Species D Adenoviruses as Oncolytic Viral Vectors
Round 1
Reviewer 1 Report
The authors have cloned species D subgroup serotypes 26, 28, 45 and 48 adenoviruses and have modified the E3-region to contain hNIS gene for virus-directed radioisotope therapy. They characterised the viruses using various in vitro methods. They show that the four species D adenoviruses can infect and can replicate in six different cancer cell lines. The experiments are well presented and of high quality.
Major concerns:
- The authors do not show any data on how these viruses are able to infect healthy tissues/primary cells. Without this data, the manuscript has little significance to the field.
- And if there is toxicity, the viruses should be modified to conditionally replicate in cancer cells. Developing viruses for systemic use this is of utmost importance.
Minor comment:
1. different serotypes can have different VP to IU ratios, it would be preferred if the infections would have been done with IU (as MOI) instead of VPs to rule out the differences in VP/IU ratios.
Author Response
Please see the attachment.
Author Response File: Author Response.pdf
Reviewer 2 Report
The manuscript by Bullard and co-workers studies oncolytic adenoviruses (Ad) with the aim to evaluate the species D Ad types 26, 28,45,48 as alternative virus types to the classical Ad5 in oncolytic virotherapy. Their generated replication competent species D viruses were tested in their ability to transduce, replicate, cel lysis in six different cancer cell lines. The results indicated that species D viruses showed the best transduction and cytotoxicity in SKBR3 breast cancer cells, followed by 293, A549, and HepG2 cells. Further, when the aforementioned viruses were modified to express the hNIS gene during infection and were evaluated for iodide uptake in multiple cancer cell lines. As shown by the authors all infected cell lines had high uptake of iodine, with the exception of the SKBR3 cell line.
The study is very well conducted, with appropriate controls and statistics in place. The manuscript is rare in this sense that it is aesthethically pleasing to read- perfect english, nicely polished figures and easy to follow the red line of the study throughout the manuscript. Furthermore, the results clearly show the promising potential of the Ad26, 28,45,48 as potential replacement for the troublesome Ad5 as the oncogenic vector. Hence, the reviewer does not have any major concerns with manuscript and considers it as an important study in oncogenic adenovirology. However, I still have a minor comments about the paper:
1) using word serotypes is not favored among the adenovirologists anymore. Rather types, since there are currently more that 110 Ad types (http://hadvwg.gmu.edu) characterized with more that half of them characterized by their genotype and not by serological features.
2) It remains unclear for me why the GFP-luciferase transgene was used if only the GFP was used as the read-out? Can the authors explain it?
3) I am a bit confused about the experiment shown in Fig.2. The species D viruses with GFP-Luc are replication competent, but as I understood from the M&M section the HAdV-5 was generated in Adeasy background which should not replicate in any other cell lines than 293. Can the authors clarify this? Even though the authors look at the transduction activity, it is important that in this comparison all the vectors are either replication competent or deficient. Since the CMV promoter can be activated by E1A (Olive et al., 1990), it is possible that GFP expression is enhanced by species D encoded E1A, whereas in the case Ad5 Adeasy vector there is no E1A which will not impact on CMV and GFP expression. Can the authors clarify that?
4)In line with my previous comment and Fig.5, can the authors clearify if they have used wild type Ad5 and where this particular refernce virus was obtained since there are different Ad5 viruses circulating in the community.
5) ther is obviously less iodine uptake in Ad26-hINIS virus infected cells compared to other virus infected cells. Can that be due to the reduced expression hNIS protein expression and not due to lower transduction? A simple western blot detecting hNIS protein could answer this question.
Anyhow, the manuscript message is clear and data solid! Congratulations to a nice work!
Author Response
Please see the attachment.
Author Response File: Author Response.pdf
Round 2
Reviewer 1 Report
The authors did not address the concerns adequately. There are plenty of published data on species D infectivity with various cell lines. Also, some published data show some cytotoxicity of Ad26 in non-transformed cells. When designing viruses for systemic use it is important to show conditional replication/cytotoxicity. The manuscript in the current form does not add significantly to the oncolytic virus field.