Soluble CD4 and low molecular weight CD4-mimetic compounds sensitize cells to be killed by anti-HIV cytotoxic immunoconjugates.

IMPORTANCE: HIV infection can be effectively treated to prevent the development of AIDS, but it cannot be cured. We have attached poisons to anti-HIV antibodies to kill the infected cells that persist even after years of effective antiviral therapy. Here we show that the killing of infected cells can be markedly enhanced by the addition of soluble forms of the HIV receptor CD4 or by mimics of CD4.

Identification of Human Anti-HIV gp160 Monoclonal Antibodies That Make Effective Immunotoxins.

The envelope (Env) glycoprotein of HIV is the only intact viral protein expressed on the surface of both virions and infected cells. Env is the target of neutralizing antibodies (Abs) and has been the subject of intense study in efforts to produce HIV vaccines. Therapeutic anti-Env Abs can also exert antiviral effects via Fc-mediated effector mechanisms or as cytotoxic immunoconjugates, such as immunotoxins (ITs). In the course of screening monoclonal antibodies (MAbs) for their ability to deliver cytotoxic agents to infected or Env-transfected cells, we noted disparities in their functional activities. Different MAbs showed diverse functions that did not correlate with each other. For example, MAbs against the external loop region of gp41 made the most effective ITs against infected cells but did not neutralize virus and bound only moderately to the same cells that they killed so effectively when they were used in ITs. There were also differences in IT-mediated killing among transfected and infected cell lines that were unrelated to the binding of the MAb to the target cells. Our studies of a well-characterized antigen demonstrate that MAbs against different epitopes have different functional activities and that the binding of one MAb can influence the interaction of other MAbs that bind elsewhere on the antigen. These results have implications for the use of MAbs and ITs to kill HIV-infected cells and eradicate persistent reservoirs of HIV infection. IMPORTANCE: There is increased interest in using antibodies to treat and cure HIV infection. Antibodies can neutralize free virus and kill cells already carrying the virus. The virus envelope (Env) is the only HIV protein expressed on the surfaces of virions and infected cells. In this study, we examined a panel of human anti-Env antibodies for their ability to deliver cell-killing toxins to HIV-infected cells and to perform other antiviral functions. The ability of an antibody to make an effective immunotoxin could not be predicted from its other functional characteristics, such as its neutralizing activity. Anti-HIV immunotoxins could be used to eliminate virus reservoirs that persist despite effective antiretroviral therapy.

Design and In Vivo Characterization of Immunoconjugates Targeting HIV gp160.

The envelope (Env) glycoprotein of HIV is expressed on the surface of productively infected cells and can be used as a target for cytotoxic immunoconjugates (ICs), in which cell-killing moieties, including toxins, drugs, or radionuclides, are chemically or genetically linked to monoclonal antibodies (MAbs) or other targeting ligands. Such ICs could be used to eliminate persistent reservoirs of HIV infection. We have found that MAbs which bind to the external loop of gp41, e.g., MAb 7B2, make highly effective ICs, particularly when used in combination with soluble CD4. We evaluated the toxicity, immunogenicity, and efficacy of the ICs targeted with 7B2 in mice and in simian-human immunodeficiency virus-infected macaques. In the macaques, we tested immunotoxins (ITs), consisting of protein toxins bound to the targeting agent. ITs were well tolerated and initially efficacious but were ultimately limited by their immunogenicity. In an effort to decrease immunogenicity, we tested different toxic moieties, including recombinant toxins, cytotoxic drugs, and tubulin inhibitors. ICs containing deglycosylated ricin A chain prepared from ricin toxin extracted from castor beans were the most effective in killing HIV-infected cells. Having identified immunogenicity as a major concern, we show that conjugation of IT to polyethylene glycol limits immunogenicity. These studies demonstrate that cytotoxic ICs can target virus-infected cells in vivo but also highlight potential problems to be addressed. IMPORTANCE: It is not yet possible to cure HIV infection. Even after years of fully effective antiviral therapy, a persistent reservoir of virus-infected cells remains. Here we propose that a targeted conjugate consisting of an anti-HIV antibody bound to a toxic moiety could function to kill the HIV-infected cells that constitute this reservoir. We tested this approach in HIV-infected cells grown in the lab and in animal infections. Our studies demonstrated that these immunoconjugates are effective both in vitro and in test animals. In particular, ITs constructed with the deglycosylated A chain prepared from native ricin were the most effective in killing cells, but their utility was blunted because they provoked immune reactions that interfered with the therapeutic effects. We then demonstrated that coating of the ITs with polyethylene glycol minimized the immunogenicity, as has been demonstrated with other protein therapies.

