Biologics

Complex Description

Rationale Researchers at the Medical Research Council (UK) (MRC) in Glasgow discovered that, herpes simplex virus infected cells produce both infectious virus particles and non infectious light particles (L-particles). These are effectively “empty” virus particles, that have the same outer coating as a live virus, but totally lack the inner nuclear material (the nucleocapsid) and are therefore unable to replicate. They are produced in equal quantities to virus particles throughout the virus replication cycle. In terms of structure, these are non-infectious particles are composed of the virion tegument and envelope proteins only (the two outer layers of the virus). They are produced by all α-herpesviruses,(HSV 1 and 2 and VZV) and they adsorb and penetrate host cells as if they were viruses. Since these preparations contain only trace amounts of DNA, L-particles are unable to replicate (typically, L-particle preparations contain contaminating virions at a ratio of less than 1 per 100,000).
	HSV1 L-particles are composed of the virus envelope and tegument components but lack the capsid and the DNA of the virion particle. As a consequence L-particles can be readily separated from virions by centrifugation through ficoll gradients. This finding has been reproduced in all of the alphaherpesviridae :- pseudorabies virus, equine herpes virus, bovine herpes virus, varicella zoster and HSV 2. Studies of these L-particles, comparing them with wild isolates has demonstrated that L-particles possess most if not all, of the virus tegument and envelope proteins but lack the nucleocapsid proteins. The role played by L-particles in nature is not fully understood, however it has been demonstrated that HSV 1 L-particles were as effective as virions in supplying tegument proteins to target cells. This indicates that L-particles might be involved in boosting the infectivity of viruses by elevating the levels of such proteins in infected cells. It is anticipated that humoural and cellular immunity will be displayed in response to these particles as all surface glycoproteins are expressed.
Researchers also found that in the absence of viral DNA synthesis (and hence of the production of infectious virus) HSV 1 infected cells release into the growth medium virus related particles that are essentially similar to L-particles but differ in terms of protein composition. These particles have been named pre-viral DNA replication enveloped particles (PREPS).
Experimental Data
L-Particles Early studies on L-particles using a herpes simplex type 1 temperature sensitive mutant (ts1201) which fails to produce mature virions demonstrated that L-particle production is independent of virion formation. It also demonstrated that the quantity and protein composition of L-particles generated by this mutant at the non permissive temperature are indistinguishable from those produced in wild type HSV 1 infections. A typical preparation of HSV 1 L-particles contains between one in 10,000 and one in 100,000 virions. The initial interactions between virions and cells (binding to the cell surface and fusion with the cytoplasmic membrane) are believed to be directed by components of the envelope. This process results in the release into the cytoplasm of the nucleocapsid and of the tegument proteins several of which are known to function during the early stages of the infection. It is likely therefore due to the close similarity between the structure and composition of L-particles and those of the virion tegument and envelope that L-particles play a role in these processes. PREPS PREPS can be developed as delivery vectors for other proteinaceous immunogens. In this case foreign proteins are engineered into the virus stock genome under the control of a suitable promoter. The protein is then incorporated into the PREPS, which are excreted into culture medium and readily purified. This has already been successfully demonstrated with glycoproteins B, the major immunognenic protein of human cytomegalovirus. Cell culture experiments have shown that PREPS are non-toxic to recipient cells up to a loading of 10,000 particles per cell, and are able to deliver proteins at levels similar to those found in virulent infections. Neither PREPS nor L-particles express the HSV-1 US-12 gene protein which impairs normal cellular antigen processing pathways
Potential Competitive Advantages L-particles and PREPS express all of the major antigenic surface glycoproteins of wild virus particles, but do not contain DNA. The risk of herpes virus DNA as a potential mutagen is overcome, whilst the ability to induce humoral and cellular responses is retained. The large scale production of PREPS will be possible by utilising recombinants, allowing a cheap and scaleable manufacture. PREPS and L-particle Development Programme Several disease areas are being studied with regard the application of PREPS and L-particles. These are summarised below in the form of a table

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