Biology subjects

Kimata, J. T.

Publications and source records attributed to Kimata, J. T..

7 recordsLinked to original sources

bioRxiv2022

Ultra-long-acting refillable nanofluidic implant confers full protection against SHIV infection in non-human primates

The impact of pre-exposure prophylaxis (PrEP) on slowing the global human immunodeficiency virus (HIV) epidemic hinges on effective drugs and delivery platforms. Oral regimens have represented the pillar of HIV PrEP for years. However, variable adherence has spurred development of long-acting delivery systems, which also aim at increasing PrEP access, uptake and persistence. Here we present an ultra-long-acting and transcutaneously refillable subcutaneous nanofluidic implant for constant and sustained release of islatravir (ISL), a nucleoside reverse transcriptase translocation inhibitor, for HIV PrEP. In rhesus macaques, the ISL-eluting implants (nISL) achieved constant plasma ISL levels (median 3.14 nM) and peripheral blood mononuclear cells (PBMCs) ISL-triphosphate levels (ISL-TP) (median 0.16 pmol/106 cells) for over 20 months uninterrupted. These drug concentrations are above the established PrEP protection threshold. In two non-blinded, placebo-controlled studies with repeated low-dose rectal and vaginal SHIVSF162P3 challenges in male and female rhesus macaques, respectively, nISL implants conferred 100% protection against infection (p=0.0005 and 0.0009, respectively between nISL and placebo control groups). The nISL implants were well tolerated with mild local tissue inflammation and no signs of systemic toxicity over the 20-month period. Overall, our refillable nISL implant is a promising ultra-long-acting delivery technology for HIV PrEP. One Sentence SummaryAn ultra-long-acting and subcutaneous refillable nanofluidic implant achieved preventive levels of islatravir in non-human primates for 20 months without refilling and conferred 100% protection against rectal and vaginal SHIV transmission.

bioengineering↗

bioRxiv2022

A Rapid, High Throughput, Viral Infectivity Assay using Automated Brightfield Microscopy with Machine Learning

Infectivity assays are essential for the development of viral vaccines, antiviral therapies and the manufacture of biologicals. Traditionally, these assays take 2-7 days and require several manual processing steps after infection. We describe an automated assay (AVIA), using machine learning (ML) and high-throughput brightfield microscopy on 96 well plates that can quantify infection phenotypes within hours, before they are manually visible, and without sample preparation. ML models were trained on HIV, influenza A virus, coronavirus 229E, vaccinia viruses, poliovirus, and adenoviruses, which together span the four major categories of virus (DNA, RNA, enveloped, and non-enveloped). A sigmoidal function, fit to virus dilution curves, yielded an R2 higher than 0.98 and a linear dynamic range comparable to or better than conventional plaque or TCID50 assays. Because this technology is based on sensitizing AIs to specific phenotypes of infection, it may have potential as a rapid, broad-spectrum tool for virus identification.

microbiology↗

bioRxiv2021

In vivo serial passaging of human-simian immunodeficiency virus clones identifies viral characteristics that enhance persistent viral replication