Animal models of ricin toxicosis.

Animal models of ricin toxicosis are necessary for testing the efficacy of therapeutic measures, as well studying the mechanisms by which ricin exerts its toxicity in intact animals. Because ricin can serve as a particularly well-characterized model of tissue damage, and the host response to that damage, studies of the mechanisms of ricin toxicity may have more general applicability. For example, our studies of the molecular mechanisms underlying the development of ricin-induced hypoglycemia may help elucidate the relationship of type II diabetes, insulin resistance, and inflammation. Studies in non-human primates are most relevant for testing and developing agents having clinical utility. But these animals are expensive and limited in quantity, and so rodents are used for most mechanistic studies.

Improved guanide compounds which bind the CXCR4 co-receptor and inhibit HIV-1 infection.

The G-protein coupled receptor CXCR4 is a co-receptor for HIV-1 infection and is involved in signaling cell migration and proliferation. In a previous study of non-peptide, guanide-based CXCR4-binding compounds, spermine and spermidine phenylguanides inhibited HIV-1 entry at low micromolar concentrations. Subsequently, crystal structures of CXCR4 were used to dock a series of naphthylguanide derivatives of the polyamines spermidine and spermine. Synthesis and evaluation of the naphthylguanide compounds identified our best compound, spermine tris-1-naphthylguanide, which bound CXCR4 with an IC(50) of 40 nM and inhibited the infection of TZM-bl cells with X4, but not R5, strains of HIV-1 with an IC(50) of 50-100 nM.

Identification of Anti-gp41 Monoclonal Antibodies That Effectively Target Cytotoxic Immunoconjugates to Cells Infected with Human Immunodeficiency Virus, Type 1.

We are developing cytotoxic immunoconjugates (CICs) targeting the envelope protein (Env) of the Human Immunodeficiency Virus, type 1 (HIV) to purge the persistent reservoirs of viral infection. We have previously studied the ability of multiple monoclonal antibodies (mAbs) to deliver CICs to an HIV-infected cell. We have found that CICs targeted to the membrane-spanning gp41 domain of Env are most efficacious, in part because their killing is enhanced in the presence of soluble CD4. The ability of a mAb to deliver a CIC does not correlate with its ability to neutralize nor mediate Ab-dependent cellular cytotoxicity. In the current study, we seek to define the most effective anti-gp41 mAbs for delivering CICs to HIV-infected cells. To do this, we have evaluated a panel of human anti-gp41 mAbs for their ability to bind and kill two different Env-expressing cell lines: persistently infected H9/NL4-3 and constitutively transfected HEK293/92UG. We measured the binding and cytotoxicity of each mAb in the presence and absence of soluble CD4. We found that mAbs to the immunodominant helix-loop-helix region (ID-loop) of gp41 are most effective, whereas neutralizing mAbs to the fusion peptide, gp120/gp41 interface, and the membrane proximal external region (MPER) are relatively ineffective at delivering CICs. There was only a weak correlation between antigen exposure and killing activity. The results show that the ability to deliver an effective IC and neutralization are distinct functions of mAbs.

Human CD1D gene expression is regulated by LEF-1 through distal promoter regulatory elements.

CD1d-expressing cells present lipid Ag to CD1d-restricted NKT cells, which play an important role in immune regulation and tumor rejection. Lymphoid enhancer-binding factor-1 (LEF-1) is one of the regulators of the Wnt signaling pathway, which is a powerful regulator in cellular growth, differentiation, and transformation. There is little evidence connecting Wnt signaling to CD1d expression. In this study, we have identified LEF-1 as a regulator of the expression of the gene encoding the human CD1d molecule (CD1D). We found that LEF-1 binds specifically to the CD1D promoter. Overexpression of LEF-1 in K562 or Jurkat cells suppresses CD1D promoter activity and downregulates endogenous CD1D transcripts, whereas knockdown of LEF-1 using LEF-1-specific small interfering RNA increases CD1D transcripts in K562 and Jurkat cells but there are different levels of surface CD1d on these two cell types. Chromatin immunoprecipitation showed that the endogenous LEF-1 is situated at the CD1D promoter and interacts with histone deacetylase-1 to facilitate the transcriptional repressor activity. Knockdown of LEF-1 using small interfering RNA potentiates an acetylation state of histone H3/H4, supporting the notion that LEF-1 acts as a transcriptional repressor for the CD1D gene. Our finding links LEF-1 to CD1D and suggests a role of Wnt signaling in the regulation of the human CD1D gene.

