Prevalence and Significance of Substitutions in the Fusion Protein of Respiratory Syncytial Virus Resulting in Neutralization Escape From Antibody MEDI8897.

Background: Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract infection among infants and young children. To date, no vaccine is approved for the broad population of healthy infants. MEDI8897, a potent anti-RSV fusion antibody with extended serum half-life, is currently under clinical investigation as a potential passive RSV vaccine for all infants. As a ribonucleic acid virus, RSV is prone to mutation, and the possibility of viral escape from MEDI8897 neutralization is a potential concern. Methods: We generated RSV monoclonal antibody (mAb)-resistant mutants (MARMs) in vitro and studied the effect of the amino acid substitutions identified on binding and viral neutralization susceptibility to MEDI8897. The impact of resistance-associated mutations on in vitro growth kinetics and the prevalence of these mutations in currently circulating strains of RSV in the United States was assessed. Results: Critical residues identified in MARMs for MEDI8897 neutralization were located in the MEDI8897 binding site defined by crystallographic analysis. Substitutions in these residues affected the binding of mAb to virus, without significant impact on viral replication in vitro. The frequency of natural resistance-associated polymorphisms was low. Conclusions: Results from this study provide insights into the mechanism of MEDI8897 escape and the complexity of monitoring for emergence of resistance.

Neutralizing antibody affords comparable protection against vaginal and rectal simian/human immunodeficiency virus challenge in macaques.

OBJECTIVE: Passive administration of broadly neutralizing antibodies has been shown to protect against both vaginal and rectal challenge in the simian/human immunodeficiency virus (SHIV)/macaque model of HIV transmission. However, the relative efficacy of antibody against the two modes of exposure is unknown and, given differences in the composition and immunology of the two tissue compartments, this is an important gap in knowledge. To investigate the significance of the challenge route for antibody-mediated protection, we performed a comparative protection study in macaques using the highly potent human monoclonal antibody, PGT126. DESIGN: Animals were administered PGT126 at three different doses before challenged either vaginally or rectally with a single dose of SHIVSF163P3. METHODS: Viral loads, PGT126 serum concentrations, and serum neutralizing titers were monitored. RESULTS: In vaginally challenged animals, sterilizing immunity was achieved in all animals administered 10 mg/kg, in two of five animals administered 2 mg/kg and in one of five animals administered 0.4 mg/kg PGT126. Comparable protection was observed for the corresponding groups challenged rectally as sterilizing immunity was achieved in three of four animals administered 10 mg/kg, in two of four animals administered 2 mg/kg and in none of four animals administered 0.4 mg/kg PGT126. Serological analysis showed similar serum concentrations of PGT126 and serum neutralization titers in animals administered the same antibody dose. CONCLUSION: Our data suggest that broadly neutralizing antibody-mediated protection is not strongly dependent on the mucosal route of challenge, which indicates that a vaccine aimed to induce a neutralizing antibody response would have broadly similar efficacy against both primary transmission routes for HIV.

Passive transfer of modest titers of potent and broadly neutralizing anti-HIV monoclonal antibodies block SHIV infection in macaques.

It is widely appreciated that effective human vaccines directed against viral pathogens elicit neutralizing antibodies (NAbs). The passive transfer of anti-HIV-1 NAbs conferring sterilizing immunity to macaques has been used to determine the plasma neutralization titers, which must be present at the time of exposure, to prevent acquisition of SIV/HIV chimeric virus (SHIV) infections. We administered five recently isolated potent and broadly acting anti-HIV neutralizing monoclonal antibodies (mAbs) to rhesus macaques and challenged them intrarectally 24 h later with either of two different R5-tropic SHIVs. By combining the results obtained from 60 challenged animals, we determined that the protective neutralization titer in plasma preventing virus infection in 50% of the exposed monkeys was relatively modest (∼1:100) and potentially achievable by vaccination.

DNA transposition by protein transduction of the piggyBac transposase from lentiviral Gag precursors.

