Plant cell packs: a scalable platform for recombinant protein production and metabolic engineering

Industrial plant biotechnology applications include the production of sustainable fuels, complex metabolites and recombinant proteins, but process development can be impaired by a lack of reliable and scalable screening methods. Here, we describe a rapid and versatile expression system which involves the infusion of Agrobacterium tumefaciens into three‐dimensional, porous plant cell aggregates deprived of cultivation medium, which we have termed plant cell packs (PCPs). This approach is compatible with different plant species such as Nicotiana tabacum BY2, Nicotiana benthamiana or Daucus carota and 10‐times more effective than transient expression in liquid plant cell culture. We found that the expression of several proteins was similar in PCPs and intact plants, for example, 47 and 55 mg/kg for antibody 2G12 expressed in BY2 PCPs and N. tabacum plants respectively. Additionally, the expression of specific enzymes can either increase the content of natural plant metabolites or be used to synthesize novel small molecules in the PCPs. The PCP method is currently scalable from a microtiter plate format suitable for high‐throughput screening to 150‐mL columns suitable for initial product preparation. It therefore combined the speed of transient expression in plants with the throughput of microbial screening systems. Plant cell packs therefore provide a convenient new platform for synthetic biology approaches, metabolic engineering and conventional recombinant protein expression techniques that require the multiplex analysis of several dozen up to hundreds of constructs for efficient product and process development

HIV-1 Superinfection in an HIV-2-Infected Woman with Subsequent Control of HIV-1 Plasma Viremia

A human immunodeficiency virus type 2 (HIV-2)-infected woman experienced asymptomatic superinfection with HIV-1 subtype AG. She did not have cross-neutralizing autologous HIV-1 antibodies before and shortly after HIV-1 superinfection. This evidence supports a mechanism other than cross-neutralizing antibodies for the mild course of HIV-1 infection in this woma

Mathematical models: a key to understanding HIV envelope interactions?

The spikes of the human immunodeficiency virus (HIV) mediate viral entry and are the most important targets for neutralizing antibodies. Each spike consists of three identical subunits. The role of the spike's subunits in antibody binding is not fully understood. One experimental approach to analyze trimer function uses assays with mixed envelope trimer expressing cells or viruses. As these experiments do not allow direct observation of subunit functions, mathematical models are required to interpret them. Here we describe a modeling framework to study (i) the interaction of the V1V2 loop with epitopes on the V3 loop and (ii) the composition of quaternary epitopes. In a first step we identify which trimers can form in these assays and how they function under antibody binding. We then derive the behavior of an average trimer. We contrast two experimental reporting systems and list their advantages and disadvantages. In these experiments trimer formation might not be perfectly random and we show how these effects can be tested. As we still lack a potent vaccine against HIV, and this vaccine surely has to stimulate the production of neutralizing antibodies, mixed trimer approaches in combination with mathematical models will help to identify vulnerable sites of the HIV spike

Cellulose acetate phthalate, a common pharmaceutical excipient, inactivates HIV-1 and blocks the coreceptor binding site on the virus envelope glycoprotein gp120

BACKGROUND: Cellulose acetate phthalate (CAP), a pharmaceutical excipient used for enteric film coating of capsules and tablets, was shown to inhibit infection by the human immunodeficiency virus type 1 (HIV-1) and several herpesviruses. CAP formulations inactivated HIV-1, herpesvirus types 1 (HSV-1) and 2 (HSV-2) and the major nonviral sexually transmitted disease (STD) pathogens and were effective in animal models for vaginal infection by HSV-2 and simian immunodeficiency virus. METHODS: Enzyme-linked immunoassays and flow cytometry were used to demonstrate CAP binding to HIV-1 and to define the binding site on the virus envelope. RESULTS: 1) CAP binds to HIV-1 virus particles and to the envelope glycoprotein gp120; 2) this leads to blockade of the gp120 V3 loop and other gp120 sites resulting in diminished reactivity with HIV-1 coreceptors CXCR4 and CCR5; 3) CAP binding to HIV-1 virions impairs their infectivity; 4) these findings apply to both HIV-1 IIIB, an X4 virus, and HIV-1 BaL, an R5 virus. CONCLUSIONS: These results provide support for consideration of CAP as a topical microbicide of choice for prevention of STDs, including HIV-1 infection

Positive In Vivo Selection of the HIV-1 Envelope Protein gp120 Occurs at Surface-Exposed Regions

The rapid evolution of human immunodeficiency virus (HIV) envelope represents a major challenge to vaccine and drug development, particularly because the underlying mechanisms are not completely understood. To explore whether distinct patterns of positive selection within the envelope protein glycoprotein (gp) 120 exist and are associated with functionally relevant domains, we conducted a long-term survey of sequence evolution in 20 HIV-1-infected persons who interrupted antiretroviral therapy. In total, 1753 clonal sequences encompassing the C2-V3-C3 region of gp120 were derived. Strikingly, positively selected amino acids mapped almost exclusively (P=.0003) to externally accessible residues on the gp120 crystal structure. The current understanding of envelope structure and function associates the main determinants of viral entry and the targets for neutralizing antibodies with these exterior regions of gp120, strongly suggesting that the observed adaptive evolution of these sites occurs in response to respective selective force

