The manuscript presents meticulously executed work, a clear explanation, and attention to detail in the methods employed.
The authors demonstrate the generation of fibrillar assemblies from rPrP of various species, evaluating their ability to propagate in vivo in a transgenic model overexpressing hamster PrPC. They show that the majority, but not all, of the assemblies were pathogenic, exhibiting divergent adaptation processes. This study is highly relevant as it focuses on transmission barriers, offering insights into prion transmission and strain diversity.
While I appreciate the significance of this study to the scientific community, I have some concerns that I would like the authors to address.
Major concerns:
Lines 220-226: The authors mention the appearance of spherical objects in their preparations visualized by AFM. However, they do not document any in-depth study on these objects. Therefore, it is an overstatement to attribute the low abundance of these spherical objects in the preparation made with bovine rPrP to the absence of infectivity. There are many other reasons that could justify why only the bovine sample does not infect the hamster model.
It is well known that the PMCA technique, commonly used by the authors of this study, is a good technique for reproducing serial passages in vivo but in a faster and in vitro manner. On the other hand, it seems to be a fact that the conversion to a higher molecular weight pattern seems to depend, in some assemblies more than others, on the number of passages given to them in vivo. Therefore, the authors, familiar with the PMCA technique, should show whether this pattern change occurs after the application of serial rounds of PMCA.
The authors show how the seeding activities of the synthetic prions were equivalent or superior to those of hamster-adapted 263K scrapie prions. However, they do not show what the seeding activities of the original seeds were, nor if this activity changes with passages, especially those where there has been a pattern change.
Minor concerns:
In the abstract: "recapitulated" is an overstatement that should be changed as it could be misinterpreted to mean that it recapitulates the species barriers between mouse, human, and hamster, which is not the case.
In the abstract: "through a mutation-like process" is a concept that requires prior explanation as provided throughout the text. However, neither the term nor the concept is very appropriate given that sensu stricto no mutation occurs, nor is it accepted by everyone. Therefore, I suggest not using it in the abstract.
Lines 251-254: It is surprising how the misfolded murine recombinant, on a second passage, is capable of inducing disease in just 80 days. Given the peculiarity of this behavior, which differs from the other two samples from humans and hamsters, has this second passage been repeated with a new preparation from the beginning? The authors show the repetition of the first passage using a misfolded rPrP generated at another time, but they do not show that this dramatic change also occurs in the second passage.

Lines 293-297: It should be stated in how many animals the change to higher molecular weight occurs in the fourth passage.
Do the authors have any data showing that any of these synthetic prions can propagate in a wt hamster or wt mouse model? To what extent do the authors believe that the overexpression of the hamster protein distorts the reality of a transmission barrier? This information should be reflected in the discussion.
Line 378: I miss a reference for "and has been extended to 379 other protein misfolding neurodegenerative diseases such as Alzheimer’s and Parkinson’s diseases".
Discussion: Given the similarity between the hamster and the mouse and the significant difference in behavior after inoculation of the assemblies of these species, the authors could make some comments on the implication of some amino acids, of the few that these two proteins differ, that could provide an explanation for such peculiar behavior.
There is a lack of comment on why the recombinant bovine has not propagated in this model.
Line 464: I could not understand the phrase "We proposed that their synergetic action is essential for prion adaptation and overcoming species barriers".
Lines 465-467: In order to understand the hypothesis that the authors propose, a drawing/cartoon would be very helpful.
Lines 468-472: Given the limited knowledge available about the spherical particles mentioned by the authors, it is difficult to comprehend their implications in explaining the differential behavior exhibited by the hamster, mouse, or human assemblies, all of which are infectious and capable of crossing the species barrier.
Comments:
Line 35: The article https://doi.org/10.1073/pnas.1120076109 should be added since it is the one that is really related to the statement regarding the rabbit.
Lines 61-63: It refers to a situation that could also be explained with a "nonadaptive prion amplification (NAPA)" (https://www.pnas.org/doi/full/10.1073/pnas.1611891114) and therefore, it is suggested to include it as another option.
Lines 77-79: It refers to the generation of bona fide infectivity using recombinant protein, so a recent work should be included that not only talks about this but also about the generation of different strains that might help to understand the work of this manuscript (https://actaneurocomms.biomedcentral.com/articles/10.1186/s40478-023-01640-8).
Similarly, a recent publication demonstrating the generation of hundreds of infectious recombinant proteins would also be a good reference to include in this context (https://www.nature.com/articles/s41467-024-46360-2).

I do not think it is appropriate to reference an unpublished article from 2012 (BioArchives) - reference 87.
Have the authors been able to propagate the recombinant murine strain in a hamster recombinant? What would be expected after its inoculation in the Tg7 model; behavior similar to hamster or mouse assemblies?

The authors have addressed all my concerns.