DNA transposon-based vectors have emerged as gene vehicles with a wide biomedical and therapeutic potential. So far, genomic insertion of such vectors has relied on the co-delivery of genetic material encoding the gene-inserting transposase protein, raising concerns related to persistent expression, insertional mutagenesis and cytotoxicity. This report describes potent DNA transposition achieved by direct delivery of transposase protein. By adapting integrase-deficient lentiviral particles (LPs) as carriers of the hyperactive piggyBac transposase protein (hyPBase), we demonstrate rates of DNA transposition that are comparable with the efficiency of a conventional plasmid-based strategy. Embedded in the Gag polypeptide, hyPBase is robustly incorporated into LPs and liberated from the viral proteins by the viral protease during particle maturation. We demonstrate lentiviral co-delivery of the transposase protein and vector RNA carrying the transposon sequence, allowing robust DNA transposition in a variety of cell types. Importantly, this novel delivery method facilitates a balanced cellular uptake of hyPBase, as shown by confocal microscopy, and allows high-efficiency production of clones harboring a single transposon insertion. Our findings establish engineered LPs as a new tool for transposase delivery. We believe that protein transduction methods will increase applicability and safety of DNA transposon-based vector technologies.

The Neonatal Fc receptor (FcRn) enhances human immunodeficiency virus type 1 (HIV-1) transcytosis across epithelial cells.

The mechanisms by which human immunodeficiency virus type 1 (HIV-1) crosses mucosal surfaces to establish infection are unknown. Acidic genital secretions of HIV-1-infected women contain HIV-1 likely coated by antibody. We found that the combination of acidic pH and Env-specific IgG, including that from cervicovaginal and seminal fluids of HIV-1-infected individuals, augmented transcytosis across epithelial cells as much as 20-fold compared with Env-specific IgG at neutral pH or non-specific IgG at either pH. Enhanced transcytosis was observed with clinical HIV-1 isolates, including transmitted/founder strains, and was eliminated in Fc neonatal receptor (FcRn)-knockdown epithelial cells. Non-neutralizing antibodies allowed similar or less transcytosis than neutralizing antibodies. However, the ratio of total:infectious virus was higher for neutralizing antibodies, indicating that they allowed transcytosis while blocking infectivity of transcytosed virus. Immunocytochemistry revealed abundant FcRn expression in columnar epithelia lining the human endocervix and penile urethra. Acidity and Env-specific IgG enhance transcytosis of virus across epithelial cells via FcRn and could facilitate translocation of virus to susceptible target cells following sexual exposure.

Familial hypercholesterolemia and atherosclerosis in cloned minipigs created by DNA transposition of a human PCSK9 gain-of-function mutant.

Lack of animal models with human-like size and pathology hampers translational research in atherosclerosis. Mouse models are missing central features of human atherosclerosis and are too small for intravascular procedures and imaging. Modeling the disease in minipigs may overcome these limitations, but it has proven difficult to induce rapid atherosclerosis in normal pigs by high-fat feeding alone, and genetically modified models similar to those created in mice are not available. D374Y gain-of-function mutations in the proprotein convertase subtilisin/kexin type 9 (PCSK9) gene cause severe autosomal dominant hypercholesterolemia and accelerates atherosclerosis in humans. Using Sleeping Beauty DNA transposition and cloning by somatic cell nuclear transfer, we created Yucatan minipigs with liver-specific expression of human D374Y-PCSK9. D374Y-PCSK9 transgenic pigs displayed reduced hepatic low-density lipoprotein (LDL) receptor levels, impaired LDL clearance, severe hypercholesterolemia, and spontaneous development of progressive atherosclerotic lesions that could be visualized by noninvasive imaging. This model should prove useful for several types of translational research in atherosclerosis.

A panel of IgG1 b12 variants with selectively diminished or enhanced affinity for Fcγ receptors to define the role of effector functions in protection against HIV.

Passive transfer of neutralizing antibodies is effective in protecting rhesus macaques against simian/human immunodeficiency virus (SHIV) challenge. In addition to neutralization, effector functions of the crystallizable fragment (Fc) of antibodies are involved in antibody-mediated protection against a number of viruses. We recently showed that interaction between the Fc fragment of the broadly neutralizing antibody IgG1 b12 and cellular Fcγ receptors (FcγRs) plays an important role in protection against SHIV infection in rhesus macaques. The specific nature of this Fc-dependent protection is largely unknown. To investigate, we generated a panel of 11 IgG1 b12 antibody variants with selectively diminished or enhanced affinity for the two main activating FcγRs, FcγRIIa and FcγRIIIa. All 11 antibody variants bind gp120 and neutralize virus as effectively as does wild-type b12. Binding studies using monomeric (enzyme-linked immunosorbent assay [ELISA] and surface plasmon resonance [SPR]) and cellularly expressed Fcγ receptors show decreased (up to 5-fold) and increased (up to 90-fold) binding to FcγRIIa and FcγRIIIa with this newly generated panel of antibodies. In addition, there was generally a good correlation between b12 variant affinity for Fcγ receptor and variant function in antibody-dependent cell-mediated virus inhibition (ADCVI), phagocytosis, NK cell activation assays, and antibody-dependent cellular cytotoxicity (ADCC) assays. In future studies, these b12 variants will enable the investigation of the protective role of individual FcγRs in HIV infection.

