Induction of antibodies against epitopes inaccessible on the HIV type 1 envelope oligomer by immunization with recombinant monomeric glycoprotein 120

An N-glycan (N306) at the base of the V3 loop of HIV-BRU gp120 is shielding a linear neutralization epitope at the tip of the V3 loop on oligomeric Env. In contrast, this epitope is readily antigenic on monomeric gp120. Immunization with recombinant monomeric HIV-BRU gp120 may thus be expected to elicit antibodies preferentially neutralizing mutant variants of HIV-BRU lacking the N306 glycan. Therefore, two guinea pigs were immunized with monomeric wild-type HIV-BRU gp120 possessing the N306 glycan and immune sera were tested for neutralization against target viruses HIV-BRU, -A308, and -A308T321. HIV-A308 and HIV-A308T321 lack the N306 glycan; HIV-A308T321 contains an additional mutation at the tip of V3 rendering it resistant to MAb binding at this epitope. Both immune sera preferentially neutralized the two mutant virus variants lacking the N306 glycan, with a 10- to 20-fold increase in neutralization titer compared with the wild-type HIV-BRU. Thus, immunization with monomeric HIV-BRU gp120 elicited antibodies preferentially neutralizing HIV variants lacking the N306 glycan. In addition to antibodies directed against the tip of V3, other antibodies directed against epitopes shielded by the N306 glycan on the envelope oligomer were elicited by the immunization, as demonstrated by the ability of the immune sera to neutralize HIV-A308T321. One such epitope was overlapping the NEA-9284 epitope located at the amino-terminal flank of the V3 loop. Our results demonstrate that monomeric gp120 contains immunogenic structures inaccessible on the envelope oligomer. The limited ability of recombinant gp120 vaccines to induce neutralizing antibodies against primary isolates may thus not exclusively reflect genetic variation.     

Interactions of polyclonal and monoclonal anti-glycoprotein 120 antibodies with oligomeric glycoprotein 120-glycoprotein 41 complexes of a primary HIV type 1 isolate: relationship to neutralization

The arterial ketone body ratio (AKBR) is considered to be an accurate index of the functional reserve of the liver, and the validity of this idea has been confirmed in the field of abdominal surgery. We found low AKBR value intracerebral hemorrhage patients and discussed the clinical significance of this finding in this paper. Twenty-five patients with intracerebral hemorrhage treated at our institution were included in this study. Their ages ranged from 42 to 86 years old (average 68.5 years). There were 13 cases of putaminal hemorrhage and 12 cases of thalamic hemorrhage. Evacuation of the hematoma or ventricle drainage was performed in 20 of these cases within 3 days after symptoms of intracerebral hemorrhage appeared. There were 12 cases with intraventricular hemorrhage. The outcome of these patients was as follows; 17 cases survived, eight cases died. We collected blood samples on days 1, 2, 3, 7 and 10 after the onset of symptoms (day 0) and measured the following: 1, beta-hydroxybutyrate; 2, acetoacetate; 3, epinephrine; 4, norepinephrine. On day 0 total ketone body levels were higher (246.3 +/- 231.7 mumol/l), AKBR values (0.60 +/- 0.18) were significantly lower than in the control group (2.05 +/- 1.35) (p < 0.001). However, both epinephrine and norepinephrine levels were significantly higher, 638.4 +/- 229.0 pg/ml and 1036.5 +/- 288.2 pg/ml, respectively. The AKBR value was 0.76 +/- 0.19 on day 1, 1.04 +/- 0.30 on day 2, and increased thereafter. In addition, the relation between sequential changes of AKBR in patients with intraventricular hemorrhage and outcome were also discussed. AKBR values are known to decrease not only in cases of hepatic failure, but in cases in which the liver energy charge is reduced, such as shock and hypoxemia, but no investigations have ever been performed to determine whether AKBR is altered in cerebrovascular disease. In this study, we found that AKBR values were lower in intracerebral hemorrhage, presumably due to reduced hepatic blood flow causes by increased levels of epinephrine and norepinephrine. In addition, our findings suggest that the fluctuations in AKBR values correlated with the outcome of intracerebral hemorrhage patients.     

