 5% of the neutralizers identified in our large collection of 185 unique gp140-specific antibodies in terms of breadth and potency.     

Calcium modulation of monoclonal antibody binding to phosphatidylinositol phosphate

The binding characteristics of two monoclonal antibodies (mAb) to phosphatidylinositol-4-phosphate (PIP) were examined: a murine IgM mAb to PIP; and a human IgG mAb (4E10) that binds both to HIV-1 envelope protein and also to neutral and anionic phospholipids, including PIP. Binding of each mAb to pure PIP was inhibited by Ca(2+) as determined by ELISA. When studied by surface plasmon resonance, liposomes containing PIP could be stripped (i.e., removed) by either Ca(2+) or phosphorylated haptens after binding of the liposomes to the murine anti-PIP antibody attached to a BIAcore chip. In contrast, the binding of liposomal PIP to 4E10 was irreversible and could not be stripped. We therefore conclude that Ca(2+) and phosphate can modulate the initial binding of both types of antibodies to PIP. However, 4E10 binds to liposomal PIP in a two-stage process involving first Ca(2+)-modulated binding to the PIP polar headgroup, followed by irreversible binding to liposomal hydrophobic groups.     

Liposome-stabilized oil-in-water emulsions as adjuvants: increased emulsion stability promotes induction of cytotoxic T lymphocytes against an HIV envelope antigen

Protective or therapeutic immunity against HIV infection is currently believed to require both antibody and CTL responses against the envelope protein. In the present study, the adjuvant activity of a unique oil-in-water emulsion, in which liposomes containing lipid A (LA) and encapsulated antigen served as the emulsifying agent, was examined in mice using oligomeric gp140 (ogp140) derived from the HIV-1 envelope as the antigen. Emulsions rendered either highly stable or unstable by altering the ratio of liposomes to oil were used to examine the effect of stability of the emulsion on adjuvant activity. Stable and unstable emulsions had similar potencies for inducing both IgG antibodies to ogp140 and antigen-specific T-lymphocyte proliferation. Stable emulsions, but not unstable emulsions, induced antigen-specific CTL responses, possibly because of the depot effect of the stable emulsions. Furthermore, stable emulsions induced lower IgG2a/IgG1 ratios than the unstable emulsions. We conclude that stable liposomal oil-in-water emulsions provide an effective means of obtaining both antibody and CTL responses against an HIV envelope antigen.     

Liposomes containing glucosyl ceramide specifically bind T4 bacteriophage: a self-assembling nanocarrier formulation

A unique formulation is described comprising liposomes containing glucosyl ceramide (GluCer) in the lipid bilayer to which bacteriophage T4 was attached. Binding of the phage T4 did not occur to glycolipids, such as galactosyl ceramide, containing an aldose in which the C-2 or C-4 conformations were not identical to glucose. These results strongly support previous proposals that glucose is a major receptor moiety for T4 binding to Escherichia coli. By using the binding of T4 to liposomal GluCer, we further describe a formulation that can be used as a self-assembling combined antigen and adjuvant carrier. A peptide antigen derived from C-trimer (Ct) of HIV-1 gp41 was fused to the highly antigenic outer capsid protein (Hoc), a nonessential protein of T4 that spontaneously binds to the T4 capsid. This resulted in display of the Ct-Hoc construct on the T4 capsid, and specific binding of a human monoclonal antibody that recognizes a peptide sequence of Ct was demonstrated. Liposomes containing monophosphoryl lipid A (MPLA) have been demonstrated to have potent adjuvant activities for experimental vaccines both in humans and animals, and because of this, mice were immunized with the Ct-Hoc-T4 construct that was bound to liposomes containing both GluCer and MPLA, resulting in the induction of high titers of Ct-specific antibodies. We conclude that liposomes containing both GluCer and MPLA can spontaneously bind to a construct of T4 that displays antigens that spontaneously binds to the capsid of T4 bacteriophage. This formulation could be utilized as an easily manufactured self-assembling antigen and adjuvant carrier.     