Parenteral Exposure of Mice to Ricin Toxin Induces Fatal Hypoglycemia by Cytokine-Mediated Suppression of Hepatic Glucose-6-Phosphatase Expression.

Ricin toxin is an agent of biodefense concern and we have been developing countermeasures for ricin threats. In doing so, we sought biomarkers of ricin toxicosis and found that in mice parenteral injection of ricin toxin causes profound hypoglycemia, in the absence of other clinical laboratory abnormalities. We now seek to identify the mechanisms underlying this hypoglycemia. Within the first hours following injection, while still normoglycemic, lymphopenia and pro-inflammatory cytokine secretion were observed, particularly tumor necrosis factor (TNF)-α. The cytokine response evolved over the next day into a complex storm of both pro- and anti-inflammatory cytokines. Evaluation of pancreatic function and histology demonstrated marked islet hypertrophy involving predominantly ß-cells, but only mildly elevated levels of insulin secretion, and diminished hepatic insulin signaling. Drops in blood glucose were observed even after destruction of ß-cells with streptozotocin. In the liver, we observed a rapid and persistent decrease in the expression of glucose-6-phosphatase (G6Pase) RNA and protein levels, accompanied by a drop in glucose-6-phosphate and increase in glycogen. TNF-α has previously been reported to suppress G6Pase expression. In humans, a genetic deficiency of G6Pase results in glycogen storage disease, type-I (GSD-1), a hallmark of which is potentially fatal hypoglycemia.

Distinct Metabolic States Are Observed in Hypoglycemia Induced in Mice by Ricin Toxin or by Fasting.

Hypoglycemia may be induced by a variety of physiologic and pathologic stimuli and can result in life-threatening consequences if untreated. However, hypoglycemia may also play a role in the purported health benefits of intermittent fasting and caloric restriction. Previously, we demonstrated that systemic administration of ricin toxin induced fatal hypoglycemia in mice. Here, we examine the metabolic landscape of the hypoglycemic state induced in the liver of mice by two different stimuli: systemic ricin administration and fasting. Each stimulus produced the same decrease in blood glucose and weight loss. The polar metabolome was studied using 1H NMR, quantifying 59 specific metabolites, and untargeted LC-MS on approximately 5000 features. Results were analyzed by multivariate analyses, using both principal component analysis (PCA) and partial least squares-discriminant analysis (PLS-DA), to identify global metabolic patterns, and by univariate analyses (ANOVA) to assess individual metabolites. The results demonstrated that while there were some similarities in the responses to the two stimuli including decreased glucose, ADP, and glutathione, they elicited distinct metabolic states. The metabolite showing the greatest difference was O-phosphocholine, elevated in ricin-treated animals and known to be affected by the pro-inflammatory cytokine TNF-α. Another difference was the alternative fuel source utilized, with fasting-induced hypoglycemia primarily ketotic, while the response to ricin-induced hypoglycemia involves protein and amino acid catabolism.

Novel compounds containing multiple guanide groups that bind the HIV coreceptor CXCR4.

The G-protein-coupled receptor CXCR4 acts as a coreceptor for human immunodeficiency virus type 1 (HIV-1) infection, as well as being involved in signaling cell migration and proliferation. Compounds that block CXCR4 interactions have potential uses as HIV entry inhibitors to complement drugs such as maraviroc that block the alternate coreceptor CCR5 or in cancer therapy. The peptide T140, which contains five arginine residues, is the most potent antagonist of CXCR4 developed to date. In a search for nonpeptide CXCR4 ligands that could inhibit HIV entry, three series of compounds were synthesized from 12 linear and branched polyamines with 2, 3, 4, 6, or 8 amino groups, which were substituted to produce the corresponding guanidines, biguanides, or phenylguanides. The resulting compounds were tested for their ability to compete with T140 for binding to the human CXCR4 receptor expressed on mammalian cells. The most effective compounds bound CXCR4 with a 50% inhibitory concentration of 200 nM, and all of the compounds had very low cytotoxicity. Two series of compounds were then tested for their ability to inhibit the infection of TZM-bl cells with X4 and R5 strains of HIV-1. Spermine phenylguanide and spermidine phenylguanide inhibited infection by X4 strains, but not by R5 strains, at low micromolar concentrations. These results support further investigation and development of these compounds as HIV entry inhibitors.