Limited or no protection by weakly or nonneutralizing antibodies against vaginal SHIV challenge of macaques compared with a strongly neutralizing antibody.

To guide vaccine design, we assessed whether human monoclonal antibodies (MAbs) b12 and b6 against the CD4 binding site (CD4bs) on HIV-1 gp120 and F240 against an immundominant epitope on gp41 could prevent vaginal transmission of simian HIV (SHIV)-162P4 to macaques. The two anti-gp120 MAbs have similar monomeric gp120-binding properties, measured in vitro, but b12 is strongly neutralizing and b6 is not. F240 is nonneutralizing. Applied vaginally at a high dose, the strongly neutralizing MAb b12 provided sterilizing immunity in seven of seven animals, b6 in zero of five animals, and F240 in two of five animals. Compared with control animals, the protection by b12 achieved statistical significance, whereas that caused by F240 did not. For two of three unprotected F240-treated animals there was a trend toward lowered viremia. The potential protective effect of F240 may relate to the relatively strong ability of this antibody to capture infectious virions. Additional passive transfer experiments also indicated that the ability of the administered anti-gp120 MAbs to neutralize the challenge virus was a critical influence on protection. Furthermore, when data from all of the experiments were combined, there was a significant increase in the number of founder viruses establishing infection in animals receiving MAb b6, compared with other nonprotected macaques. Thus, a gp120-binding, weakly neutralizing MAb to the CD4bs was, at best, completely ineffective at protection. A nonneutralizing antibody to gp41 may have a limited capacity to protect, but the results suggest that the central focus of HIV-1 vaccine research should be on the induction of potently neutralizing antibodies.

Targeting of human interleukin-12B by small hairpin RNAs in xenografted psoriatic skin.

BACKGROUND: Psoriasis is a chronic inflammatory skin disorder that shows as erythematous and scaly lesions. The pathogenesis of psoriasis is driven by a dysregulation of the immune system which leads to an altered cytokine production. Proinflammatory cytokines that are up-regulated in psoriasis include tumor necrosis factor alpha (TNFα), interleukin-12 (IL-12), and IL-23 for which monoclonal antibodies have already been approved for clinical use. We have previously documented the therapeutic applicability of targeting TNFα mRNA for RNA interference-mediated down-regulation by anti-TNFα small hairpin RNAs (shRNAs) delivered by lentiviral vectors to xenografted psoriatic skin. The present report aims at targeting mRNA encoding the shared p40 subunit (IL-12B) of IL-12 and IL-23 by cellular transduction with lentiviral vectors encoding anti-IL12B shRNAs. METHODS: Effective anti-IL12B shRNAs are identified among a panel of shRNAs by potency measurements in cultured cells. The efficiency and persistency of lentiviral gene delivery to xenografted human skin are investigated by bioluminescence analysis of skin treated with lentiviral vectors encoding the luciferase gene. shRNA-expressing lentiviral vectors are intradermally injected in xenografted psoriatic skin and the effects of the treatment evaluated by clinical psoriasis scoring, by measurements of epidermal thickness, and IL-12B mRNA levels. RESULTS: Potent and persistent transgene expression following a single intradermal injection of lentiviral vectors in xenografted human skin is reported. Stable IL-12B mRNA knockdown and reduced epidermal thickness are achieved three weeks after treatment of xenografted psoriatic skin with lentivirus-encoded anti-IL12B shRNAs. These findings mimic the results obtained with anti-TNFα shRNAs but, in contrast to anti-TNFα treatment, anti-IL12B shRNAs do not ameliorate the psoriatic phenotype as evaluated by semi-quantitative clinical scoring and by immunohistological examination. CONCLUSIONS: Our studies consolidate the properties of lentiviral vectors as a tool for potent gene delivery and for evaluation of mRNA targets for anti-inflammatory therapy. However, in contrast to local anti-TNFα treatment, the therapeutic potential of targeting IL-12B at the RNA level in psoriasis is questioned.

