Posts Tagged ‘Biotechnology’

Remember what happened last time we “cured” AIDS?

Thursday, March 6th, 2014

Immune Timothy Brown, the “Berlin patient” was freed of HIV infection via a bone marrow transfusion from a compatible CCR5Δ32 donor in 2007.

Last year there was a report of a newborn baby cured by very early drug therapy. The news today carries reports of a second case in a baby which confirms that this approach can work in newborns, although not in adults with established HIV infections.

Also in the news today is the story of the phase I clinical trial of gene-editing technology to control (but not eliminate) HIV infection using autologous donation to create CCR5Δ32 in the patient’s own cells (Gene Editing of CCR5 in Autologous CD4 T Cells of Persons Infected with HIV. (2014) N Engl J Med 2014; 370: 901-910 doi: 10.1056/NEJMoa1300662). But as Nature News correctly points out, the big story here is the relatively crude zinc-finger nuclease (ZFN) technology used in this study as opposed to the much more powerful transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced palindromic repeats (CRISPRs) technologies under development to edit the somatic genome.

Watch this space for further updates.

 

Life in the Post-Antibiotic Era

Thursday, November 7th, 2013

Record-High Antibiotic Sales for Meat and Poultry Production Last week I was struck by this article in The Verge, Dead meat: how to raise livestock in a post-antibiotic era. I was particularly interested in this because only a week earlier I had been teaching our first year students about emerging infectious diseases and the growing feeling that we are entering the post-antibiotic era. As this paper published in 2005 put it:

“The indiscriminate and inappropriate use of antibiotics in outpatient clinics, hospitalized patients and in the food industry is the single largest factor leading to antibiotic resistance. In recent years, the number of new antibiotics licensed for human use in different parts of the world has been lower than in the recent past. In addition, there has been less innovation in the field of antimicrobial discovery research and development. The pharmaceutical industry, large academic institutions or the government are not investing the necessary resources to produce the next generation of newer safe and effective antimicrobial drugs. In many cases, large pharmaceutical companies have terminated their anti-infective research programs altogether due to economic reasons. The potential negative consequences of all these events are relevant because they put society at risk for the spread of potentially serious MDR bacterial infections.”
Alanis, A. J. (2005) Resistance to antibiotics: are we in the post-antibiotic era? Archives of medical research, 36(6), 697-705

The medical profession has rightly received a lot of criticism for undermining antibiotics by handing them out far too freely for trivial infections which though inconvenient, are self-limiting. That situation is now largely historic, and in most countries over-prescribing of antibiotics has ended, or at least decreased, although it is still worrying that in some countries antibiotics are still available without prescription to anyone who can afford to buy them. But medics don’t deserve a fraction of the criticism due to the worst offenders – the agricultural industry, where antibiotics have been used for decades as “growth promoters” in livestock production. The term “growth promoter” means that these valuable compounds are not being used for animal welfare to treat veterinary infections – which is entirely justified – but in a blanket fashion to make animals put on weight faster so they can be sold at a younger age, increasing profit.

I first became aware of this problem many years ago when I was visiting a student on an industrial placement with a major pharmaceutical company who told me proudly about their production of growth promoters and how many thousands of tons and antibiotics they sold to farmers each year. The recent report from Johns Hopkins University puts this ongoing problem into perspective. After decades of this misuse, we are paying the price, with multidrug-resistant bacteria now common in foodstuffs.

For decades we kept ahead of this impending catastrophe by discovering and marketing new antibiotics, but that pipeline has essentially run dry. Although we may find a few useful new compounds from coral reefs or deep sea hydrothermal vents, we are having to admit that we can no longer run fast enough to outpace bacterial evolution. And as future prospects decrease, so economics pays us back.

“In many cases, large pharmaceutical companies have terminated their anti-infective research programs altogether due to economic reasons.”

This is not a problem that the free market is likely to solve. So where do we go from here? Personally I’m not optimistic about the prospects for probiotics making all our problems go away. I don’t think persuading cows to gargle garlic tea is going to save us at this point. So what will (if anything?). The only realistic hope I can see on the horizon is the application of nanotechnology to build new anti-infective compounds rather than relying on snatching them from nature. Realistically, that prospect is decades away. Less realistically, it is also not without risks, such as the grey goo scenario of runaway nano-machines which keeps Prince Charles awake at night.

So are we all going to die? Yes, of course we are – such is the nature of life. But we have a choice of how and when depending on how hard we work on the problem. If future generations of microbiologists can learn enough about the molecular mechanisms which pathogens need to function, then we will have the opportunity to build a new generation of nanobiotics which will keep us ahead of the bacteria. For a little while. But never forget that bacteria have been around a lot longer than we have, and they’re not about to give up just yet.