Biophysical Evaluation of Rhesus Macaque Fc Gamma Receptors Reveals Similar IgG Fc Glycoform Preferences to Human Receptors.

Rhesus macaques are a common non-human primate model used in the evaluation of human monoclonal antibodies, molecules whose effector functions depend on a conserved N-linked glycan in the Fc region. This carbohydrate is a target of glycoengineering efforts aimed at altering antibody effector function by modulating the affinity of Fcγ receptors. For example, a reduction in the overall core fucose content is one such strategy that can increase antibody-mediated cellular cytotoxicity by increasing Fc-FcγRIIIa affinity. While the position of the Fc glycan is conserved in macaques, differences in the frequency of glycoforms and the use of an alternate monosaccharide in sialylated glycan species add a degree of uncertainty to the testing of glycoengineered human antibodies in rhesus macaques. Using a panel of 16 human IgG1 glycovariants, we measured the affinities of macaque FcγRs for differing glycoforms via surface plasmon resonance. Our results suggest that macaques are a tractable species in which to test the effects of antibody glycoengineering.

Bispecific Anti-HIV Immunoadhesins That Bind Gp120 and Gp41 Have Broad and Potent HIV-Neutralizing Activity.

We have constructed bispecific immunoglobulin-like immunoadhesins that bind to both the HIV-envelope glycoproteins: gp120 and gp41. These immunoadhesins have N terminal domains of human CD4 engrafted onto the N-terminus of the heavy chain of human anti-gp41 mAb 7B2. Binding of these constructs to recombinant Env and their antiviral activities were compared to that of the parental mAbs and CD4, as well as to control mAbs. The CD4/7B2 constructs bind to both gp41 and gp140, as well as to native Env expressed on the surface of infected cells. These constructs deliver cytotoxic immunoconjugates to HIV-infected cells, but not as well as a mixture of 7B2 and sCD4, and opsonize for antibody-mediated phagocytosis. Most surprisingly, given that 7B2 neutralizes weakly, if at all, is that the chimeric CD4/7B2 immunoadhesins exhibit broad and potent neutralization of HIV, comparable to that of well-known neutralizing mAbs. These data add to the growing evidence that enhanced neutralizing activity can be obtained with bifunctional mAbs/immunoadhesins. The enhanced neutralization activity of the CD4/7B2 chimeras may result from cross-linking of the two Env subunits with subsequent inhibition of the pre-fusion conformational events that are necessary for entry.

Post-exposure targeting of specific epitopes on ricin toxin abrogates toxin-induced hypoglycemia, hepatic injury, and lethality in a mouse model.

Effects in the liver of fatal intoxication with the binary toxin ricin are unclear. We report a robust neutrophil influx into the liver of C57BL/6 mice after lethal parenteral ricin challenge, occurring in peri-portal and centro-lobular hepatic areas within 2 h, followed by the abrupt disappearance of hepatic macrophages/Kupffer cells. Chemokine profiles determined by microarray, ribonuclease protection assays, northern blotting, and enzyme-linked immunosorbent assays showed rapid (2 h) upregulation and persistence of those for neutrophils (CXCL1/KC, CXCL2/MIP-2) and monocytes (CCL2/MCP-1). Red blood cell pooling (8-12 h), loss of hepatocyte glycogen (8-48 h) associated with progressive hypoglycemia, fibrin deposition (24-48 h), and death (72-96 h) followed. Monoclonal antibody to ricin A chain, administered intravenously, blunted hypoglycemia, and abrogated death. This outcome was observed when anti-ricin antibody was given before toxin exposure as well as when administered approximately 10 h after toxin exposure. Targeting antibody to specific amino-acid sequences on the ricin A chain (HAEL and QXXWXXA) was critical to the therapeutic effect. Re-emergence of liver macrophages/Kupffer cells and replenishment of glycogen in previously depleted hepatocytes preceded full recovery of the host. These data identify critical events for liver injury and healing in ricin intoxication, as well as a new means and specific targets for post-exposure therapeutic intervention.