Amelioration of psoriasis by anti-TNF-alpha RNAi in the xenograft transplantation model.

Tumor necrosis factor-alpha (TNF-alpha) is upregulated in psoriatic skin and represents a prominent target in psoriasis treatment. The level of TNF-alpha-encoding mRNA, however, is not increased in psoriatic skin, and it remains unclear whether intervention strategies based on RNA interference (RNAi) are therapeutically relevant. To test this hypothesis the present study describes first the in vitro functional screening of a panel of short hairpin RNAs (shRNAs) targeting human TNF-alpha mRNA and, next, the transfer of the most potent TNF-alpha shRNA variant, as assessed in vitro, to human skin in the psoriasis xenograft transplantation model by the use of lentiviral vectors. TNF-alpha shRNA treatment leads to amelioration of the psoriasis phentotype in the model, as documented by reduced epidermal thickness, normalization of the skin morphology, and reduced levels of TNF-alpha mRNA as detected in skin biopsies 3 weeks after a single vector injection of lentiviral vectors encoding TNF-alpha shRNA. Our data show efficient lentiviral gene delivery to psoriatic skin and therapeutic applicability of anti-TNF-alpha shRNAs in human skin. These findings validate TNF-alpha mRNA as a target molecule for a potential persistent RNA-based treatment of psoriasis and establish the use of small RNA effectors as a novel platform for target validation in psoriasis and other skin disorders.

Shielding of sleeping beauty DNA transposon-delivered transgene cassettes by heterologous insulators in early embryonal cells.

The Sleeping Beauty (SB) transposon system represents an important alternative to viral integrating vector systems but may, as its viral counterparts, be subject to transcriptional silencing. To investigate shielding of SB-delivered transgene cassettes against transcriptional repression, we establish silencing assays in which SB vector-containing F9 murine teratocarcinoma cell clones are identified by strategies that include or exclude selection for transgene expression. Among clones carrying one or more SB transposon vectors, more than one-third are immediately silenced, and most of the remaining clones move toward silencing during prolonged passage. In line with the lack of an intrinsic ability of SB to resist silencing, we show that the stable transfection rate of SB vectors in F9 cells is significantly improved by flanking the transgene with heterologous 5'-HS4 chicken beta-globin (cHS4) insulators. In approaches based on drug selection and subsequent flow-cytometric detection of transgene expression, clones containing cHS4-insulated vectors are to a much higher degree protected against transcriptional silencing, resulting in long-term expression of the fluorescent marker. Our findings demonstrate that SB vectors, prone for transcriptional silencing by positional effects in F9 cells, are protected by insulators. We believe that insulated SB-derived vectors will become useful tools in transposon-based transgenesis and therapeutic gene transfer.

Regulated gene insertion by steroid-induced PhiC31 integrase.

Nonviral integration systems are widely used genetic tools in transgenesis and play increasingly important roles in strategies for therapeutic gene transfer. Methods to efficiently regulate the activity of transposases and site-specific recombinases have important implications for their spatiotemporal regulation in live transgenic animals as well as for studies of their applicability as safe vectors for genetic therapy. In this report, strategies for posttranslational induction of a variety of gene-inserting proteins are investigated. An engineered hormone-binding domain, derived from the human progesterone receptor, hPR891, and specifically recognized by the synthetic steroid mifepristone, is fused to the Sleeping Beauty, Frog Prince, piggyBac and Tol2 transposases as well as to the Flp and PhiC31 recombinases. By analyzing mifepristone-directed inducibility of gene insertion in cultured human cells, efficient posttranslational regulation of the Flp recombinase and the PhiC31 integrase is documented. In addition, fusion of the PhiC31 integrase with the ER(T2) modified estrogen receptor hormone-binding domain results in a protein, which is inducible by a factor of 22-fold and retains 75% of the activity of the wild-type protein. These inducible PhiC31 integrase systems are important new tools in transgenesis and in safety studies of the PhiC31 integrase for gene therapy applications.