Reef Fish Survey Techniques: Assessing the Potential for Standardizing Methodologies.

Dramatic changes in populations of fishes living on coral reefs have been documented globally and, in response, the research community has initiated efforts to assess and monitor reef fish assemblages. A variety of visual census techniques are employed, however results are often incomparable due to differential methodological performance. Although comparability of data may promote improved assessment of fish populations, and thus management of often critically important nearshore fisheries, to date no standardized and agreed-upon survey method has emerged. This study describes the use of methods across the research community and identifies potential drivers of method selection. An online survey was distributed to researchers from academic, governmental, and non-governmental organizations internationally. Although many methods were identified, 89% of survey-based projects employed one of three methods-belt transect, stationary point count, and some variation of the timed swim method. The selection of survey method was independent of the research design (i.e., assessment goal) and region of study, but was related to the researcher's home institution. While some researchers expressed willingness to modify their current survey protocols to more standardized protocols (76%), their willingness decreased when methodologies were tied to long-term datasets spanning five or more years. Willingness to modify current methodologies was also less common among academic researchers than resource managers. By understanding both the current application of methods and the reported motivations for method selection, we hope to focus discussions towards increasing the comparability of quantitative reef fish survey data.

Genetically Encoded Azide Containing Amino Acid in Mammalian Cells Enables Site-Specific Antibody-Drug Conjugates Using Click Cycloaddition Chemistry.

Antibody-drug conjugates (ADC) have emerged as potent antitumor drugs that provide increased efficacy, specificity, and tolerability over chemotherapy for the treatment of cancer. ADCs generated by targeting cysteines and lysines on the antibody have shown efficacy, but these products are heterogeneous, and instability may limit their dosing. Here, a novel technology is described that enables site-specific conjugation of toxins to antibodies using chemistry to produce homogeneous, potent, and highly stable conjugates. We have developed a cell-based mammalian expression system capable of site-specific integration of a non-natural amino acid containing an azide moiety. The azide group enables click cycloaddition chemistry that generates a stable heterocyclic triazole linkage. Antibodies to Her2/neu were expressed to contain N6-((2-azidoethoxy)carbonyl)-l-lysine at four different positions. Each site allowed over 95% conjugation efficacy with the toxins auristatin F or a pyrrolobenzodiazepine (PBD) dimer to generate ADCs with a drug to antibody ratio of >1.9. The ADCs were potent and specific in in vitro cytotoxicity assays. An anti Her2/neu conjugate demonstrated stability in vivo and a PBD containing ADC showed potent efficacy in a mouse tumor xenograph model. This technology was extended to generate fully functional ADCs with four toxins per antibody. The high stability of the azide-alkyne linkage, combined with the site-specific nature of the expression system, provides a means for the generation of ADCs with optimized pharmacokinetic, biological, and biophysical properties.

Engineering, expression, purification, and characterization of stable clade A/B recombinant soluble heterotrimeric gp140 proteins.

The envelope glycoprotein (Env) of human immunodeficiency virus type 1 (HIV-1) is composed of two noncovalently associated subunits: an extracellular subunit (gp120) and a transmembrane subunit (gp41). The functional unit of Env on the surface of infectious virions is a trimer of gp120/gp41 heterodimers. Env is the target of anti-HIV neutralizing antibodies. A considerable effort has been invested in the engineering of recombinant soluble forms of the virion-associated Env trimer as vaccine candidates to elicit anti-HIV neutralizing antibody responses. These soluble constructs contain three gp120 subunits and the extracellular segments of the corresponding gp41 subunits. The individual gp120/gp41 protomers on these soluble trimers are identical in amino acid sequence (homotrimers). Here, we engineered novel soluble trimeric gp140 proteins that are formed by the association of gp140 protomers that differ in amino acid sequence and glycosylation patterns (heterotrimers). Specifically, we engineered soluble heterotrimeric proteins composed of clade A and clade B Env protomers. The clade A gp140 protomers were derived from viruses isolated during acute infection (Q168a2, Q259d2.17, and Q461e2), whereas the clade B gp140 protomers were derived from a virus isolated during chronic infection (SF162). The amino acid sequence divergence between the clade A and the clade B Envs is approximately 24%. Neutralization epitopes in the CD4 binding sites and coreceptor binding sites, as well as the membrane-proximal external region (MPER), were differentially expressed on the heterotrimeric and homotrimeric proteins. The heterotrimeric gp140s elicited broader anti-tier 1 isolate neutralizing antibody responses than did the homotrimeric gp140s.