Anti-HIV type 1 activity of wild-type and functional defective RANTES intrakine in primary human lymphocytes

We have developed a genetic "intrakine" strategy to inactivate the CC-chemokine receptor 5 (CCR-5), the principal coreceptor for macrophage (M)-tropic HIV-1 viruses (Yang et al, 1997). The inactivation of CCR5 was achieved by targeting a modified CC-chemokine (RANTES) to the lumen of the endoplasmic reticulum (ER) to block the transport of the newly synthesized CCR-5. The transduced lymphocytes with the phenotypic CCR5 knockout were shown to be resistant to M-tropic HIV-1 infection. This study illustrated the feasibility of the intrakine strategy to block HIV-1 infection. In our current study, the potential clinical application of the intrakine approach was further evaluated in human peripheral blood lymphocytes (PBLs). PBLs were transduced with the RANTES intrakine gene by using retroviral vectors with the truncated low-affinity human nerve growth factor receptor (deltaNGFR) marker, and then isolated by an anti-NGFR antibody/magnetic bead method. The surface expression of CCR-5 in the transduced lymphocytes was dramatically inhibited, as demonstrated by flow cytometric assays. The transduced PBLs were shown to resist various types of M-tropic HIV-1 virus infection. The cell viability, cell proliferation rates, and cell surface markers of the intrakine-transduced PBLs were shown to be comparable to those of control PBLs. The transduced PBLs were also found to respond to the stimulation of various CXC- and CC-chemokines, other than RANTES. The transduced PBLs responded to tetanus antigen stimulation by increasing IL-2 production and cell proliferation. In addition, a functionally defective mutant of RANTES that retains its binding activity to CCR-5, but loses its signaling ability, was used to generate a mutant RANTES intrakine. The primary lymphocytes transduced with the mutant RANTES intrakine were found to be resistant to M-tropic HIV-1 infection. From these results, we conclude that the primary human lymphocytes transduced with either the wild-type or functionally defective RANTES intrakine are resistant to M-tropic HIV-1 infection, and maintain their basic biological functions. This study, therefore, indicates the potential clinical application of the intrakine approach for HIV-1 gene therapy.     

The antigenic structure of the HIV gp120 envelope glycoprotein

The human immunodeficiency virus HIV-1 establishes persistent infections in humans which lead to acquired immunodeficiency syndrome (AIDS). The HIV-1 envelope glycoproteins, gp120 and gp41, are assembled into a trimeric complex that mediates virus entry into target cells. HIV-1 entry depends on the sequential interaction of the gp120 exterior envelope glycoprotein with the receptors on the cell, CD4 and members of the chemokine receptor family. The gp120 glycoprotein, which can be shed from the envelope complex, elicits both virus-neutralizing and non-neutralizing antibodies during natural infection. Antibodies that lack neutralizing activity are often directed against the gp120 regions that are occluded on the assembled trimer and which are exposed only upon shedding. Neutralizing antibodies, by contrast, must access the functional envelope glycoprotein complex and typically recognize conserved or variable epitopes near the receptor-binding regions. Here we describe the spatial organization of conserved neutralization epitopes on gp120, using epitope maps in conjunction with the X-ray crystal structure of a ternary complex that includes a gp120 core, CD4 and a neutralizing antibody. A large fraction of the predicted accessible surface of gp120 in the trimer is composed of variable, heavily glycosylated core and loop structures that surround the receptor-binding regions. Understanding the structural basis for the ability of HIV-1 to evade the humoral immune response should assist in the design of a vaccine.     

Inhibition of platelet aggregation by alpha1-acid glycoprotein

In view of known abnormalities of plasma proteins in diseases showing changes in platelet aggregation, the effect of one of the major serum proteins, alpha1-acid glycoprotein, on platelet aggregation was evaluated. This protein, when added to platelet-rich plasma, markedly inhibited platelet aggregation induced by both adenosine diphosphate (ADP) and epinephrine. Transferrin, similarly studied, had no effect. These results are consistent with the hypothesis that the relative concentration of alpha1-acid glycoprotein may influence platelet aggregation in diseases associated with abnormal concentrations of this protein.     

