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In vitro vaccine testing.. problems!


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Hi all! I'm a PhD student in the Netherlands and I'm in my second year... hence I'm really starting to worry about my PhD project.

It's about developing an in vitro testing method for a vaccine that is already commercialized and in use - only they need to test the batch-to-batch variation on mice, and they'd like to switch to cells. Long story short: the products I've been given have proved to be toxic on my cells. One product is the final vaccine, which has a very low viral protein concentration (3 ug/ml) and high alum concentration (2 g/l); the other is the non-adsorbed vaccine (no alum), with a higher protein concentration (60 ug/ml) but also high sucrose concentration (42% v/v). For one reason (too high alum) or another (too high sucrose) these products not only fail to activate my dendritic cells cultures, but they cause a high amount of debris, cell death, and the few results are hardly reproducible given the low viability. Also, the controls (excipient with alum and 42% sucrose in medium) cause the same effects, so it's obvious the problems are caused by the formulation and aren't specific of the inactivated virus (i wished...). I've tried to remove the sucrose/concentrate the proteins with dialysis cassettes, but it hasn't really worked - my first product has really really few proteins and they stick to the cassette, so it's not really concentrating, more like losing :( I'm trying with the second product now, but I'm quite skeptical as well.

These troubles seem something that I can do little about - basically the company thought "let's just give what we use on grown up humans and that should work as well on a 100.000 cells well!", well it doesn't. It has affected my motivation a lot, since I've been trying for long (several dilutions.. timepoints.. cells) and with no good results.

Does anybody have any suggestions?

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My preferred method of desalting and concentrating protein samples is simple ultracentrifugation using low MWCO filters. Use materials that have low protein adsorption. Dialysis tends to have low yields in my hands, too.

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3 minutes ago, CharonY said:

My preferred method of desalting and concentrating protein samples is simple ultracentrifugation using low MWCO filters. Use materials that have low protein adsorption. Dialysis tends to have low yields in my hands, too.

Thanks Charon, do you think a 10kDa filter would work?

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That depends on the MW and shape of your protein or protein fragment that you use. Also note that at low concentrations it may be better to use a smaller volume filter and go several times over it rather than with a large volume, as in the latter case you may have higher loss due to adhesion to a larger surface area on the plastic material.

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  • 1 month later...

Suppose the company could supply inactivated virus without the excipient but alum is thought to function as adjuvant at the dendritic cell level.  In your dilutions, have you tried to anticipate the in vivo concentrations presented to dendiritc cells?

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  • 5 months later...
On 09/01/2018 at 11:56 AM, polinares said:

Hi all! I'm a PhD student in the Netherlands and I'm in my second year... hence I'm really starting to worry about my PhD project.

It's about developing an in vitro testing method for a vaccine that is already commercialized and in use - only they need to test the batch-to-batch variation on mice, and they'd like to switch to cells. Long story short: the products I've been given have proved to be toxic on my cells. One product is the final vaccine, which has a very low viral protein concentration (3 ug/ml) and high alum concentration (2 g/l); the other is the non-adsorbed vaccine (no alum), with a higher protein concentration (60 ug/ml) but also high sucrose concentration (42% v/v). For one reason (too high alum) or another (too high sucrose) these products not only fail to activate my dendritic cells cultures, but they cause a high amount of debris, cell death, and the few results are hardly reproducible given the low viability. Also, the controls (excipient with alum and 42% sucrose in medium) cause the same effects, so it's obvious the problems are caused by the formulation and aren't specific of the inactivated virus (i wished...). I've tried to remove the sucrose/concentrate the proteins with dialysis cassettes, but it hasn't really worked - my first product has really really few proteins and they stick to the cassette, so it's not really concentrating, more like losing :( I'm trying with the second product now, but I'm quite skeptical as well.

These troubles seem something that I can do little about - basically the company thought "let's just give what we use on grown up humans and that should work as well on a 100.000 cells well!", well it doesn't. It has affected my motivation a lot, since I've been trying for long (several dilutions.. timepoints.. cells) and with no good results.

Does anybody have any suggestions?

I would quote your second post, but this belongs to the original.

The best way to test vaccines is to get a blood sample with them in it. Then, you see if it gives you back blood you can test with the purity test I will show you. This is where you need to see if mosquitoes attack it, yes? If not,then too bad, it doesn't work. On the other hand, if you breed flies in them, there will be more flies bred in the sickened samples, as, this is where the disease will aid the flies, of course. Alternatively, you could filter the blood, and, see if there is, with a sliding scale, 'drops' left over. This would be a sign of the disease, with more density, due to it's parasitic nature, where it bonds with the blood, clinging onto the 'mesh.'

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Gather your method is an attempt to simplify QC.  Understand your issues as you must use finished vaccine drug and adjuvants screw up your attempts.   What parameters are most critical to your vaccine efficacy?  Perhaps you can focus on those rather than jumping into in vitro model for immunity.

 

Curious.  How do you propose to validate your in vitro cell model?      It's a leap as even vaccines that generate antibody response in vivo are not always effective in disease.  As you point out - finished drug (with adjuant) makes it even more of a challenge.   prevention.http://www.pnas.org/content/pnas/85/18/6944.full.pdf

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On 8/3/2018 at 3:11 PM, Brett Nortj said:

The best way to test vaccines is to get a blood sample with them in it. Then, you see if it gives you back blood you can test with the purity test I will show you. This is where you need to see if mosquitoes attack it, yes? If not,then too bad, it doesn't work. On the other hand, if you breed flies in them, there will be more flies bred in the sickened samples, as, this is where the disease will aid the flies, of course. Alternatively, you could filter the blood, and, see if there is, with a sliding scale, 'drops' left over. This would be a sign of the disease, with more density, due to it's parasitic nature, where it bonds with the blood, clinging onto the 'mesh.'

Just in case it was not obvious. None of that makes any biological or medical sense.

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