Lentiviral vectors

[myriam]

Hi everyone.

 

I recently had an accident in the lab. I swallowed a couple of lentiviral supernatants drops from 293 cells when my dish fell down inside the hood and spilled all over my face. It was the day when collecting infectious media with a human p53 mutant construct. I´m so scared, I reported the accident and I´m under medical follow-up but I am uncertain of what might happen in the future to me. I am aware my titer of lentiviral vector in the supernatant was very high, between 1E+5 and 1E+6 per ml.

 

Do you guys believe my stomach pH might have killed all the viral particles before the attached any of my cells in the gastric tract?. I´m going crazy!

 

Thank you for your answers.

 

Myriam.

[CharonY]

Note: not medical advice!

 

First, I have to put up my biosafety hat on and comment that your work procedure needs revising. If you work with BSL2 (or above) you are required to work with face protection and in a hood in such a way that dropping your sample will not splash your face. I.e. the sash has to be closed that spills will not reach exposed body parts.

 

That being said, infection processes are not initiated by ingestion. Direct contact with mucosal tissue (e.g. eyes, nostrils, mouth). The stomach acid is not going to factor in much.

The rest depends on the type of vector you used and the inserts it carries. Normally, they are replication incompetent and if the insert is relatively harmless, it should have no effect (though I will not pretend to know what fiddling with p53 may actually do outside of cell culture).

Considering that no precautions above BSL2 have been taken, I assume that it is mostly harmless. Still, inform yourself about the vector and associated risks. In fact, this is something you should always do before starting work. It is a prerequisite for working in a lab (and failure to do so counts as a safety violation).

 

Regardless, for the future there are several things you should keep in mind: a) inform yourself about potential risks associated with your work (i..e know precisely the health risks and b) protect yourself properly. Revise procedures so that these kind of accidents do not happen (Googles, face masks, proper use of biosafety cabinets, etc.).

 

Still, serious consequences from these incidents are rare. Maintain follow-ups and you should be fine. But again (and this is for everyone who may be working in a lab and reading this): inform yourself about the risk. I am aware that some labs have a lax safety culture, but it is your own frigging health on the line.

[myriam]

Hi Charon. I really appreciate your advice and recognize I made a huge careless mistake. I know the lentiviral vector will not replicate. My concern is getting the P53 oncogene inside my DNA and being express from there. Do you think stomach pH killed all the particles before they were internalized?

[CharonY]

As I said, stomach acid does little (unless you started to drink a lot of it). The worrisome interactions will well happen before that (epidermis to some point, but mostly mucous surfaces in eyes, mouth, oesophagus).

As I mentioned, you should figure out what it really was. However, p53 is not an oncogene and if you had one the BSL should have been bumped to 2+-3. So I assume there is little to worry there. The only question is really what kind of mutation your construct has.

Edited August 29, 2012 by CharonY

[John Cuthber]

This a million miles from my field of expertise so I hope someone looks at it and tells me if I'm talking nonsense. but, as I understand it, P53 is a tumour suppressor gene.

A mutation that stopped it working might be viewed as an oncogene but I don't think it's a big problem here.

Each cell in you already has a couple of copies of it. If at least one of them works you shouldn't be at heightened risk of cancer (If both work then you should have a lowered risk).

Adding a copy of a gene that codes for a version that doesn't work will just waste a bit of effort making a faulty protein.

Your endogenous genes will still keep producing the same working versions as they always did.

It shouldn't affect your risk of getting caner.

[CharonY]

The potential risk lies in the precise construct. With recombination it may be possible to introduce defects or deletion into p53 which may increase the chance of the transfected cell to start multiplying. The chances are low overall, though.

As a whole, other stuff that we deal with on a daily basis are much more likely to increase cancer risk than a bit of a splash.

[jp255]

The terms oncogene and tumour supressor genes are, in many cases, a misleading description. When p53 is described as a tumour supressor gene, it is misleading because p53 is known to have oncogenic gain-of function mutations which gives the mutant p53 new oncogenic functions. There are many genes like p53 that display properties of both tumor supressor genes and oncogenes. So it entirely depends on the construct as Charon said. However Miriam described it as oncogenic, if she meant that she was handling an oncogenic p53 mutant then that isn't good news.

 

I'm not sure if there is a term to describe cancer contributing genes that have both loss-of-function and gain-of-function mutations. It is important to remember that even though a gene might be described as a tumour supressor gene, it can still have oncogenic gain-of-function mutations and therefore be described as an oncogene also. I think p53 is primarily described as a tumour supressor gene because the mutation spectrum mostly consists of loss-of-function mutations, this is just a guess though.

 

I think it could increase the risk for the cells which were infected, as long as the the mutation was a gain-of-function or a loss of function mutation in a construct which would replace the functioning one as Charon suggested.

Edited August 30, 2012 by jp255

[CharonY]

Well, explained. That is why I urge myriam to figure out what precisely she had as a construct. Based on the description of the events it is not clear to me whether naming it an oncogene was based actual knowledge of the nature of the construct, or not.

