Increased Antibody Affinity Confers Broad In Vitro Protection against Escape Mutants of Severe Acute Respiratory Syndrome Coronavirus

Even though the effect of antibody affinity on neutralization potency is well documented, surprisingly, its impact on neutralization breadth and escape has not been systematically determined. Here, random mutagenesis and DNA shuffling of the single-chain variable fragment of the neutralizing antibody 80R followed by bacterial display screening using anchored periplasmic expression (APEx) were used to generate a number of higher-affinity variants of the severe acute respiratory syndrome coronavirus (SARS-CoV)-neutralizing antibody 80R with equilibrium dissociation constants (K(D)) as low as 37 pM, a >270-fold improvement relative to that of the parental 80R single-chain variable fragment (scFv). As expected, antigen affinity was shown to correlate directly with neutralization potency toward the icUrbani strain of SARS-CoV. Additionally, the highest-affinity antibody fragment displayed 10-fold-increased broad neutralization in vitro and completely protected against several SARS-CoV strains containing substitutions associated with antibody escape. Importantly, higher affinity also led to the suppression of viral escape mutants in vitro. Escape from the highest-affinity variant required reduced selective pressure and multiple substitutions in the binding epitope. Collectively, these results support the hypothesis that engineered antibodies with picomolar dissociation constants for a neutralizing epitope can confer escape-resistant protection.     

Cloning of aquaporin-1 of the blue crab, Callinectes sapidus: its expression during the larval development in hyposalinity

ABSTRACT: BACKGROUND: Ontogenetic variation in salinity adaptation has been noted for the blue crab, Callinectes sapidus, which uses the export strategy for larval development: females migrate from the estuaries to the coast to spawn, larvae develop in the ocean, and postlarvae (megalopae) colonize estuarine areas. We hypothesized that C. sapidus larvae may be stenohaline and have limited osmoregulatory capacity which compromises their ability to survive in lower salinity waters. We tested this hypothesis using hatchery-raised larvae that were traceable to specific life stages. In addition, we aimed to understand the possible involvement of AQP-1 in salinity adaptation during larval development and during exposure to hyposalinity. RESULTS: A full-length cDNA sequence of aquaporin (GenBank JQ970426) was isolated from the hypodermis of the blue crab, C. sapidus, using PCR with degenerate primers and 5[PRIME] and 3[PRIME] RACE. The open reading frame of CasAQP-1 consists of 238 amino acids containing six helical structures and two NPA motifs for the water pore. The expression pattern of CasAQP-I was ubiquitous in cDNAs from all tissues examined, although higher in the hepatopancreas, thoracic ganglia, abdominal muscle, and hypodermis and lower in the antennal gland, heart, hemocytes, ovary, eyestalk, brain, hindgut, Y-organs, and gill. Callinectes larvae differed in their capacity to molt in hyposalinity, as those at earlier stages from Zoea (Z) 1 to Z4 had lower molting rates than those from Z5 onwards, as compared to controls kept in 30 ppt water. No difference was found in the survival of larvae held at 15 and 30 ppt. CasAQP-1 expression differed with ontogeny during larval development, with significantly higher expression at Z1-2, compared to other larval stages. The exposure to 15 ppt affected larval-stage dependent CasAQP-1 expression which was significantly higher in Z2- 6 stages than the other larval stages. CONCLUSIONS: We report the ontogenetic variation in CasAQP-1 expression during the larval development of C. sapidus and the induction of its expression at early larval stages in the exposure of hyposalinity. However, it remains to be determined if the increase in CasAQP-1 expression at later larval stages may have a role in adaptation to hyposalinity.     

Facilitation and Dominance in a Schooling Predator: Foraging Behavior of Florida Pompano,Trachinotus carolinus

Presumably an individual’s risk of predation is reduced by group membership and this ‘safety in numbers’ concept has been readily applied to investigations of schooling prey; however, foraging in groups may also be beneficial. We tested the hypothesis that, when feeding in groups, foraging of a coastal fish (Florida Pompano, Trachinotus carolinus) on a benthic prey source would be facilitated (i.e. fish feeding in groups will consume more prey items). Although this question has been addressed for other fish species, it has not been previously addressed for Florida Pompano, a fish known to exhibit schooling behavior and that is used for aquaculture, where understanding the feeding ecology is important for healthy and efficient grow-out. In this experiment, juvenile Florida Pompano were offered a fixed number of coquina clams (Donax spp.) for one hour either in a group or as individuals. The following day they were tested in the opposite configuration. Fish in groups achieved greater consumption (average of 26 clams consumed by the entire group) than the individuals comprising the group (average of 14 clams consumed [sum of clams consumed by all individuals of the group]). Fish in groups also had fewer unsuccessful foraging attempts (2.75 compared to 4.75 hr^-1) and tended to have a shorter latency until the first feeding activity. Our results suggest fish in groups were more comfortable feeding and more successful in their feeding attempts. Interestingly, the consumption benefit of group foraging was not shared by all – not all fish within a group consumed equal numbers of clams. Taken together, the results support our hypothesis that foraging in a group provides facilitation, but the short-term benefits are not equally shared by all individuals.     

