Pharmacokinetic serum concentrations of VRC01 correlate with prevention of HIV-1 acquisition.

BACKGROUND: The phase 2b proof-of-concept Antibody Mediated Prevention (AMP) trials showed that VRC01, an anti-HIV-1 broadly neutralising antibody (bnAb), prevented acquisition of HIV-1 sensitive to VRC01. To inform future study design and dosing regimen selection of candidate bnAbs, we investigated the association of VRC01 serum concentration with HIV-1 acquisition using AMP trial data. METHODS: The case-control sample included 107 VRC01 recipients who acquired HIV-1 and 82 VRC01 recipients who remained without HIV-1 during the study. We measured VRC01 serum concentrations with a qualified pharmacokinetic (PK) Binding Antibody Multiplex Assay. We employed nonlinear mixed effects PK modelling to estimate daily-grid VRC01 concentrations. Cox regression models were used to assess the association of VRC01 concentration at exposure and baseline body weight, with the hazard of HIV-1 acquisition and prevention efficacy as a function of VRC01 concentration. We also compared fixed dosing vs. body weight-based dosing via simulations. FINDINGS: Estimated VRC01 concentrations in VRC01 recipients without HIV-1 were higher than those in VRC01 recipients who acquired HIV-1. Body weight was inversely associated with HIV-1 acquisition among both placebo and VRC01 recipients but did not modify the prevention efficacy of VRC01. VRC01 concentration was inversely correlated with HIV-1 acquisition, and positively correlated with prevention efficacy of VRC01. Simulation studies suggest that fixed dosing may be comparable to weight-based dosing in overall predicted prevention efficacy. INTERPRETATION: These findings suggest that bnAb serum concentration may be a useful marker for dosing regimen selection, and operationally efficient fixed dosing regimens could be considered for future trials of HIV-1 bnAbs. FUNDING: Was provided by the National Institutes of Health, National Institute of Allergy and Infectious Diseases (NIAID) (UM1 AI068614, to the HIV Vaccine Trials Network [HVTN]; UM1 AI068635, to the HVTN Statistical Data and Management Center [SDMC], Fred Hutchinson Cancer Center [FHCC]; 2R37 054165 to the FHCC; UM1 AI068618, to HVTN Laboratory Center, FHCC; UM1 AI068619, to the HPTN Leadership and Operations Center; UM1 AI068613, to the HIV Prevention Trials Network [HPTN] Laboratory Center; UM1 AI068617, to the HPTN SDMC; and P30 AI027757, to the Center for AIDS Research, Duke University (AI P30 AI064518) and University of Washington (P30 AI027757) Centers for AIDS Research; R37AI054165 from NIAID to the FHCC; and OPP1032144 CA-VIMC Bill & Melinda Gates Foundation.

RNA silencing suppressor-influenced performance of a virus vector delivering both guide RNA and Cas9 for CRISPR gene editing.

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.

Reproducible genomic DNA preparation from diverse crop species for molecular genetic applications.

BACKGROUND: Several 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. RESULTS: We 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. CONCLUSION: To 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.