Structure and Function Analysis of Therapeutic Monoclonal Antibodies against Dengue Virus Type 2

Dengue virus (DENV) is the most prevalent insect-transmitted viral disease in humans globally, and currently no specific therapy or vaccine is available. Protection against DENV and other related flaviviruses is associated with the development of antibodies against the viral envelope (E) protein. Although prior studies have characterized the neutralizing activity of monoclonal antibodies (MAbs) against DENV type 2 (DENV-2), none have compared simultaneously the inhibitory activity against a genetically diverse range of strains in vitro, the protective capacity in animals, and the localization of epitopes. Here, with the goal of identifying MAbs that can serve as postexposure therapy, we investigated in detail the functional activity of a large panel of new anti-DENV-2 mouse MAbs. Binding sites were mapped by yeast surface display and neutralization escape, cell culture inhibition assays were performed with homologous and heterologous strains, and prophylactic and therapeutic activity was evaluated with two mouse models. Protective MAbs localized to epitopes on the lateral ridge of domain I (DI), the dimer interface, lateral ridge, and fusion loop of DII, and the lateral ridge, C-C′ loop, and A strand of DIII. Several MAbs inefficiently inhibited at least one DENV-2 strain of a distinct genotype, suggesting that recognition of neutralizing epitopes varies with strain diversity. Moreover, antibody potency generally correlated with a narrowed genotype and serotype specificity. Five MAbs functioned efficiently as postexposure therapy when administered as a single dose, even 3 days after intracranial infection of BALB/c mice. Overall, these studies define the structural and functional complexity of antibodies against DENV-2 with protective potential.Dengue virus (DENV), a member of the Flaviviridae family of RNA viruses, is related to several other human pathogens of global concern, including yellow fever and tick-borne, West Nile, and Japanese encephalitis viruses. DENV infection in humans occurs after Aedes aegypti or Aedes albopictus mosquito inoculation and results in clinical disease, ranging from a febrile illness (dengue fever [DF]) to a life-threatening hemorrhagic and capillary leak syndrome (dengue hemorrhagic fever [DHF]/dengue shock syndrome [DSS]). Globally, there is significant diversity among DENV strains, including four distinct serotypes (DENV type 1 [DENV-1], DENV-2, DENV-3, and DENV-4) that differ at the amino acid level by 25 to 40%. Additional complexity occurs within each serotype, as genotypes vary from one another by up to 3% at the amino acid level (21, 49). No approved antiviral treatment is currently available, and several candidate tetravalent vaccines remain in clinical development (reviewed in reference 11). Because of the increased geographic range of its mosquito vectors, urbanization, and international travel, DENV continues to spread worldwide and now causes an estimated 50 to 100 million infections and 250,000 to 500,000 cases of DHF/DSS per year, with 2.5 billion people at risk (68).DENV is an enveloped icosahedral virus with a single-stranded, positive-polarity RNA genome. The 10.7-kb genome is translated as a single polyprotein, which is cleaved into three structural proteins (capsid [C], premembrane/membrane [prM/M], and envelope [E]) and seven nonstructural (NS) proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) by host and viral proteases. The mature DENV virion is ∼500 Å in diameter, with a highly organized outer protein shell, a 50-Å lipid membrane bilayer, and a nucleocapsid core (26). Mature DENV virions are covered by 90 anti-parallel E protein homodimers, arranged flat along the surface with quasi-icosahedral symmetry. The immature virion, which lacks cleavage of the prM protein, has a rough surface with 60 spikes each composed of three prM-E heterodimers (7, 73). Exposure to mildly acidic conditions in the trans-Golgi network promotes virus maturation through a structural rearrangement of the flavivirus E proteins and cleavage of prM to M by a furin-like protease (29, 66, 69, 70). The ectodomain of DENV E protein is comprised of three discrete domains (34-36, 39). Domain I (DI) is a central, eight-stranded β-barrel, which contains a single N-linked glycan in most DENV strains. DII is a long, finger-like protrusion from DI, with the highly conserved fusion peptide at its distal end and a second N-linked glycan that recognizes DC-SIGN (37, 38, 46, 59). DIII, which adopts an immunoglobulin-like fold, has been suggested to contain cell surface receptor recognition sites (5, 64, 71). Several groups have recently defined contact residues for type-specific, subcomplex-specific, and cross-reactive monoclonal antibodies (MAbs) that recognize DIII of DENV-2 (16, 17, 31, 47, 57, 61). Type-specific MAbs with neutralizing activity against DENV-2 localized to the BC, DE, and FG loops on the lateral ridge of DIII, whereas subcomplex-specific MAbs recognized an adjacent epitope centered on the connecting A strand of DIII at residues K305, K307, and K310.To date, no study has compared the in vitro inhibitory activity of MAbs in cells against a genetically diverse range of DENV-2 strains and their protective capacity in animals. Here, we had the goal of generating strongly neutralizing MAbs that would recognize virtually all DENV-2 strains and function as a possible postexposure therapy. Twenty-four new anti-DENV-2 mouse MAbs were generated with moderate or strong neutralizing activity against the homologous virus in cell culture assays. Binding sites were mapped for the majority of these by yeast surface display, identifying distinct epitopes in regions in DI (lateral ridge), DII (dimer interface, lateral ridge, and fusion loop), and DIII (lateral ridge, C-C′ loop, and A strand). Several MAbs failed to neutralize efficiently at least one DENV-2 strain of a distinct genotype, suggesting that antibody recognition of neutralizing epitopes varies among DENV-2 genotypes.To begin to assess the utility of this new panel of inhibitory MAbs as possible therapeutics against DENV-2, we evaluated their protective capacity in a stringent intracranial challenge model in BALB/c mice. Among the 16 neutralizing MAbs tested in mice, most were protective when given as prophylaxis. Seven of these had postexposure therapeutic activity when administered as a single dose by intraperitoneal route even 3 days after intracranial infection. For the MAbs with the greatest therapeutic potential, protection was confirmed with an antibody-enhanced vascular leakage mouse model (2, 72) of DENV-2 infection.     