 

 

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BTV VLPs, OMG

Thursday, May 23rd, 2013

BTV VLPs Bluetongue is a severe disease of ruminants, notably sheep and cattle. The causal agent, the dsRNA Bluetongue virus, is spread by an insect vector and occurs in its vector’s habitat in temperate climates throughout much of the world. BTV is the type member of genus Orbivirus in the family Reoviridae, with 26 known serotypes. When bluetongue first broke out in the United Kingdom in autumn of 2007, the disease was already rapidly spreading throughout continental Europe, causing high mortality rates in sheep and having a detrimental effect on the livestock trade through trade restrictions and loss of stock. The only effective weapon against the disease is control of the spread of BTV through rigorous vaccination programmes. Currently available commercial vaccines are based on both inactivated virus and live, attenuated strains and protect against a single serotype or multiple serotypes when provided as a cocktail. However, the possibility of recombination between the live vaccine strain(s) and wild-type virus in infected animals, leading to the emergence of new infectious strains has motivated efforts to develop safer vaccines.

One approach in the development of an inherently safe vaccine has been the production of Bluetongue virus-like particles (VLPs). BTV has a nonenveloped icosahedral structure, with four main structural proteins (VP3, VP7, VP5 and VP2) arranged in concentric shells around the segmented double-stranded RNA genome and minor structural and nonstructural proteins involved in virus replication. French et al. have shown that these four structural proteins, expressed in insect cells using a baculovirus expression system, assemble into virus-like particles devoid of nucleic acid.

This paper describes plant-based high-level expression of assembled subcore-, core- and virus-like particles of BTV serotype 8. Purified preparations of the VLPs, consisting of all four structural proteins, elicited an immune response in sheep and provided protective immunity against challenge with a South African BTV-8 field isolate. This demonstrates that plant expression provides an economically viable method for producing complex VLPs, such as those of BTV, with the desired biological properties. It represents a significant advance in the use of plant-based systems for the production of complex biopharmaceuticals. The methods employed could also be applied to other situations where the expression of multiple proteins is required, such as the reconstruction of metabolic pathways.

 

A method for rapid production of heteromultimeric protein complexes in plants: assembly of protective bluetongue virus-like particles. Plant Biotechnol J. 06 May 2013 doi: 10.1111/pbi.12076
Plant expression systems based on nonreplicating virus-based vectors can be used for the simultaneous expression of multiple genes within the same cell. They therefore have great potential for the production of heteromultimeric protein complexes. This work describes the efficient plant-based production and assembly of Bluetongue virus-like particles (VLPs), requiring the simultaneous expression of four distinct proteins in varying amounts. Such particles have the potential to serve as a safe and effective vaccine against Bluetongue virus (BTV), which causes high mortality rates in ruminants and thus has a severe effect on the livestock trade. Here, VLPs produced and assembled in Nicotiana benthamiana using the cowpea mosaic virus-based HyperTrans (CPMV-HT) and associated pEAQ plant transient expression vector system were shown to elicit a strong antibody response in sheep. Furthermore, they provided protective immunity against a challenge with a South African BTV-8 field isolate. The results show that transient expression can be used to produce immunologically relevant complex heteromultimeric structures in plants in a matter of days. The results have implications beyond the realm of veterinary vaccines and could be applied to the production of VLPs for human use or the coexpression of multiple enzymes for the manipulation of metabolic pathways.

 

Plant viruses as gene delivery vehicles

Wednesday, May 1st, 2013

Cowpea Chlorotic Mottle Virus The use of animal virus-like particles (VLPs) as vectors for the delivery of genes to mammalian cells has been explored for many years. Plant viruses are almost without exception “just” genetic material (DNA or RNA) surrounded by a shell composed of the capsid protein (CP) – with no membrane envelope. Plant-derived VLPs have not been used for direct gene delivery and expression. There have been no attempts to use a spherical plant viral capsid to deliver heterologous genes for expression in mammalian cells, even though there are several independent demonstrations of the internalization of plant virus by cells.

In light of there being no direct demonstration of spherical plant viruses disassembling and thereby releasing their contents in animal cells, this paper asks the question: Can heterologous genes in spherical plant VLPs be made available to a mammalian cell and their protein products synthesized?

Getting RNA into cells is a major barrier to future theraputic approaches, so robust systems are urgently needed.

 

Reconstituted plant viral capsids can release genes to mammalian cells. Virology. 19 April 2013 doi: 10.1016/j.virol.2013.03.001
The nucleocapsids of many plant viruses are significantly more robust and protective of their RNA contents than those of enveloped animal viruses. In particular, the capsid protein (CP) of the plant virus Cowpea Chlorotic Mottle Virus (CCMV) is of special interest because it has been shown to spontaneously package, with high efficiency, a large range of lengths and sequences of single-stranded RNA molecules. In this work we demonstrate that hybrid virus-like particles, assembled in vitro from CCMV CP and a heterologous RNA derived from a mammalian virus (Sindbis), are capable of releasing their RNA in the cytoplasm of mammalian cells. This result establishes the first step in the use of plant viral capsids as vectors for gene delivery and expression in mammalian cells. Furthermore, the CCMV capsid protects the packaged RNA against nuclease degradation and serves as a robust external scaffold with many possibilities for further functionalization and cell targeting.