CCR5 coreceptor utilization involves a highly conserved arginine residue of HIV type 1 gp120

The seven-transmembrane CCR5 was recently found to double as a coreceptor for a genetically diverse family of human and nonhuman primate lentiviruses. Paradoxically, the main region of the envelope protein believed to be involved in CCR5 utilization was mapped to hypervariable region 3, or V3, of the envelope glycoprotein gp120. In this study, we addressed the question of whether functional convergence in CCR5 utilization is mediated by certain V3 residues that are highly conserved among HIV type 1 (HIV-1), HIV type 2, and simian immunodeficiency virus. Site-directed mutagenesis carried out on three such V3 residues revealed that the Arg-298 of HIV-1 gp120 has an important role in CCR5 utilization. In contrast, no effect was observed for the other residues we tested. The inability of Arg-298 mutants to use CCR5 was not attributed to global alteration of gp120 conformation. Neither the expression, processing, and incorporation of mutant envelope proteins into virions, nor CD4 binding were significantly affected by the mutations. This interpretation is further supported by the finding that alanine substitutions of five residues immediately adjacent to the arginine residue had no effect on CCR5 utilization. Taken together, our data strongly suggests that the highly conserved Arg-298 residue identified in the V3 of HIV-1 has a significant role in CCR5 utilization, and may represent an unusually conserved target for future anti-viral designs.     

An investigation of the high-avidity antibody response to glycoprotein 120 of human immunodeficiency virus type 1

The avidity of antibodies for antigens can be measured by determining what remains bound after exposing the antibody-antigen complex to a chaotropic agent such as urea. This method has been gaining popularity for assessing the immune response to the human immunodeficiency virus type 1 (HIV-1) surface glycoprotein gp120 (or its counterpart from simian immunodeficiency virus), during natural infection or after subunit vaccination. High-avidity antibodies have been considered to be a possible correlate of protection. We have examined the avidity assay to determine what it, in fact, measures. First, we studied the development of the anti-gp120 response in seroconverting individuals. Urea elution reduced the polyclonal anti-gp120 titers by 3- to 10-fold. After allowing for the consequent reduction in assay sensitivity, there was no obvious change in the rate of development of the high-avidity and unfractionated antibody responses. Furthermore, in the one individual who developed a strong autologous, virus-neutralizing response, the appearance of neutralizing antibodies and high-avidity antibodies did not coincide. Antibodies to the V3 loop, when present, comprised a major fraction of the polyclonal response that survives urea elution. We next examined the effect of urea elution on the binding to gp120 of a panel of monoclonal antibodies (MAbs). Urea treatment preferentially eluted MAbs to discontinuous rather than continuous epitopes, independent of their affinities. Furthermore, these patterns of epitope stability were unaltered by the presence of polyclonal anti-gp120 antibodies. As most broadly neutralizing anti-gp120 antibodies recognize discontinuous epitopes, this skewing effect must be taken into account when interpreting studies using polyclonal sera.     

Human submandibular saliva inhibits human immunodeficiency virus type 1 infection by displacing envelope glycoprotein gp120 from the virus

Actin is a highly conserved, ubiquitous cytoskeletal protein, which is essential for multiple cellular functions. Despite its small size (Mr = 42 000), unpolymerized forms of actin, as well as polymerized forms, exist primarily in the cytoplasm, excluded from the nucleus. Although spatial control of actin is crucially important, the molecular mechanisms ensuring the cytoplasmic localization of unpolymerized actin have not been revealed so far. In this paper we report that actin contains two leucine-rich type nuclear export signal (NES) sequences in the middle part of the molecule, which are both shown to be functional. Monomeric actin, when injected into the nucleus, was rapidly exported in a manner which was sensitive to leptomycin B (LMB), a specific inhibitor of NES-dependent nuclear export. LMB treatment of cells prevented nuclear exclusion of endogenous actin, inducing its nuclear accumulation. Furthermore, actin mutants with disrupted NESs accumulated in the nucleus. Expression of these NES-disrupted actin mutants, but not of wild-type actin, induced a decrease in the proliferative potential of the cell. These results reveal a novel molecular mechanism controlling the subcellular distribution of actin.     