Dissemination of Microbial Agents Using an Autoinoculating Device and Several Insect Species as Vectors

A device for autoinoculating insects with marker dye and active agents, such as biocompetitors of plant pathogens (e.g., Trichoderma harzianum, Trichoderma polysporum, Bacillus subtilis) and entomopathogens (e.g., Bacillus thuringiensis, Beauveria bassiana) is described. Laboratory tests using a powdered dye indicated that the device worked for sap beetles, house flies, pomace dies, and moths. Quantitative studies with blue dye (as an indicator of dispersal) and some bioactive agents demonstrated that a high percentage of sap beetles became contaminated with the material placed inside the autoinoculator in a short period of time (minutes). In the laboratory, sap beetles carried B. bassiana from the autoinoculator to unexposed sap beetles, causing high mortality. In the field, sap beetles entered the baited traps attached to the autoinoculator, became contaminated with the dye, and carried it to damaged corn or apples.     

Differential effects of calcium on progesterone production in small and large bovine luteal cells

We studied the effects of calcium (Ca2+) ions in progesterone (P) production by separated small and large luteal cells. Corpora lutea were collected from 31 heifers between days 10 and 12 of the estrous cycle. Purified small and large cells were obtained by unit gravity sedimentation and flow cytometry. P accumulation in cells plus media was determined after incubating 1 x 10(5) small and 5 x 10(3) large cells for 2 and 4 h respectively. Removal of Ca2+ from the medium did not influence basal P production in the small cells (P greater than 0.05). However, stimulation of P by luteinizing hormone (LH), prostaglandin E2 (PGE2), 8-bromo-cyclic 3',5' adenosine monophosphate (8-Br-cAMP) and prostaglandin F2 alpha (PGF2 alpha) was impaired (P less than 0.05) by low Ca2+ concentrations. LH and PGE2-stimulated cAMP production was not altered by low extracellular Ca2+ concentrations, and PGF2 alpha had no effect on cAMP. In contrast, basal as well as LH and forskolin-stimulated P production were attenuated (P less than 0.05) in Ca2(+)-deficient medium in the large cells. However, P production stimulated by 8-Br-cAMP was not altered in Ca2(+)-deficient medium. Steroidogenesis in large cells was also dependent on intracellular Ca2+, since 8-N, N-diethylamineocytyl-3,4,5-trimethoxybenzoate (TMB-8), an inhibitor of intracellular Ca2+ release and/or action, suppressed (P less than 0.05) basal, LH and 8-Br-cAMP stimulated P. In contrast, basal P in small cells was not altered by TMB-8; whereas LH-stimulated P was reduced 2-fold (P less than 0.05). The calcium ionophore, A23187, inhibited LH-stimulated P in small cells and both basal and agonist-stimulated P in large cells. These studies show that basal P production in small cells does not require Ca2+ ions, while hormone-stimulated P production in small cells and both basal and hormone-stimulated P in large cells do require Ca2+. The inhibitory effect of Ca2+ ion removal was exerted prior to the generation of cAMP in the large cells, but distal to cAMP generation in hormone-stimulated small cells. The calmodulin/protein kinase C antagonist, W-7, also inhibited both basal and hormone-stimulated P production in both small and large luteal cells, indicating that P production in luteal cells also involves Ca2(+)-calmodulin/protein kinase C-dependent mechanisms.     