Peptides selected from a phage display library with an HIV-neutralizing antibody elicit antibodies to HIV gp120 in rabbits, but not to the same epitope.

Monoclonal antibodies specific for the conserved CD4 binding site region of the HIV envelope protein gp120 were used to select phage from two different random peptide display libraries. Synthetic peptides were made with sequences corresponding to those displayed on the selected phage, and peptide-protein fusions were expressed that contained the selected phage-displayed peptide sequence and either the N-terminal domain of the phage pIII protein or the small heat shock protein of Methanococcus jannaschii or both. For monoclonal antibody 5145A, these constructs containing the selected peptide sequences were all capable of specifically inhibiting the binding of 5145A to HIV-1 gp120. Rabbits immunized with peptide-protein fusions produced antisera that bound to recombinant HIV-1 gp120, but did not bind to HIV-infected cells nor neutralize HIV. The antisera also did not compete with CD4 or antibodies to the CD4 binding site for binding to gp120.

Selective cytotoxicity of a novel immunotoxin based on pulchellin A chain for cells expressing HIV envelope.

Immunotoxins (ITs), which consist of antibodies conjugated to toxins, have been proposed as a treatment for cancer and chronic infections. To develop and improve the ITs, different toxins such as ricin, have been used, aiming for higher efficacy against target cells. The toxin pulchellin, isolated from the Abrus pulchellus plant, has similar structure and function as ricin. Here we have compared two plant toxins, recombinant A chains from ricin (RAC) and pulchellin (PAC) toxins, for their ability to kill HIV Env-expressing cells. In this study, RAC and PAC were produced in E. coli, and chromatographically purified, then chemically conjugated to two different anti-HIV monoclonal antibodies (MAbs), anti-gp120 MAb 924 or anti-gp41 MAb 7B2. These conjugates were characterized biochemically and immunologically. Cell internalization was studied by flow cytometry and confocal microscopy. Results showed that PAC can function within an effective IT. The ITs demonstrated specific binding against native antigens on persistently HIV-infected cells and recombinant antigens on Env-transfected cells. PAC cytotoxicity appears somewhat less than RAC, the standard for comparison. This is the first report that PAC may have utility for the design and construction of therapeutic ITs, highlighting the potential role for specific cell targeting.

Coupling Peptide Antigens to Virus-Like Particles or to Protein Carriers Influences the Th1/Th2 Polarity of the Resulting Immune Response.

We have conjugated the S9 peptide, a mimic of the group B streptococcal type III capsular polysaccharide, to different carriers in an effort to elicit an optimal immune response. As carriers, we utilized the soluble protein keyhole limpet hemocyanin and virus-like particles (VLPs) from two plant viruses, Cowpea Chlorotic Mottle Virus and Cowpea Mosaic Virus. We have found that coupling the peptide to the soluble protein elicits a Th2 immune response, as evidenced by the production of the peptide-specific IgG1 antibody and IL-4/IL-10 production in response to antigen stimulation, whereas the peptide conjugated to VLPs elicited a Th1 response (IgG2a, IFN-γ). Because the VLPs used as carriers package RNA during the assembly process, we hypothesize that this effect may result from the presence of nucleic acid in the immunogen, which affects the Th1/Th2 polarity of the response.

Recent advances in the development of vaccines against ricin.

Several promising subunit vaccines against ricin toxin (RT) have been developed during the last decade and are now being tested for safety and immunogenicity in humans and for efficacy in nonhuman primates. The incentive to develop a preventive vaccine as a countermeasure against RT use as a bioweapon is based on the high toxicity of RT after aerosol exposure, its environmental stability, abundance, and ease of purification. RT is the second most lethal biological toxin and is considered a "universal toxin" because it can kill all eukaryotic cells through binding to ubiquitous cell surface galactosyl residues. RT has two subunits conjoined by a single disulfide linkage: RTB, which binds galactosyl residues and RTA which enzymatically inactivates ribosomes intracellularly by cleavage ribosomal RNA. Attenuation of toxicity by elimination of the active site or introduction of other structural mutations of RTA has generated two similar clinical subunit vaccine candidates which induce antibodies in both humans and nonhuman primates. In rhesus macaques, inhaled RT causes rapid lung necrosis and fibrosis followed by death. After parenteral vaccination with RTA vaccine, macaques can be protected against aerosol RT exposure, suggesting that circulating antibodies can protect lung mucosa. Vaccination induces RT-neutralizing antibodies, the most likely correlate of protection. Macaques responded to conformational determinants in an RTA vaccine formulation, indicating preservation of RTA structure during initial manufacture. Comparative mapping studies have also demonstrated that macaques and humans recognize the same epitopes, significant in the study of macaques as a model during development of vaccines which cannot be tested for efficacy in humans.