Modified bacteria turn waste into fuel 

Friday, March 1st, 2013
“Green” chemistry turns plant waste into fatty acids, and then into fuel.

Genetically modified E. coli bacteria are being used to to produce fatty acids from hydrolysates of biomass products such as  switchgrass and forestry residues. There are two ways to make fuel from biomass – either you make alcohol, or you make petroleum-like fuels that can go into current infrastructure. This program is for infrastructure-compatible transportation fuels. Since the project began, the researchers have increased fuel production 100-fold.

Source: http://goo.gl/Ka6Ja

#MicrobiologyBytes

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Just a bag of enzymes?

Thursday, February 14th, 2013

Mycoplasma With synthetic biology racing ahead with the creation of “artificial” organisms such as Mycoplasma laboratorium, this recent review provides a nice level headed summary of the potential and difficulties of such research.

 

Semi-synthetic minimal cells: origin and recent developments. (2013) Curr Opin Biotechnol. pii: S0958-1669(13)00005-0. doi: 10.1016/j.copbio.2013.01.002
The notion of minimal cells refers to cellular structures that contain the minimal and sufficient complexity to still be defined as living, or at least capable to display the most important features of biological cells. Here we briefly describe the laboratory construction of minimal cells, a project within the broader field of synthetic biology. In particular we discuss the advancements in the preparation of semi-synthetic cells based on the encapsulation of biochemicals inside liposomes, illustrating from the one hand the origin of this research and the most recent developments; and from the other the difficulties and limits of the approach. The role of physicochemical understandings is greatly emphasized.

See: Mycoplasma laboratorium, the first synthetic organism

Say cheese

Wednesday, January 9th, 2013

Lactobacillus casei It has been known since the 1960s that lactic acid bacteria are essential for the development of cheese flavor. In the ensuing 50 years significant research has been directed at understanding the microbiology, genetics and biochemistry of this process. This review briefly covers the current status of cheese flavor development and then provides our vision for approaches which will enhance our understanding of this process. The long-term goal of this area of research is to enable technology (i.e. cultures and enzymes) that results in consistent rapid development of cheese variety-specific characteristic flavors.

As a big cheese fan, I say – more of this sort of thing!

 

Perspectives on the contribution of lactic acid bacteria to cheese flavor development. Curr Opin Biotechnol. 29 Dec 29 2012. pii: S0958-1669(12)00210-8. doi: 10.1016/j.copbio.2012.12.001

A reverse genetic system for Schmallenberg virus

Monday, January 7th, 2013

Schmallenberg virus Schmallenberg virus (SBV) is a newly emerged orthobunyavirus that has caused widespread disease in cattle, sheep and goats in Europe. Like other orthobunyaviruses SBV is characterized by a tripartite negative sense RNA genome that encodes four structural and two nonstructural proteins. in vitro, SBV has a wide in vitro host range, and BHK-21 cells are a convenient host for both SBV propagation and assay by plaque titration.

In this paper, the three SBV genome segments were cloned as cDNA and a three-plasmid rescue system was established to recover infectious virus. Recombinant virus behaved similarly in cell culture to authentic virus. The open reading frame for the nonstructural NSs protein, encoded on the smallest genome segment, was disrupted by introduction of translation stop codons in the appropriate cDNA, and when this plasmid was used in reverse genetics, a recombinant virus that lacks NSs expression was recovered. This virus had reduced capacity to shut-off host cell protein synthesis compared to wild type virus. In addition, the NSs-deleted virus induced interferon in cells, indicating that like other orthobunyaviruses, NSs functions as an interferon antagonist most probably by globally inhibiting host cell metabolism.

Development of a reverse genetic system for SBV will aid investigation of its pathogenic mechanisms as well as creation of attenuated strains that could be candidate vaccines.

 

Establishment of a reverse genetic system for Schmallenberg virus, a newly emerged orthobunyavirus in Europe. J Gen Virol.19 Dec 2012

Vaccines: From Empirical Development to Rational Design

Wednesday, November 28th, 2012

Vaccination Infectious diseases are responsible for an overwhelming number of deaths worldwide and their clinical management is often hampered by the emergence of multi-drug-resistant strains. Therefore, prevention through vaccination currently represents the best course of action to combat them. However, immune escape and evasion by pathogens often render vaccine development difficult. Furthermore, most currently available vaccines were empirically designed.

This review discusses why rational design of vaccines is not only desirable but also necessary. It describes recent developments towards specifically tailored antigens, adjuvants, and delivery systems, and discuss the methodological gaps and lack of knowledge still hampering true rational vaccine design, and addresses the potential and limitations of different strategies and technologies for advancing vaccine development.

 

Vaccines: From Empirical Development to Rational Design. (2012) PLoS Pathog 8(11): e1003001. doi:10.1371/journal.ppat.1003001