Effect of transgenic plants expressing high levels of a tobacco anionic peroxidase on the toxicity of Anagrapha falcifera nucleopolyhedrovirus to Helicoverpa zea (Lepidoptera: Noctuidae)

Wild type and corresponding transgenic tomato (Lycopersicon esculentum Miller) and two tobacco (Nicotiana spp.) plants that express high levels of a tobacco anionic peroxidase were used to determine what type of interactions occurred between peroxidase altered plant chemistry and the baculovirus Anagrapha falcifera nucleopolyhedrovirus (AfMNPV) for control of neonate corn earworms, Helicoverpa zea (Boddie). Transgenic plants expressed approximately five to 400 times higher peroxidase activity than corresponding tissues of wild type plants. The H. zea larvae typically fed 1.5 times less on transgenic compared with wild type leaf disks. There was only one experiment (of three with tomato leaves) where the larvae that fed on transgenic leaves were less susceptible to the virus based on nonoverlapping 95% confidence intervals for LC50 values. When the exposure dose was corrected for reduced feeding on the transgenic leaf disks, the insecticidal activity of the virus was not significantly different for larvae fed on transgenic versus wild type plants. Eight other experiments (with tomato and two species of tobacco) indicated either no significant effect or enhanced susceptibility (when corrected for feeding rates) to the virus of larvae fed on the transgenic leaves. These results indicate enhanced insect resistance in plants expressing high levels of a specific anionic peroxidase may be compatible with applications of AfMNPV. Potential reasons for this compatibility are discussed.     

Heterologous expression of bacteriocin E-760 in Chlamydomonas reinhardtii and functional analysis

The use of antimicrobial peptides (AMPs) synthesized by bacteria (bacteriocins) is an alternative for combating multidrug resistant bacterial strains and their production by recombinant route is a viable option for their mass production. The bacteriocin E-760 isolated from the genus Enterococcus sp. has been shown to possess inhibitory activity against Gram-negative and Gram-positive bacteria. In this study, the expression of a chimeric protein coding for E-760 in the nucleus of C. reinhardtii was evaluated, as well as, its antibacterial activity. The synthetic gene E-760S was inserted into the genome of C. reinhardtii using Agrobacterium tumefaciens. A transgenic line was identified in TAP medium with hygromycin and also by PCR. The increment in the culture medium temperature of the transgenic strain at 35 °C for 10 minutes, increased the production level of the recombinant protein from 0.14 (Noninduced culture, NIC) to 0.36% (Induced culture, IC) of total soluble proteins (TSP); this was quantified by an ELISA assay. Recombinant E-760 possesses activity against Staphylococcus aureus in 0.34 U log, Streptococcus agalactiae in 0.48 U log, Enterococcus faecium in 0.36 U log, Pseudomonas aeruginosa in 2 U log and for Klebsiella pneumoniae, the activity was 0.07 U log. These results demonstrate that the nucleus transformation of C. reinhardtii can function as a stable expression platform for the production of the synthetic gene E-760 and it can potentially be used as an antibacterial agent.     