Clinical and Pathological Findings Associated with Aerosol Exposure of Macaques to Ricin Toxin.

Ricin is a potential bioweapon that could be used against civilian and military personnel. Aerosol exposure is the most likely route of contact to ricin toxin that will result in the most severe toxicity. Early recognition of ricin exposure is essential if specific antidotes are to be applied. Initial diagnosis will most likely be syndromic, i.e., fitting clinical and laboratory signs into a pattern which then will guide the choice of more specific diagnostic assays and therapeutic interventions. We have studied the pathology of ricin toxin in rhesus macaques exposed to lethal and sublethal ricin aerosols. Animals exposed to lethal ricin aerosols were followed clinically using telemetry, by clinical laboratory analyses and by post-mortem examination. Animals exposed to lethal aerosolized ricin developed fever associated with thermal instability, tachycardia, and dyspnea. In the peripheral blood a marked neutrophilia (without immature bands) developed at 24 h. This was accompanied by an increase in monocytes, but depletion of lymphocytes. Red cell indices indicated hemoconcentration, as did serum chemistries, with modest increases in sodium and blood urea nitrogen (BUN). Serum albumin was strikingly decreased. These observations are consistent with the pathological observations of fluid shifts to the lungs, in the form of hemorrhages, inflammatory exudates, and tissue edema. In macaques exposed to sublethal aerosols of ricin, late pathologic consequences included chronic pulmonary fibrosis, likely mediated by M2 macrophages. Early administration of supportive therapy, specific antidotes after exposure or vaccines prior to exposure have the potential to favorably alter this outcome.

A modified SCID mouse model of HIV infection with utility for testing anti-HIV therapies.

Using human tumor cells we have developed a mouse model of active HIV infection that may be used for testing antiviral agents, although it does not reflect the pathogenesis of human infection. Irradiated SCID/NOD mice are injected with a tumor of human CD4+ lymphoma cells susceptible to infection and at a separate site, tumor cells persistently infected with either primary or T cell line-adapted strains of HIV. The spread of infection from the infected to the susceptible tumor is monitored as plasma p24 and the presence of HIV-infected cells in the spleen. We have used this model to examine the relative efficacy of neutralizing anti-HIV antibodies to halt the spread of infection. We have found that the tetrameric CD4-antibody fusion protein, CD4-IgG2, is highly effective compared to an anti-V3 loop antibody. This animal model, while not replicating the human disease, allows for the simultaneous testing of efficacy, toxicity, and pharmacokinetics of potential new antiviral therapies. The model can easily be powered to enable comparisons between different therapeutic agents and dosing regimens.

Comparison of different microarray data analysis programs and description of a database for microarray data management.

Data analysis and management represent a major challenge for gene expression studies using microarrays. Here, we compare different methods of analysis and demonstrate the utility of a personal microarray database. Gene expression during HIV infection of cell lines was studied using Affymetrix U-133 A and B chips. The data were analyzed using Affymetrix Microarray Suite and Data Mining Tool, Silicon Genetics GeneSpring, and dChip from Harvard School of Public Health. A small-scale database was established with FileMaker Pro Developer to manage and analyze the data. There was great variability among the programs in the lists of significantly changed genes constructed from the same data. Similarly choices of different parameters for normalization, comparison, and standardization greatly affected the outcome. As many probe sets on the U133 chip target the same Unigene clusters, the Unigene information can be used as an internal control to confirm and interpret the probe set results. Algorithms used for the determination of changes in gene expression require further refinement and standardization. The use of a personal database powered with Unigene information can enhance the analysis of gene expression data.