Nonneutralizing HIV-1 gp41 envelope cluster II human monoclonal antibodies show polyreactivity for binding to phospholipids and protein autoantigens

HIV-1 gp41 envelope antibodies, which are frequently induced in HIV-1-infected individuals, are predominantly nonneutralizing. The rare and difficult-to-induce neutralizing antibodies (2F5 and 4E10) that target gp41 membrane-proximal epitopes (MPER) are polyspecific and require lipid binding for HIV-1 neutralization. These results raise the questions of how prevalent polyreactivity is among gp41 antibodies and how the binding properties of gp41-nonneutralizing antibodies differ from those of antibodies that are broadly neutralizing. In this study, we have characterized a panel of human gp41 antibodies with binding specificities within the immunodominant cluster I (gp41 amino acids [aa] 579 to 613) or cluster II (gp41 aa 644 to 667) for reactivity to autoantigens, to the gp140 protein, and with MPER peptide-lipid conjugates. We report that while none of the gp41 cluster I antibodies studied were polyspecific, all three gp41 cluster II antibodies bound either to lipids or autoantigens, thus showing the propensity of cluster II antibodies to manifest polyreactivity. All cluster II gp41 monoclonal antibodies (MAbs), including those that were lipid reactive, failed to bind to gp41 MPER peptide-lipid complexes. Cluster II antibodies bound strongly with nanomolar binding affinity (dissociation constant [K(d)]) to oligomeric gp140 proteins, and thus, they recognize conformational epitopes on gp41 that are distinct from those of neutralizing gp41 antibodies. These results demonstrate that lipid-reactive gp41 cluster II antibodies are nonneutralizing due to their inability to bind to the relevant neutralizing epitopes on gp41.     

HIV-1, lipid rafts,and antibodies to liposomes: implications for anti-viral-neutralizing antibodies (Review)

The human immunodeficiency virus type 1 (HIV-1) is an enveloped virus with a lipid bilayer that contains several glycoproteins that are anchored in, or closely associated with, the membrane surface. The envelope proteins have complex interactions with the lipids both on the host cells and on the target cells. The processes of budding from host cells and entry into target cells occur at sites on the plasma membrane, known as lipid rafts, that represent specialized regions that are rich in cholesterol and sphingolipids. Although the envelope glycoproteins are antigenic molecules that potentially might be used for development of broadly neutralizing antibodies in a vaccine to HIV-1, the development of such antibodies that have broad specificities against primary field isolates of virus has been largely thwarted to date by the ability of the envelope proteins to evade the immune system through various mechanisms. In this review, the interactions of HIV-1 with membrane lipids are summarized. Liposomes are commonly used as models for understanding interactions of proteins with membrane lipids; and liposomes have also been used both as carriers for vaccines, and as antigens for induction of antibodies to liposomal lipids. The possibility is proposed that liposomal lipids, or liposome-protein combinations, could be useful as antigens for inducing broadly neutralizing antibodies to HIV-1.     

HIV-1, lipid rafts, and antibodies to liposomes: implications for anti-viral-neutralizing antibodies

The human immunodeficiency virus type 1 (HIV-1) is an enveloped virus with a lipid bilayer that contains several glycoproteins that are anchored in, or closely associated with, the membrane surface. The envelope proteins have complex interactions with the lipids both on the host cells and on the target cells. The processes of budding from host cells and entry into target cells occur at sites on the plasma membrane, known as lipid rafts, that represent specialized regions that are rich in cholesterol and sphingolipids. Although the envelope glycoproteins are antigenic molecules that potentially might be used for development of broadly neutralizing antibodies in a vaccine to HIV-1, the development of such antibodies that have broad specificities against primary field isolates of virus has been largely thwarted to date by the ability of the envelope proteins to evade the immune system through various mechanisms. In this review, the interactions of HIV-1 with membrane lipids are summarized. Liposomes are commonly used as models for understanding interactions of proteins with membrane lipids; and liposomes have also been used both as carriers for vaccines, and as antigens for induction of antibodies to liposomal lipids. The possibility is proposed that liposomal lipids, or liposome-protein combinations, could be useful as antigens for inducing broadly neutralizing antibodies to HIV-1.     