[myriam]

Thank you guys, some of you are right and jp255 is correct. As I talked about "oncogenic p53" it means I did mutagenesis to create my construct. In vitro, I have seen my p53 enhancing tumorigenesis of of my cell lines, so I already know what it is capable of. My real question was: what´s the likelihood this lentiviral vector survives the stomach pH. CharonY mentioned it is low but I haven´t been able to find any information on the literature about this. There are excellent reviews about oncogenes and tumor suppressor genes on PubMed if you are looking for deep knowledge about cancer.

[CharonY]

You misunderstand me. What I am saying that if you splash yourself, if it comes to viral infections it is quite likely to happen well before it actually hits the stomach acids. You have a lot of mucal surfaces that can serve as entry points. Stomach acid will only be relevant if you drink a significant amount of liquid. Droplets will just work on you epidermis and mucosal surface in mouth, nostrils. The focus on stomach acid is quite misplaced.

[myriam]

Thank you CharonY. I agree with you partly but I don´t think my question about stomach pH is misplaced. 1 ml of lentiviral vector supernatant may contain as high as 1e+8 or 1e+9 particles, so just let´s think about a couple of drops. (0,05 ul on average).

[CharonY]

The volume determines how far they can be transported through your mouth and oesophagus to your stomach, if we talk about splashes. Think about a small ingested droplet coming in contact with your mouth area, it will mostly spread out and not travel far (i.e. the viral particles will be spread nicely in your mouth area, a few may be transported to your oesophagus, but on the way there there are a lot of nice cells they can adhere to). Think of it in terms of sample loss of a sample moving through a tube or something similar with an adherent surface.

 

 

Of course, if you got a huge splash and your mouth was open (thus ingesting a significant volume), then yes, that could be an additional issue (but really what have you done to do that?). But think about it the following way, if a significant amount ends up in your stomach, quite a bit will already infect your mucosal surface. I would kind of worry about that first. So even if the portion ending up in your stomach deactivates all of them, you still have a large amount of viral particles in your mucosal surfaces. Especially if the droplets are small (and hence large surface to interact).

Though due to their inability to replicate (I hope) the overall health effect should still be minimal. A couple of infected cells are usually not sufficient for successful establishment of a tumor (increases lifetime risk, though).

Most lentiviruses are not terribly pH stable, but it can take a little while for them to be totally deactivated (30 mins to 1h). The thing is that they are generally tested for their ability to reproduce, which in your case they should not be able to, anyway.

 

 

[Dingervb]

Hi all,

What about the risk of infection with the adenovirus5 used to transform the 293 line? Aren't all 293 lines virally transformed? And does the viral DNA retained in the genome pose any risk after exposure? I was splashed in the eye today by a very dilute droplet of 293 cells in suspension at around 150,000cells/ml. I removed my contact lens and rinsed with water immediately. Should I be concerned? These cells have not been infected with any viral vectors, but what about the adenovirus they retain?

 

Trying not to freak out...

 

[CharonY]

Technically all human cell lines are considered to be potential risks (and hence BSL2), mainly due to presence of viruses. The well-known HeLa carries HPV, for example. However, the actual risk are relatively low. They viruses are generally only found in low concentrations (if at all) and a small droplet is not likely to do much. In fact, if you work long enough with them and are not careful there is a big chance that you inhaled more during pipetting (when not working under a biosafety bench), centrifugation, or other aerosol-generated processes (which you should not, but with many people working in a lab you never know).

Lab safety is all about preparing for the worst, and it is good that you are aware of potential risks.

But again, the likelihood of immediate consequences thereof are minuscule.

You should report the event, however, to have it documented regardless.

Edited January 11, 2013 by CharonY

[Dingervb]

Thanks CharonY. I went to the doc to get checked out today, and everything is filed through my employer. No eye symptoms yet, and I'll do the standard HIV, hepB, and hepC blood tests now, and in 1, 6, and 12 months. I feel like such an idiot! 12 years in human cell bio research, and you would think I would know better and not be so careless. I knew that the cells were not recently virus infected, and I think my years with primary human tissues and blood have made me lazy when it comes to caution with cell lines...

 

The thing that bugs me now is thinking about whether any of the cells might still be in my eye or in my tear duct... Can they grow? Seems like a silly question, and I'm sure the rare possibility of infection is greater than this, but I keep seeing the ATCC web page for this line, and next to "tumor forming" it says "YES".

 

Did they really have to put it in all caps???

 

[CharonY]

Complacency is unfortunately very common (I am certainly also guilty of it at times). I do have the advantage of having a mix of hazardous and non-hazardous cells (e.g. prokaryotes) and I try to slip into different mindsets for each type of work.

 

Regarding growth in the eyes, I would not think it very likely considering that most cell lines are wimps and require quite some pampering (though you will know more about your particular cell line than I do). Also there would be issues in adherence, considering that there are quite a few mechanisms to get rid of stuff from our eyes. But a check every now and then is certainly not the worst one could do.