Optimization and validation of sample preparation for metagenomic sequencing of viruses in clinical samples

Demultiplexed raw sequencing data files (fastq.gz

Metagenomic sequencing complements routine diagnostics in identifying viral pathogens in lung transplant recipients with unknown etiology of respiratory infection

BACKGROUND: Lung transplant patients are a vulnerable group of immunosuppressed patients that are prone to frequent respiratory infections. We studied 60 episodes of respiratory symptoms in 71 lung transplant patients. Almost half of these episodes were of unknown infectious etiology despite extensive routine diagnostic testing. METHODS: We re-analyzed respiratory samples of all episodes with undetermined etiology in order to detect potential viral pathogens missed/not accounted for in routine diagnostics. Respiratory samples were enriched for viruses by filtration and nuclease digestion, whole nucleic acids extracted and randomly amplified before high throughput metagenomic virus sequencing. Viruses were identified by a bioinformatic pipeline and confirmed and quantified using specific real-time PCR. RESULTS: In completion of routine diagnostics, we identified and confirmed a viral etiology of infection by our metagenomic approach in four patients (three Rhinovirus A, one Rhinovirus B infection) despite initial negative results in specific multiplex PCR. Notably, the majority of samples were also positive for Torque teno virus (TTV) and Human Herpesvirus 7 (HHV-7). While TTV viral loads increased with immunosuppression in both throat swabs and blood samples, HHV-7 remained at low levels throughout the observation period and was restricted to the respiratory tract. CONCLUSION: This study highlights the potential of metagenomic sequencing for virus diagnostics in cases with previously unknown etiology of infection and in complex diagnostic situations such as in immunocompromised hosts

ART-DEX: A novel strategy to monitor broadly neutralizing antibody activity during antiretroviral therapy of HIV-1

Therapeutic use of HIV-1 broadly neutralizing antibodies (bnAbs), passively administered or induced by therapeutic vaccines, is a focus of advanced treatment strategies under development. To enable monitoring of bnAb activity during concurrent antiretroviral therapy (ART), we developed ART-DEX, an analytic strategy that allows high-throughput detection of pure antibody-based neutralizing activity. ART-DEX combines pH-dependent dissociation of antiretrovirals (ARVs) from plasma proteins and size exclusion to effectively remove ARVs from plasma samples, reducing the confounding effects of ARVs on neutralization assays. For complete details on the use and execution of this protocol, please refer to Schwarzmüller et al.$^{1}$

Rab27a controls HIV-1 assembly by regulating plasma membrane levels of phosphatidylinositol 4,5-bisphosphate

During the late stages of the HIV-1 replication cycle, the viral polyprotein Pr55Gag is recruited to the plasma membrane (PM), where it binds phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) and directs HIV-1 assembly. We show that Rab27a controls the trafficking of late endosomes carrying phosphatidylinositol 4-kinase type 2 α (PI4KIIα) toward the PM of CD4+ T cells. Hence, Rab27a promotes high levels of PM phosphatidylinositol 4-phosphate and the localized production of PI(4,5)P2, therefore controlling Pr55Gag membrane association. Rab27a also controls PI(4,5)P2 levels at the virus-containing compartments of macrophages. By screening Rab27a effectors, we identified that Slp2a, Slp3, and Slac2b are required for the association of Pr55Gag with the PM and that Slp2a cooperates with Rab27a in the recruitment of PI4KIIα to the PM. We conclude that by directing the trafficking of PI4KIIα-positive endosomes toward the PM, Rab27a controls PI(4,5)P2 production and, consequently, HIV-1 replication.Fil: Pereyra Gerber, Federico Pehuén. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas en Retrovirus y Sida. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Biomédicas en Retrovirus y Sida; ArgentinaFil: Cabrini, Mercedes. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas en Retrovirus y Sida. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Biomédicas en Retrovirus y Sida; ArgentinaFil: Jancic, Carolina Cristina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Paoletti, Luciana Elisa. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Banchio, Claudia Elena. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Von Bilderling, Catalina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Física de Buenos Aires. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Física de Buenos Aires; ArgentinaFil: Sigaut, Lorena. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Centro de Microscopías Avanzadas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Pietrasanta, Lia. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Centro de Microscopías Avanzadas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Duette, Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas en Retrovirus y Sida. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Biomédicas en Retrovirus y Sida; ArgentinaFil: Freed, Eric O.. National Cancer Institute at Frederick; Estados UnidosFil: Basile, Genevieve de Saint. Institut National de la Santé et de la Recherche Médicale; FranciaFil: Moita, Catarina Ferreira. Instituto Gulbenkian de Ciencia; PortugalFil: Moita, Luis Ferreira. Instituto Gulbenkian de Ciencia; PortugalFil: Amigorena, Sebastian. Institute Curie; FranciaFil: Benaroch, Philippe. Institute Curie; FranciaFil: Geffner, Jorge Raúl. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas en Retrovirus y Sida. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Biomédicas en Retrovirus y Sida; ArgentinaFil: Ostrowski, Matias. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas en Retrovirus y Sida. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Biomédicas en Retrovirus y Sida; Argentin