Immune Focusing and Enhanced Neutralization Induced by HIV-1 gp140 Chemical Cross-Linking

Experimental vaccine antigens based upon the HIV-1 envelope glycoproteins (Env) have failed to induce neutralizing antibodies (NAbs) against the majority of circulating viral strains as a result of antibody evasion mechanisms, including amino acid variability and conformational instability. A potential vaccine design strategy is to stabilize Env, thereby focusing antibody responses on constitutively exposed, conserved surfaces, such as the CD4 binding site (CD4bs). Here, we show that a largely trimeric form of soluble Env can be stably cross-linked with glutaraldehyde (GLA) without global modification of antigenicity. Cross-linking largely conserved binding of all potent broadly neutralizing antibodies (bNAbs) tested, including CD4bs-specific VRC01 and HJ16, but reduced binding of several non- or weakly neutralizing antibodies and soluble CD4 (sCD4). Adjuvanted administration of cross-linked or unmodified gp140 to rabbits generated indistinguishable total gp140-specific serum IgG binding titers. However, sera from animals receiving cross-linked gp140 showed significantly increased CD4bs-specific antibody binding compared to animals receiving unmodified gp140. Moreover, peptide mapping of sera from animals receiving cross-linked gp140 revealed increased binding to gp120 C1 and V1V2 regions. Finally, neutralization titers were significantly elevated in sera from animals receiving cross-linked gp140 rather than unmodified gp140. We conclude that cross-linking favors antigen stability, imparts antigenic modifications that selectively refocus antibody specificity and improves induction of NAbs, and might be a useful strategy for future vaccine design.     

HIV-1 envelope protein gp140 binding studies to human brain microvascular endothelial cells

Human immunodeficiency virus type-1 (HIV-1) envelope glycoprotein gp140 interacts with its specific receptors on the surface of the target cells leading to cellular activation through various signaling pathways. The effect of blocking the chemokine repertoire in human brain microvascular endothelial cells in HIV dementia (HAD) disease has not been reported. Characterizing the nature of HIV-1 envelope protein gp140 (T-tropic, HXBc2) receptor binding conditions to HBMEC is critical to gain insight into the HIV dementia, and eventually to rationally design the agents to block envelope protein receptor interactions. HIV-1 gp140 oligomers were purified and separated to monomers, dimers, and trimers. The binding conditions of gp140 to HBMEC chemokine receptor, CXCR4, were optimized with an aim of understanding the structural interactions in HAD. Analysis of the interaction between HIV-1 gp140 and CXCR4 of HBMEC by saturation binding, cross-competition analysis with radiolabeled SDF and gp140, revealed a strong interaction, specificity between HIV-1 gp140 and CXCR4. Our binding data demonstrate that HIV-1 envelope protein gp140 enters cells by protein receptor mediated interactions that are regulated by the conformational state of the gp140 at physiological environment (pH and temperature). The CXCR4 antibody 12G5 inhibited SDF-1 binding to HBMEC indicating the specificity of gp140 binding to HBMEC. Scatchard analysis revealed the presence of approximately 70250 gp140 binding sites per cell with a K(d) of 4.5 nM. Cross-competition experiments using labeled SDF-1 and gp140 revealed that both unlabeled SDF-1 and gp140 are capable of displacing their radiolabeled counterparts. The binding assay conditions and radioligand binding assay are highly valuable to identify and design better HIV inhibitors for HAD.     

Induction of antibodies in rhesus macaques that recognize a fusion-intermediate conformation of HIV-1 gp41

A component to the problem of inducing broad neutralizing HIV-1 gp41 membrane proximal external region (MPER) antibodies is the need to focus the antibody response to the transiently exposed MPER pre-hairpin intermediate neutralization epitope. Here we describe a HIV-1 envelope (Env) gp140 oligomer prime followed by MPER peptide-liposomes boost strategy for eliciting serum antibody responses in rhesus macaques that bind to a gp41 fusion intermediate protein. This Env-liposome immunization strategy induced antibodies to the 2F5 neutralizing epitope ⁶⁶⁴DKW residues, and these antibodies preferentially bound to a gp41 fusion intermediate construct as well as to MPER scaffolds stabilized in the 2F5-bound conformation. However, no serum lipid binding activity was observed nor was serum neutralizing activity for HIV-1 pseudoviruses present. Nonetheless, the Env-liposome prime-boost immunization strategy induced antibodies that recognized a gp41 fusion intermediate protein and was successful in focusing the antibody response to the desired epitope.     

