The Antibody Mediated Prevention trials showed that the broadly neutralizing antibody (bnAb) VRC01 prevented acquisition of human immunodeficiency virus-1 (HIV-1) sensitive to VRC01. Using AMP trial data, here we show that the predicted serum neutralization 80% inhibitory dilution titer (PT80) biomarker—which quantifies the neutralization potency of antibodies in an individual’s serum against an HIV-1 isolate—can be used to predict HIV-1 prevention efficacy. Similar to the results of nonhuman primate studies, an average PT80 of 200 (meaning a bnAb concentration 200-fold higher than that required to reduce infection by 80% in vitro) against a population of probable exposing viruses was estimated to be required for 90% prevention efficacy against acquisition of these viruses. Based on this result, we suggest that the goal of sustained PT80 <200 against 90% of circulating viruses can be achieved by promising bnAb regimens engineered for long half-lives. We propose the PT80 biomarker as a surrogate endpoint for evaluatinon of bnAb regimens, and as a tool for benchmarking candidate bnAb-inducing vaccines.

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We report on further development of the agroinfiltratable Tobacco mosaic virus (TMV)-based overexpression (TRBO) vector to deliver CRISPR/Cas9 components into plants. First, production of a Cas9 (HcoCas9) protein from a binary plasmid increased when co-expressed in presence of suppressors of gene silencing, such as the TMV 126-kDa replicase or the Tomato bushy stunt virus P19 protein. Such suppressor-generated elevated levels of Cas9 expression translated to efficient gene editing mediated by TRBO-G-3′gGFP expressing GFP and also a single guide RNA targeting the mgfp5 gene in the Nicotiana benthamiana GFP-expressing line 16c. Furthermore, HcoCas9 encoding RNA, a large cargo insert of 4.2 kb, was expressed from TRBO-HcoCas9 to yield Cas9 protein again at higher levels upon co-expression with P19. Likewise, co-delivery of TRBO-HcoCas9 and TRBO-G-3′gGFP in the presence of P19 also resulted in elevated levels percentages of indels (insertions and deletions). These data also revealed an age-related phenomenon in plants whereby the RNA suppressor P19 had more of an effect in older plants. Lastly, we used a single TRBO vector to express both Cas9 and a sgRNA. Taken together, we suggest that viral RNA suppressors could be used for further optimization of single viral vector delivery of CRISPR gene editing parts.

BackgroundSeveral high-throughput molecular genetic analyses rely on high-quality genomic DNA. Copurification of other molecules can negatively impact the functionality of plant DNA preparations employed in these procedures. Isolating DNA from agronomically important crops, such as sugarcane, rice, citrus, potato and tomato is a challenge due to the presence of high fiber, polysaccharides, or secondary metabolites. We present a simplified, rapid and reproducible SDS-based method that provides high-quality and -quantity of DNA from small amounts of leaf tissue, as required by the emerging biotechnology and molecular genetic applications.ResultsWe developed the TENS-CO method as a simplified SDS-based isolation procedure with sequential steps of purification to remove polysaccharides and polyphenols using 2-mercaptoethanol and potassium acetate, chloroform partitioning, and sodium acetate/ethanol precipitation to yield high-quantity and -quality DNA consistently from small amounts of tissue (0.15 g) for different plant species. The method is simplified and rapid in terms of requiring minimal manipulation, smaller extraction volume, reduced homogenization time (20 s) and DNA precipitation (one precipitation for 1 h). The method has been demonstrated to accelerate screening of large amounts of plant tissues from species that are rich in polysaccharides and secondary metabolites for Southern blot analysis of reporter gene overexpressing lines, pathogen detection by quantitative PCR, and genotyping of disease-resistant plants using marker-assisted selection.ConclusionTo facilitate molecular genetic studies in major agronomical crops, we have developed the TENS-CO method as a simple, rapid, reproducible and scalable protocol enabling efficient and robust isolation of high-quality and -quantity DNA from small amounts of tissue from sugarcane, rice, citrus, potato, and tomato, thereby reducing significantly the time and resources used for DNA isolation.Electronic supplementary materialThe online version of this article (10.1186/s13007-017-0255-6) contains supplementary material, which is available to authorized users.