When I observe the raw data in the V2plus4 software both versions there is always a comma separator displayed in the raw data window independent from the existance of the nanovna.ini.
From the manual, LabView changes how the comma is handled when talking to external peripherals depending how the INI is set. The INI does not effect how the data is presented on the display.
I would have no way of knowing what you are doing. Every detail is important. For all I know you may feel that the 1.x and 2.x software shares the same calibration file format and are trying to use a 1.x cal with the 2.x software. I would have to guess as my crystal ball is out for service. Every detail matters. You may feel that firmware for the same hardware should use the same calibration.
For the original NanoVNA
If you read the posts in this forum, you know that DisLord was attempting to add a feature to the firmware to handle the comma. Does other firmware handle the comma differently? I have no desire to look into it. There is a reason I had settled on a version of firmware I use. Of course, if you took the time to read the posts here, you saw the whole first and second discussion about the regional settings. You are experimenting with the latest firmware available for your original NanoVNA, which I have said had problems and also why I abandoned them.
Why is the V2plus4 working in any case even without the nanovna.ini and with a comma selected in the regional settings ?
Let's assume you took the time to read the documentation they published for it. So you know that the V2Plus is using a completely different protocol and where the original NanoVNA uses ASCII, the V2Plus uses? Are the decimal and comma ASCII characters? This should have been obvious.
To sum it all up,
Read more, post less.
Create new calibration files for the software/firmware/hardware/cables/standards you are using at the time.
If you are trying to solve problems like this, don't make your problem even more complex by adding more variables like calibration. Learn to simplify your problems. If possible, learn to split the problem. In your example with the original NanoVNA, while you left out the fact you had changed your regional settings, it was pretty obvious you had. You could have used the default regional settings and standard INI (or no INI) and see if the problem went away. This is what I mean by cutting a problem in half.
If you can't solve your problem, detail every step so someone else can reproduce your findings.
If you like playing with the firmware of the week, expect to run into problems.
If you use one of the regional settings that supports the comma, add the useLocaleDecimalPt=False to the INI file. If you use the decimal, then leave this line out.