Envelope deglycosylation enhances antigenicity of HIV-1 gp41 epitopes for both broad neutralizing antibodies and their unmutated ancestor antibodies

The HIV-1 gp41 envelope (Env) membrane proximal external region (MPER) is an important vaccine target that in rare subjects can elicit neutralizing antibodies. One mechanism proposed for rarity of MPER neutralizing antibody generation is lack of reverted unmutated ancestor (putative naive B cell receptor) antibody reactivity with HIV-1 envelope. We have studied the effect of partial deglycosylation under non-denaturing (native) conditions on gp140 Env antigenicity for MPER neutralizing antibodies and their reverted unmutated ancestor antibodies. We found that native deglycosylation of clade B JRFL gp140 as well as group M consensus gp140 Env CON-S selectively increased the reactivity of Env with the broad neutralizing human mAbs, 2F5 and 4E10. Whereas fully glycosylated gp140 Env either did not bind (JRFL), or weakly bound (CON-S), 2F5 and 4E10 reverted unmutated ancestors, natively deglycosylated JRFL and CON-S gp140 Envs did bind well to these putative mimics of naive B cell receptors. These data predict that partially deglycoslated Env would bind better than fully glycosylated Env to gp41-specific naïve B cells with improved immunogenicity. In this regard, immunization of rhesus macaques demonstrated enhanced immunogenicity of the 2F5 MPER epitope on deglyosylated JRFL gp140 compared to glycosylated JRFL gp140. Thus, the lack of 2F5 and 4E10 reverted unmutated ancestor binding to gp140 Env may not always be due to lack of unmutated ancestor antibody reactivity with gp41 peptide epitopes, but rather, may be due to glycan interference of binding of unmutated ancestor antibodies of broad neutralizing mAb to Env gp41.     

Lipid binding properties of 4E10, 2F5, and WR304 monoclonal antibodies that neutralize HIV-1

Two human mAbs (2F5 and 4E10), originally derived from HIV-1-infected patients, are important, but rare, mAbs that exhibit broad cross-clade neutralizing activities against HIV-1. In addition to peptide sequences on the gp41 envelope protein, both antibodies reportedly also bound specifically to several phospholipid antigens. However, the phospholipid binding property of 2F5 has been disputed and, because of uncertainly regarding phospholipid binding, the modeling of neutralizing mechanisms has been difficult. To explore this issue, we examined the binding of 4E10 and 2F5 to a broad range of lipid antigens by ELISA. 4E10 and 2F5 both bound to a variety of purified phospholipids, and 4E10 bound, but 2F5 did not bind, to cardiolipin. Both mAbs also bound to a sulfated glycolipid, sulfogalactosyl ceramide (sulfatide), and to two neutral glycolipids, galactosyl ceramide and glucosyl ceramide, but not to other galactosyl glycolipids. 4E10, but not 2F5, also bound to cholesterol, although both mAbs bound to squalene. Interestingly, 4E10, but not 2F5, exhibited striking binding to lipid A, the lipid moiety of Gram-negative bacterial lipopolysaccharide. The binding properties of 4E10 to phospholipids, sulfatide, cholesterol, squalene, and lipid A were similar to those of a neutralizing murine mAb (WR304) induced by liposomes containing phosphatidylinositol phosphate and lipid A, although WR304 did not bind to neutral glycolipids. The discovery of a binding specificity of 4E10 for lipid A, a widely used vaccine adjuvant, suggests that innate immunity stimulated by lipid A could have played a role for induction of multispecific antibodies that simultaneously recognize both HIV-1 protein and lipid antigens.     