Binding interactions between soluble HIV envelope glycoproteins and quaternary-structure-specific monoclonal antibodies PG9 and PG16.

PG9 and PG16 are antibodies isolated from a subject infected with HIV-1 and display broad anti-HIV neutralizing activities. They recognize overlapping epitopes, which are preferentially expressed on the membrane-anchored trimeric form of the HIV envelope glycoprotein (Env). PG9 and PG16 were reported not to bind to soluble mimetics of Env. The engineering of soluble Env proteins on which the PG9 and PG16 epitopes are optimally exposed will support efforts to elicit broad anti-HIV neutralizing antibodies by immunization. Here, we identified several soluble gp140 Env proteins that are recognized by PG9 and PG16, and we investigated the molecular details of those binding interactions. The IgG versions of PG9 and PG16 recognize the soluble trimeric gp140 form less efficiently than the corresponding monomeric gp140 form. In contrast, the Fab versions of PG9 and PG16 recognized the monomeric and trimeric gp140 forms with identical binding kinetics and with binding affinities similar to the high binding affinity of the anti-V3 antibody 447D to its epitope. Our data also indicate that, depending on the Env backbone, the interactions of PG9 and PG16 with gp140 may be facilitated by the presence of the gp41 ectodomain and are independent of the proper enzymatic cleavage of gp140 into gp120 and gp41. The identification of soluble Env proteins that express the PG9 and PG16 epitopes and the detailed characterization of the molecular interactions between these two antibodies and their ligands provide important and novel information that will assist in improving the engineering of future Env immunogens.

Improving the expression of recombinant soluble HIV Envelope glycoproteins using pseudo-stable transient transfection.

The Envelope glycoprotein (Env) of the human immunodeficiency virus (HIV) is the target of neutralizing antibodies (NAbs). So far, HIV Env-derived immunogens have not been able to elicit broad neutralizing antibody responses against primary isolates. Identifying conditions that will permit the efficient production of different soluble HIV Env proteins will facilitate a high throughput comparative analysis of the immunogenicity of diverse Env constructs, potentially identifying Env forms that are more conducive to the elicitation of anti-HIV NAbs. Here we compared different cell types, transfection reagents, transfection conditions and different DNA expression vectors on soluble HIV Envelope expression levels. We identified optimal expression conditions and developed a protocol to streamline and maximize production of diverse HIV Env constructs. Using this optimized platform, milligram quantities of purified soluble HIV Env trimer can be routinely achieved in a rapid and cost-effective manner.

Factors associated with the development of cross-reactive neutralizing antibodies during human immunodeficiency virus type 1 infection.

The characterization of the cross-reactive, or heterologous, neutralizing antibody responses developed during human immunodeficiency virus type 1 (HIV-1) infection and the identification of factors associated with their generation are relevant to the development of an HIV vaccine. We report that in healthy HIV-positive, antiretroviral-naïve subjects, the breadth of plasma heterologous neutralizing antibody responses correlates with the time since infection, plasma viremia levels, and the binding avidity of anti-Env antibodies. Anti-CD4-binding site antibodies are responsible for the exceptionally broad cross-neutralizing antibody responses recorded only in rare plasma samples. However, in most cases examined, antibodies to the variable regions and to the CD4-binding site of Env modestly contributed in defining the overall breadth of these responses. Plasmas with broad cross-neutralizing antibody responses were identified that targeted the gp120 subunit, but their precise epitopes mapped outside the variable regions and the CD4-binding site. Finally, although several plasmas were identified with cross-neutralizing antibody responses that were not directed against gp120, only one plasma with a moderate breadth of heterologous neutralizing antibody responses contained cross-reactive neutralizing antibodies against the 4E10 epitope, which is within the gp41 transmembrane subunit. Overall, our study indicates that more than one pathway leads to the development of broad cross-reactive neutralizing antibodies during HIV infection and that the virus continuously escapes their action.