Cat allergens are generated when cats lick and groom themselves. The saliva dries and small particles become airborne. More than 95% of patients suffering from cat allergies show a reaction to major allergen, Fel d 1. The following steps were used to generate mutant proteins of Fel d 1 then IgE binding capacity were analyzed:
1. Error-prone PCR (epPCR) was done on the Fel D 1 to generate mutants that retained epitope function were then expressed in E. Coli.
2. The proteins were then extracted and eluted by size exclusion chromatography, 30 kDa, similar to Fel D 1 wild type.
The IgE binding capacity and T cell activations were then analyzed and showed that these mutants actually reduced IgE binding affinity and reduced T cell activation. This method can definitely serve as a means of generating vaccines for the various types of allergens.
Source:
Nilsson OB, Adedoyin J, Rhyner C, Neimert-Andersson T, Grundström J, Berndt KD, Crameri R, Grönlund H.
PLoS One. 2011;6(9):e24558. Epub 2011 Sep 13.
- PMID:
- 21931754
This is a pretty interesting paper. Essentially, as you described, they induced mutations in the gene rFel d1 (which encodes for Fel d1, the primary cat allergen) and assessed the ability of the mutated product to function as an allergen. I find it to be fascinating that after seven rounds of inducing mutations via "error-prone PCR" that no differences were observed in Fel d1 expression or surface topology. The authors then assessed the ability of these mutants to function as an allergen by looking at IgE binding, anaphylactic activity, T-cell reactivity, and other factors. In conclusion, they were able to create an in vitro model for quick allergy vaccine development.
ReplyDeleteI just had a quick question for you:
What exactly is error-prone polymerase-chain reaction (PCR)? Is that just PCR using a low-fidelity polymerase (the enzyme that actually replicates the single-stranded DNA templates) or is it PCR under sub-optimal conditions (e.g. incorrect cycling temperatures or less than ideal buffer concentrations, etc.)?
Citations:
Nilsson OB, Adedoyin J, Rhyner C, Neimert-Andersson T, Grundström J, et al. 2011 In Vitro Evolution of Allergy Vaccine Candidates, with Maintained Structure, but Reduced B Cell and T Cell Activation Capacity. PLoS ONE 6(9): e24558. doi:10.1371/journal.pone.0024558
That is a great question. I had also had to look up what erPCR was and this article covers it very well. One method uses mutagenic (overhanging) primers even before the use of PCR. However, in the erPCR the reaction conditions are altered like adding Mn+2 or Mg+2 to the reaction mixture.
ReplyDeleteA general model of error-prone PCR.
Pritchard L, Corne D, Kell D, Rowland J, Winson M.
J Theor Biol. 2005 Jun 21;234(4):497-509.
There are some people who seem to be allergic to kittens and not cats, or vice versa. Do you know why this may be the case? Does it have to do with the allergen you mentioned,Fel D 1 and it possibly not being expressed in the kitten? Or do people developing some sort of immunological tolerance against the kitten so that once it matures into a cat, they can live with it? Thanks.
ReplyDeleteThis is very interesting! Much of my family is allergic to cats BUT NOT kittens or dogs. I have always wondered what would cause someone to have an allergic reaction to one but not the others. Could it passibly be due to a slightly different version of Fel D 1 in cats than in kittens? Maybe as they mature, the posttranslational modifications are altered leading to an altered Fel D 1 IgE? Also, do they have any data on how long T cell activation was reduced after vaccination? Is it a perfect vaccine or do some subjects still have a minor allergic response? Finally, is the binding affinity of IgE decreased for all antigens it can bind or just IgE for Fel D 1?
ReplyDeleteGreat questions guys! I did some searching and found that there is NO clear reason to why some people are allergic to cats but not to kittens. Some studies have shown that Fel D1 is expressed less in kittens compared to mature cats.
ReplyDeleteAlex, you bring up a good point on the post-translational modifications, but I could not find any support for this. As for the T cell activation they took different concentrations of the Fel D1 and mutants incubated them with PBMCs and compared the (cpm) counts per minute for stimulated T-cells. These were then expressed as a stimulation index (SI) and any value greater or equal to 2.0 were considered positive.
In some patients the vaccination did have a minor allergic response. The IgE affinity for Fel D1 was decreased along with some of the other antigens. Depending on the mutant the affinity of IgE was effected.
I am one of those who are allergic to cats but not kittens at all. This was very interesting to read and I really enjoyed the facts that you threw out there. In addition, the questions and answers in the previous comments were definitely things I was wondering and wanted to know. Awesome job!
ReplyDelete