A Fusion Intermediate gp41 Immunogen Elicits Neutralizing Antibodies to HIV-1

The membrane-proximal external region (MPER) of the human immunodeficiency virus, type 1 (HIV-1) envelope glycoprotein subunit gp41 is targeted by potent broadly neutralizing antibodies 2F5, 4E10, and 10E8. These antibodies recognize linear epitopes and have been suggested to target the fusion intermediate conformation of gp41 that bridges viral and cellular membranes. Anti-MPER antibodies exert different degrees of membrane interaction, which is considered to be the limiting factor for the generation of such antibodies by immunization. Here we characterize a fusion intermediate conformation of gp41 (gp41_int-Cys) and show that it folds into an elongated ∼12-nm-long extended structure based on small angle x-ray scattering data. Gp41_int-Cys was covalently linked to liposomes via its C-terminal cysteine and used as immunogen. The gp41_int-Cys proteoliposomes were administered alone or in prime-boost regimen with trimeric envelope gp140_CA018 in guinea pigs and elicited high anti-gp41 IgG titers. The sera interacted with a peptide spanning the MPER region, demonstrated competition with broadly neutralizing antibodies 2F5 and 4E10, and exerted modest lipid binding, indicating the presence of MPER-specific antibodies. Although the neutralization potency generated solely by gp140_CA018 was higher than that induced by gp41_int-Cys, the majority of animals immunized with gp41_int-Cys proteoliposomes induced modest breadth and potency in neutralizing tier 1 pseudoviruses and replication-competent simian/human immunodeficiency viruses in the TZM-bl assay as well as responses against tier 2 HIV-1 in the A3R5 neutralization assay. Our data thus demonstrate that liposomal gp41 MPER formulation can induce neutralization activity, and the strategy serves to improve breadth and potency of such antibodies by improved vaccination protocols.     

HIV-1 antibodies and vaccine antigen selectively interact with lipid domains

The rare, broadly neutralizing antibodies, 4E10 and 2F5, that target the HIV-1 membrane proximal external region also associate with HIV-1 membrane lipids as part of a required first-step in HIV-1 neutralization. HIV-1 virions have high concentration of cholesterol and sphingomyelin, which are able to organize into liquid-ordered domains (i.e., lipid rafts), and could influence the interaction of neutralizing antibodies with epitopes proximal to the membrane. The objective of this research is to understand how these lipid domains contribute to 2F5/4E10 membrane interactions and to antigen presentation in liposomal form of HIV-1 vaccines. To this end we have engineered biomimetic supported lipid bilayers and are able to use atomic force microscopy to visualize membrane domains, antigen clustering, and antibody–membrane interactions. Our results demonstrate that 2F5/4E10 do not interact with highly ordered gel and liquid-ordered domains and exclusively bind to a liquid-disordered lipid phase. This suggests that vaccine liposomes that contain key viral membrane components, such as high cholesterol content, may not be advantageous for 2F5/4E10 vaccine strategies. Rather, vaccine liposomes that primarily contain a liquid-disordered phase may be more likely to elicit production of lipid reactive, 2F5- and 4E10-like antibodies.     

Identification of a conserved domain of the HIV-1 transmembrane protein gp41 which interacts with cholesteryl groups

A soluble form of the HIV-1 envelope glycoprotein gp160 devoid of the transmembrane anchor domain was found to bind to cholesteryl-hemisuccinate agarose. The external subunit gp120 failed to bind to the resin, suggesting that the site responsible for the binding to cholesterol was located in the transmembrane protein gp41. We constructed a series of maltose binding protein (MBP) fusion proteins representing overlapping fragments of the gp41 molecule and we studied their capacity to bind to cholesteryl beads. The domain responsible for binding to cholesterol was localised within the residues 668 to 684 immediately adjacent to the membrane spanning domain. We identified a short sequence (LWYIK, aa 678-683) comparable to the cholesterol interaction amino acid consensus pattern published by Li and Papadopoulos [Endocrinology 139 (1998) 4991]. We demonstrated that the sequence LWYIK synthesized fused to the MBP was able to bind to cholesteryl groups. A synthetic peptide containing the sequence LWYIK was found to inhibit the interaction between cholesteryl beads and MBP44, an MBP fusion HIV-1 envelope protein that contains the putative cholesterol binding domain. Human sera obtained from HIV-1 seropositive patients did not react in ELISA to the LWYIK sequence, suggesting that this region is not exposed to the immune system. The biological significance of the interaction between gp41 and cholesterol is discussed.     