Natural feeding influences protein expression in the dogfish shark rectal gland: A proteomic analysis

The rectal gland is the principal salt-secreting organ in elasmobranchs, yet its functional response to normal physiological variation (e.g., due to feeding, stress) has only recently been examined. To complement studies on acid-base, digestive, and osmoregulatory physiology in response to natural feeding, we investigated protein-level responses in the rectal gland of spiny dogfish (Squalus acanthias) 6 h, 20 h, and 5 days (reference control) after a meal. Our objective was to identify proteins involved in regulation of osmoregulatory and metabolic processes in response to feeding. Proteins were separated by two-dimensional gel electrophoresis, and protein spots that were significantly up- or down-regulated > 2 fold (i.e., abundance increased more than 100% or decreased more than 50%) were detected using gel image analysis software. Of 684 proteins analyzed on 2D gels, 16 proteins changed significantly 6 h after feeding vs. 5 day controls (5 decreased; 11 increased), and 12 proteins changed > 2 fold 20 h after feeding vs. 5 day controls (2 decreased; 10 increased). Thirteen of these proteins were identified using mass spectrometry and classified into functional pathways using the PANTHER bioinformatics database. Rectal gland proteins that were regulated following feeding fell into three main categories: cytoskeletal/muscular (e.g., tropomyosin alpha chain, transgelin), energy metabolism (e.g., malate dehydrogenase, ATP synthase), and nucleotide metabolism (nucleoside diphosphate kinase). The data also revealed that previously documented increases in the activity of isocitrate dehydrogenase after feeding are at least partially due to increased abundance of a cytosolic, NADP-dependent isoform of this enzyme. One of the primary components of the rectal gland's response to feeding appears to be maintenance of the cellular supply of energy, which would be necessary to fuel increased activities of enzymes involved in salt secretion and oxidative metabolism in the rectal gland following a meal.     

Responses of Carpophilus hemipterus larvae and adults to selected secondary metabolites of maize

Selected secondary metabolites produced by maize (Zea mays L.) were tested for effects on larvae and adults of the dried-fruit beetle [Carpophilus hemipterus (L.)] in no-choice and choice assays. Feeding by adults and larvae was significantly reduced by ferulic acid and 6-methoxy-2-benzoxazolinone (MBOA) in no-choice assays. In choice assays, larvae and adults generally preferred diets with trans-cinnamic acid, quercetin, rutin, and thymol, but were repelled by diets with either ferulic acid or MBOA.

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Résumé Les réactions de larves et adultes du nitidulidé C. hemipterus (L.), vecteur de champignons produisant la mycotoxine, aux composés phénoliques caractéristiques, aux flavonoïdes et aux acides hydroxamiques, métabolites secondaires qui provoquent la résistance du maïs (Zea mays L.) ont été examinées au cours d'expériences avec et sans choix. L'alimentation des adultes et des larves est généralement réduite par les acides coumarique et férulique et par la 6-méthoxy-2-benzoxazolinone dans des expériences sans choix; les insectes évitent généralement les aliments qui contiennent ces produits. Quoi qu'il en soit, les larves préfèrent consommer d'autres aliments contenant les autres phénoliques ou flavonoïdes examinés. Les adultes sont plus inconstants dans leur choix alimentaires, mais préfèrent souvent des aliments contenant de la quercetine. Ainsi, des programmes de sélection orientés contre les principaux ravageurs comme Heliothis zea (Boddie) ou Ostrinia nubilalis (Hübner), impliquant la sélection de plantes à teneur élevée en acides phénolique ou hydroxamique, augmentant probablement aussi la résistance aux nitidulidés.

     

From receptor recognition mechanisms to bioinspired mimetic antagonists in HIV-1/cell docking

Understanding the ways in which two or more proteins interact may give insight into underlying binding and activation mechanisms in biology, methods for protein separation and structure-based antagonism. This review describes ways in which protein recognition has been explored in our laboratory for the HIV-1/cell entry process. Initial contact between an HIV-1 virion particle and a human cell occurs between gp120 (an HIV-1 envelope protein) and CD4 (a human extracellular signaling protein). This interaction leads to a sequence of events which includes a conformational change in gp120, fusion of the HIV-1 and cellular membranes and eventual infection of the cell. Using an optical biosensor and a reporter antibody, we have been able to measure the conformational change in gp120 that occurs upon CD4 binding. We also have used this biosensor system to characterize CD4 mimetics, obtained by peptide synthesis in miniprotein scaffolds. Phage display techniques have been employed to identify novel miniprotein sequences. The combination of biosensor interaction kinetics analysis and phage display provides a useful approach for understanding the recognition mechanisms involved in the HIV/cell docking process. This approach may also be useful in investigating other protein complexes of importance in health and disease.