Anti-F(ab'')2 antibody in HIV type 1 infection: relationship to hypergammaglobulinemia and to antibody specific to the V3 loop region of glycoprotein 120

Cyclosporine (Sandimmune) is an effective immunosuppressive drug but may be poorly absorbed in the early postoperative period after liver transplantation, exposing the recipient to an increased risk for rejection. Neoral is a new oral formulation of cyclosporine that uses a mixture of surfactant, lipophilic, and hydrophilic solvents to permit microemulsification that leads to potentially better absorption. This oral drug has not been evaluated in children immediately posttransplantation. The aim of this study was to evaluate the pharmacokinetics, bioavailability, and safety of Neoral during the first week post-liver transplantation in children. Twelve children, 8 boys and 4 girls, with a median age of 2.6 years (range, 1 to 8 years) were administered Neoral within 12 hours posttransplantation. Pharmacokinetic profiles were performed over a 12-hour period on each child on days 1, 3, and 5 and twice-daily trough levels were obtained on days 2, 4, 6, and 7. The maximum concentration (Cmax), time to reach Cmax (Tmax), 12-hour trough levels, and area under the curve were calculated, and rejection episodes and adverse events were documented over a 12-week period. Neoral was well absorbed, even on the first postoperative day. After the introduction of enteral feeding, the peak levels increased (Cmax, 655 ng/mL) and were achieved significantly sooner (Tmax, 2 hours). There was no significant difference in drug exposure between days 1, 3, and 5 (P > .05). The incidence of acute rejection was 25% and hypertension was reported in 4 of 12 patients during the first week. Neoral was well absorbed in the early post-liver transplantation period, provided effective immunosuppression, and was not associated with a high incidence of adverse events or toxicity. The introduction of enteral feeding improved absorption.     

Membrane fusion induced by the HIV type 1 fusion peptide: modulation by factors affecting glycoprotein 41 activity and potential anti-HIV compounds

Peptides representing a sequence of 23 amino acid residues at the N terminus of human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein gp41 bind and subsequently induce fusion of large unilamellar vesicles (LUV), an activity presumably related to gp41 function in viral infection. These in vitro effects can be modulated by several factors that are known to affect HIV-1 infectivity and gp41-mediated virus-cell fusion. Peptide-induced membrane fusion but not peptide binding can be inhibited by two factors known to block gp41 activity: a polar amino acid substitution V --> E in position 2 and the presence of the N-terminal hexapeptide of gp41 in addition to the parent sequence. Whereas inclusion of the alternative gp120 receptor galactosylceramide in membranes has virtually no effect, membrane cholesterol stimulates fusion activity. In view of its putative physiological relevance, we have used the fusion activity of the peptides as a tool to evaluate the inhibitory effect of antivirals that might target this sequence. We describe three dissimilar effects: Amphotericin B inhibits in a cholesterol-independent way peptide-induced fusion but not binding, human serum albumin inhibits binding and consequently fusion, and dextran sulfate (M(r) 5000) does not affect either binding or fusion.     

Structure of an HIV gp120 envelope glycoprotein in complex with the CD4 receptor and a neutralizing human antibody

The entry of human immunodeficiency virus (HIV) into cells requires the sequential interaction of the viral exterior envelope glycoprotein, gp120, with the CD4 glycoprotein and a chemokine receptor on the cell surface. These interactions initiate a fusion of the viral and cellular membranes. Although gp120 can elicit virus-neutralizing antibodies, HIV eludes the immune system. We have solved the X-ray crystal structure at 2.5 A resolution of an HIV-1 gp120 core complexed with a two-domain fragment of human CD4 and an antigen-binding fragment of a neutralizing antibody that blocks chemokine-receptor binding. The structure reveals a cavity-laden CD4-gp120 interface, a conserved binding site for the chemokine receptor, evidence for a conformational change upon CD4 binding, the nature of a CD4-induced antibody epitope, and specific mechanisms for immune evasion. Our results provide a framework for understanding the complex biology of HIV entry into cells and should guide efforts to intervene.     

Replication and neutralization of human immunodeficiency virus type 1 lacking the V1 and V2 variable loops of the gp120 envelope glycoprotein

A human immunodeficiency virus type 1 (HIV-1) mutant lacking the V1 and V2 variable loops in the gp120 exterior envelope glycoprotein replicated in Jurkat lymphocytes with only modest delays compared with the wild-type virus. Revertants that replicated with wild-type efficiency rapidly emerged and contained only a few amino acid changes in the envelope glycoproteins compared with the parent virus. Both the parent and revertant viruses exhibited increased sensitivity to neutralization by antibodies directed against the V3 loop or a CD4-induced epitope on gp120 but not by soluble CD4 or an antibody against the CD4 binding site. This result demonstrates the role of the gp120 V1 and V2 loops in protecting HIV-1 from some subsets of neutralizing antibodies.     