Antibodies with specificity to native gp120 and neutralization activity against primary human immunodeficiency virus type 1 isolates elicited by immunization with oligomeric gp160.

Current human immunodeficiency virus type 1 (HIV-1) envelope vaccine candidates elicit high antibody binding titers with neutralizing activity against T-cell line-adapted but not primary HIV-1 isolates. Serum antibodies from these human vaccine recipients were also found to be preferentially directed to linear epitopes within gp120 that are poorly exposed on native gp120. Systemic immunization of rabbits with an affinity-purified oligomeric gp160 protein formulated with either Alhydrogel or monophosphoryl lipid A-containing adjuvants resulted in the induction of high-titered serum antibodies that preferentially bound epitopes exposed on native forms of gp120 and gp160, recognized a restricted number of linear epitopes, efficiently bound heterologous strains of monomeric gp120 and cell surface-expressed oligomeric gp120/gp41, and neutralized several strains of T-cell line-adapted HIV-1. Additionally, those immune sera with the highest oligomeric gp160 antibody binding titers had neutralizing activity against some primary HIV-1 isolates, using phytohemagglutinin-stimulated peripheral blood mononuclear cell targets. Induction of an antibody response preferentially reactive with natively folded gp120/gp160 was dependent on the tertiary structure of the HIV-1 envelope immunogen as well as its adjuvant formulation, route of administration, and number of immunizations administered. These studies demonstrate the capacity of a soluble HIV-1 envelope glycoprotein vaccine to elicit an antibody response capable of neutralizing primary HIV-1 isolates.     

Determinants of Human Immunodeficiency Virus Type 1 Envelope Glycoprotein Activation by Soluble CD4 and Monoclonal Antibodies

Infection by some human immunodeficiency virus type 1 (HIV-1) isolates is enhanced by the binding of subneutralizing concentrations of soluble receptor, soluble CD4 (sCD4), or monoclonal antibodies directed against the viral envelope glycoproteins. In this work, we studied the abilities of different antibodies to mediate activation of the envelope glycoproteins of a primary HIV-1 isolate, YU2, and identified the regions of gp120 envelope glycoprotein contributing to activation. Binding of antibodies to a variety of epitopes on gp120, including the CD4 binding site, the third variable (V3) loop, and CD4-induced epitopes, enhanced the entry of viruses containing YU2 envelope glycoproteins. Fab fragments of antibodies directed against either the CD4 binding site or V3 loop also activated YU2 virus infection. The activation phenotype was conferred on the envelope glycoproteins of a laboratory-adapted HIV-1 isolate (HXBc2) by replacing the gp120 V3 loop or V1/V2 and V3 loops with those of the YU2 virus. Infection by the YU2 virus in the presence of activating antibodies remained inhibitable by macrophage inhibitory protein 1β, indicating dependence on the CCR5 coreceptor on the target cells. Thus, antibody enhancement of YU2 entry involves neither Fc receptor binding nor envelope glycoprotein cross-linking, is determined by the same variable loops that dictate enhancement by sCD4, and probably proceeds by a process fundamentally similar to the receptor-activated virus entry pathway.     

Antibodies to lipids and liposomes: immunology and safety

Naturally occurring antibodies to phospholipids and cholesterol are widespread; they occur commonly during the course of acute infections; they are not causally related to the anti-phospholipid syndrome; they have been associated with other clinical entities only as an epiphenomenon; and they have not been implicated as causing any clinical syndrome or disease. There are theoretical and experimental reasons to believe that normal cells and tissues are protected from binding of antibodies to bilayer lipids by steric hindrance due to adjacent larger molecules, such as large or charged adjacent glycolipids or proteins on the cell surface. There are also reasons to believe that certain natural antibodies to lipids can even serve useful normal functions. Antibodies to liposomal lipids induced by liposomes containing lipid A appear to have characteristics that are similar or identical to naturally occurring antibodies to lipids, and it is therefore believed that such antibodies would not cause adverse clinical effects. Numerous Phase I and II human clinical trials of experimental vaccines containing liposomes and lipid A have shown a high level of safety.