We previously reported that a human immunodeficiency virus type 1 with a simian immunodeficiency virus vif substitution (HSIV-vifNL4-3) could replicate in pigtailed macaques (PTMs), demonstrating that Vif is a species-specific tropism factor of primate lentiviruses. However, infections did not result in high peak viremia or setpoint plasma viral loads, as observed during SIV infection of PTMs. Here, we characterized variants isolated from one of the original infected animals with CD4 depletion after nearly four years of infection to identify determinants of increased replication fitness. In our studies, we found that the HSIV-vif clones did not express the HIV-1 Vpr protein due to interference from the vpx open reading frame in singly spliced vpr mRNA. To examine whether these viral genes contribute to persistent viral replication, we generated infectious HSIV-vif clones expressing either the HIV-1 Vpr or SIV Vpx protein. And then to determine viral fitness determinants of HSIV-vif, we conducted three rounds of serial in vivo passaging in PTMs, starting with an initial inoculum containing a mixture of CXCR4-tropic (Vpr- HSIV-vifNL4-3 isolated at 196 (C/196) and 200 (C/200) weeks post-infection from a PTM with depressed CD4 counts) and CCR5-tropic HSIV (Vpr+ HSIV-vif derivatives based NL-AD8 and Bru-Yu2 and a Vpx expressing HSIV-vifYu2). Interestingly, all infected PTMs showed peak plasma viremia close to or above 105 copies/ml and persistent viral replication for more than 20 weeks. The passage 3 PTM showed peak viral loads greater than 105 viral RNA copies/ml. Infectious molecular clones (IMCs) recovered from the passage 3 PTM (HSIV-P3 IMCs) included mutations required for HIV-1 Vpr expression and those mutations encoded by the CXCR4-tropic HSIV-vifNL4-3 isolates C/196 and C/200. The data indicate that the biological isolates selected during long-term infection acquired HIV-1 Vpr expression to enhance their replication fitness in PTMs. Further passaging of HSIV-P3 IMCs in vivo may generate pathogenic variants with higher replication capacity, which will be a valuable resource as challenge virus in vaccine and cure studies.

microbiology↗

bioRxiv2021

Yeast-expressed Recombinant SARS-CoV-2 Receptor Binding Domain, RBD203-N1 as a COVID-19 Protein Vaccine Candidate

BackgroundSARS-CoV-2 protein subunit vaccines are being evaluated by multiple manufacturers to fill the need for low-cost, easy to scale, safe, and effective COVID-19 vaccines for global access. Vaccine candidates relying on the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein have been the focus of our development program. In this paper, we report on the generation of the RBD203-N1 yeast expression construct, which produces a recombinant protein that when formulated with alum and the TLR-9 agonist, CpG1826 elicits a robust immune response and protection in mice. MethodThe RBD203-N1 antigen was expressed in the yeast Pichia pastoris X33. After fermentation at the 5 L scale, the protein was purified by hydrophobic interaction chromatography followed by anion exchange chromatography. The purified protein was characterized biophysically and biochemically, and after its formulation, the immunogenicity and efficacy were evaluated in mice. Results, Conclusions, and SignificanceThe RBD203-N1 production process yielded 492.9 {+/-} 3.0 mg/L of protein in the fermentation supernatant. A two-step purification process produced a >96% pure protein with a recovery rate of 55 {+/-} 3% (total yield of purified protein: 270.5 {+/-} 13.2 mg/L fermentation supernatant). The protein was characterized as a homogeneous monomer with well-defined secondary structure, thermally stable, antigenic, and when adjuvanted on alum and CpG, it was immunogenic and induced robust levels of neutralizing antibodies against SARS-CoV-2 pseudovirus. These characteristics show that this vaccine candidate is well suited for technology transfer with feasibility of its transition into the clinic to evaluate its immunogenicity and safety in humans.

molecular biology↗

bioRxiv2021

Receptor-binding domain recombinant protein RBD219-N1C1 on alum-CpG induces broad protection against SARS-CoV-2 variants of concern

We conducted preclinical studies in mice using a yeast-produced SARS-CoV-2 RBD subunit vaccine candidate formulated with aluminum hydroxide (alum) and CpG deoxynucleotides. This formulation is equivalent to the Corbevax vaccine that recently received emergency use authorization by the Drugs Controller General of India. We compared the immune response of mice vaccinated with RBD/alum to mice vaccinated with RBD/alum+CpG. We also evaluated mice immunized with RBD/alum+CpG and boosted with RBD/alum. Mice were immunized twice intramuscularly at a 21-day interval. Compared to two doses of the /alum formulation, the RBD/alum+CpG vaccine induced a stronger and more balanced Th1/Th2 cellular immune response, with high levels of neutralizing antibodies against the original Wuhan isolate of SARS-CoV-2 as well as the B.1.1.7 (Alpha), B. 1.351 (Beta), B. 1.617.2 and (Delta) variants. Neutralizing antibody titers against the B.1.1.529 (BA.1, Omicron) variant exceeded those in human convalescent plasma after Wuhan infection but were lower than against the other variants. Interestingly, the second dose did not benefit from the addition of CpG, possibly allowing dose-sparing of the adjuvant in the future. The data reported here reinforces that the RBD/alum+CpG vaccine formulation is suitable for inducing broadly neutralizing antibodies against SARS-CoV-2 including variants of concern.