Cln3 function is linked to osmoregulation in a Dictyostelium model of Batten disease

Mutations in CLN3 cause a juvenile form of neuronal ceroid lipofuscinosis (NCL), commonly known as Batten disease. Currently, there is no cure for NCL and the mechanisms underlying the disease are not well understood. In the social amoeba Dictyostelium discoideum, the CLN3 homolog, Cln3, localizes predominantly to the contractile vacuole (CV) system. This dynamic organelle functions in osmoregulation, and intriguingly, osmoregulatory defects have been observed in mammalian cell models of CLN3 disease. Therefore, we used Dictyostelium to further study the involvement of CLN3 in this conserved cellular process. First, we assessed the localization of GFP-Cln3 during mitosis and cytokinesis, where CV system function is essential. GFP-Cln3 localized to the CV system during mitosis and cln3^? cells displayed defects in cytokinesis. The recovery of cln3^? cells from hypotonic stress and their progression through multicellular development was delayed and these effects were exaggerated when cells were treated with ammonium chloride. In addition, Cln3-deficiency reduced the viability of cells during hypotonic stress and impaired the integrity of spores. During hypertonic stress, Cln3-deficiency reduced cell viability and inhibited development. We then performed RNA sequencing to gain insight into the molecular pathways underlying the sensitivity of cln3^? cells to osmotic stress. This analysis revealed that cln3-deficiency upregulated the expression of tpp1A, the Dictyostelium homolog of human TPP1/CLN2. We used this information to show a correlated increase in Tpp1 enzymatic activity in cln3^? cells. In total, our study provides new insight in the mechanisms underlying the role of CLN3 in osmoregulation and neurodegeneration.     

Accumulation of point mutations in mitochondrial DNA of aging mice

Mitochondrial DNA (mtDNA) exists in a highly genotoxic environment created by exposure to reactive oxygen species, somewhat deficient DNA repair, and the relatively low fidelity of polymerase gamma. Given the severity of the environment, it was anticipated that mutation accumulation in the mtDNA of aging animals should exceed that of nuclear genes by several orders of magnitude. We have analyzed fragments amplified from the D-loop region of mtDNA from 2 to 22-month-old mice. The amplified 432 bp fragments were cloned into plasmid vectors, and plasmid DNAs from individual clones were purified and sequenced. None of 110 fragments from young mice contained a mutation, while 9 of 87 clones originating from old animals contained base substitutions (chi square = 11.9, P<0.001). The estimated mutation frequency in mtDNA from old mice was 11.6+/-2.7 or 25.4+/-7.8 per 10(5) nucleotides (depending on assumptions of clonality), which exceeds existing estimates for mutation frequencies for nuclear genes by approximately 1000-fold. Our data suggest that at 22 months of age, which roughly corresponds to 3/4 of the mouse natural life span, most mtDNA molecules carry multiple point mutations.     

Regulation of tumor invasion and metastasis in protein kinase C epsilon-transformed NIH3T3 fibroblasts

Protein kinase C epsilon is an oncogenic, actin nucleating protein that coordinately regulates changes in cell growth and shape. Cells constitutively expressing PKCepsilon spontaneously acquire a polarized morphology and extend long cellular membrane protrusions. Here we report that the regulatory C1 domain of PKCepsilon contains an actin binding site that is essential for the formation of elongate invadopodial-like structures, increased pericellular metalloproteinase activity, in vitro invasion of a Matrigel barrier, and the invasion and metastasis of tumors grown in vivo by PKCepsilon-transformed NIH3T3 fibroblasts in nude mice. While removing this small actin binding motif caused a dramatic reversion of tumor invasion, the deletion mutant of PKCepsilon remained oncogenic and tumorigenic in this experimental system. We propose that PKCepsilon directly interacts with actin to stimulate polymerization and the extension of membrane protrusions that transformed NIH3T3 cells use in vivo to penetrate and degrade surrounding tissue boundaries.     

Establishment and maintenance of gammaherpesvirus latency are independent of infective dose and route of infection

Gammaherpesviruses such as Epstein-Barr virus and Kaposi's sarcoma-associated herpesvirus are important human pathogens that establish long-term latent infections. Understanding of the initiation and maintenance of latent infections has important implications for the prevention and treatment of gammaherpesvirus-related diseases. Although much is known about gammaherpesvirus pathogenesis, it is unclear how the infectious dose of a virus influences its ability to establish latent infection. To examine the relationship between the infectious dose and gammaherpesvirus latency, we inoculated wild-type mice with 0.01 to 10(6) PFU of murine gammaherpesvirus 68 (gammaHV68) and quantitatively measured latency and acute-phase replication. Surprisingly, during latency, the frequencies of ex vivo reactivation were similar over a 10(7)-fold range of doses for i.p. infection and over a 10(4)-fold range of doses for intranasal infection. Further, the frequencies of cells harboring viral genome during latency did not differ substantially over similar dose ranges. Although the kinetics of acute-phase replication were delayed at small doses of virus, the peak titer did not differ significantly between mice infected with a large dose of virus and those infected with a small dose of virus. The results presented here indicate that any initiation of infection leads to substantial acute-phase replication and subsequent establishment of a maximal level of latency. Thus, infections with doses as small as 0.1 PFU of gammaHV68 result in stable levels of acute-phase replication and latent infection. These results demonstrate that the equilibrium level of establishment of gammaherpesvirus latency is independent of the infectious dose and route of infection.