Inhibition of the infectivity of influenza virus by tea polyphenols

(-)Epigallocatechin gallate (EGCg) and theaflavin digallate (TF3) (1-10 microM) inhibited the infectivity of both influenza A virus and influenza B virus in Madin-Darby canine kidney (MDCK) cells in vitro. Study by electron microscope revealed that EGCg and TF3 (1 mM) agglutinated influenza viruses as well as did antibody, and that they prevented the viruses from adsorbing to MDCK cells. EGCg and TF3 more weakly inhibited adsorption of the viruses to MDCK cells. EGCg and TF3 (1-16 microM) also inhibited haemagglutination by influenza viruses. These findings suggest that tea polyphenols bind to the haemagglutinin of influenza virus, inhibit its adsorption to MDCK cells, and thus block its infectivity.     

Induction of lymphomonocyte activation by HIV-1 glycoprotein gp120. Possible role in AIDS pathogenesis

Dysfunction of cytokine secretion pattern has been suggested to play a central role in the immunopathogenesis of HIV infection. In fact a shift of T helper cell functions from a Th1-type to TH0- or TH2-type has been observed in HIV-1 infected subjects undergoing disease progression. The inhalance of cytokine network is accompanied by persistent activation of the immune system, impaired ability to mount a proper activation response (anergy), and priming to apoptosis. Extensive investigation during the last decade has been conducted on the influence of HIV-1 gp120 or of its precursor gp160 on several lymphocyte and monocyte functions. Gp120 is able to rise intracellular calcium concentration and to induce the formation of inositol triphosphate, can block mitogen- or antigen-driven T cell activation, can induce altered cytokine production by activated PBMC subpopulations, determines impaired cytotoxicity and chemotactic response to antigens, interferes with the activity of antigen presenting cells, enhances or induces apoptosis, stimulates polyclonal B cell activation and induces or up-modulates a number of cytokines, including IL-6. TNF, IL-1-alpha and -beta, IL-10 and IL-8. Furthermore, both IFN-alpha and -gamma, as well as several markers of IFN activity, such as beta 2-microglobulin and neopterin, are induced in gp120-stimulated PBMC. However, neither IL-4 (Th2-type) nor IL-2 (Th1-type), nor DNA synthesis are activated by gp120. On the other hand gp120-stimulated PBMC express increased IL-2 receptors, and can be induced by exogenous IL-2 to proliferate, suggesting that they are in a state of at least partial activation. According to this hypothesis, other activation markers, both early (such as CD69), and late (such as CD45RO and CD71), are induced by gp120, but this even partial activation does not lead to the ability of PBMC to support productive infection by HIV-1, unless in the presence of exogenous IL-2. The HIV-induced cytokines can influence HIV infection either directly, by up- or down-modulating virus replication, or indirectly, by modulating the expression of cellular molecules. In fact, during the budding process, the HIV envelope captures a number of cell membrane proteins, including cytokine receptors such as IL-2R, adhesion molecules such as LFA-1, ICAM-1, -2, HLA Class I and II, as well as cell lineage markers. Gp120-induced cytokines, particularly IFN-gamma, upmodulate the cellular expression of intercellular adhesion molecules, such as ICAM-1. We have shown that the IFN-gamma-driven increase of the expression of ICAM-1 by cells chronically infected with HIV-1 can be transmitted to the virus progeny, resulting in phenotypic alteration of the virus, and leading to the expansion of its host cell spectrum to CD4-negative cells expressing the appropriate ligands, i.e. LFA-1. Intercellular adhesion molecules are also involved in the cell-mediated transmission of HIV infection, and the increased ICAM-1 expression induced by IFN-gamma determines a stimulation of the transmission of HIV from abortively infected endothelial cells to permissive CD4 lymphocytes. On the whole, these data indicate that HIV, or its soluble products such as gp120, can modify several PBMC functions, by inducing a number of cytokines and a partial state of immune activation. It is possible that the gp120-driven changes of PBMC functions are not only an epiphenomenon of HIV infection, but rather, it is likely that they can participate in the immunopathological events responsible for disease progression.