molecular biology↗

bioRxiv2020

Preventive efficacy of a tenofovir alafenamide fumarate nanofluidic implant in SHIV-challenged nonhuman primates

Pre-exposure prophylaxis (PrEP) using antiretroviral oral drugs is effective at preventing HIV transmission when individuals adhere to the dosing regimen. Tenofovir alafenamide (TAF) is a potent antiretroviral drug, with numerous long-acting (LA) delivery systems under development to improve PrEP adherence. However, none has undergone preventive efficacy assessment. Here we show that LA TAF using a novel subcutaneous nanofluidic implant (nTAF) confers partial protection from HIV transmission. We demonstrate that sustained subcutaneous delivery through nTAF in rhesus macaques maintained tenofovir diphosphate concentration at a median of 390.00 fmol/106 peripheral blood mononuclear cells, 9 times above clinically protective levels. In a non-blinded, placebo-controlled rhesus macaque study with repeated low-dose rectal SHIVSF162P3 challenge, the nTAF cohort had a 62.50% reduction (95% CI: 1.72% to 85.69%; p=0.068) in risk of infection per exposure compared to the control. Our finding mirrors that of tenofovir disoproxil fumarate (TDF) monotherapy, where 60.00% protective efficacy was observed in macaques, and clinically, 67.00% reduction in risk with 86.00% preventive efficacy in individuals with detectable drug in the plasma. Overall, our nanofluidic technology shows potential as a subcutaneous delivery platform for long-term PrEP and provides insights for clinical implementation of LA TAF for HIV prevention.

bioengineering↗

bioRxiv2019

Full-coverage native RNA sequencing of HIV-1 viruses

ObjectiveTo evaluate native RNA sequencing for sequencing HIV-1 viral genomes MethodsFifteen HIV-1 strains were processed with Direct RNA Sequencing (SQK-RNA002) library kits and sequenced on MinION Mk1B devices with RevD flow cells (Oxford Nanopore Technologies (ONT), Oxford, UK). Raw reads were converted to FASTQ, aligned to reference sequences, and assembled into contigs. Multi-sequence alignments of the contigs were generated and used for cladistics analysis. ResultsWe sequenced full-length HIV-1 from the transcriptional start site to 3 LTR (100% virion genome) in 3 out of 15 isolates (89.6, NLAD8, AD17), achieving majority coverage (defined as > 50%) in another 7 out of 15 isolates. Inspection of NLAD8 sequence alignments revealed splicing or deletion signatures. Despite the strong 3 bias, read coverage was sufficient to evaluate single-nucleotide variants (SNVs), insertions and deletions in 9 isolates, and to assemble HIV-1 genomes directly from viral RNA, achieving a maximum of 94% assembly coverage for NLAD8. Phylogenetic relationships were maintained at the level of contigs, as well as individual reads. ConclusionsONT native RNA sequencing performed as expected, covering full-length HIV-1 RNA without PCR or cDNA sequencing. Native single-molecule RNA sequencing supported previous models of HIV-1 replication, and samples exhibited strain-specific transcriptional signals. We propose Context Dependency Variant Classification to describe variants occurring in information-dense regions of HIV. These data provide rich resources for emerging RNA modification detection schemes. Future work will expand HIV-1 transcript profiling to infection models and clinical samples.

genomics↗