Peer review of: A broad-spectrum virus- and host-targeting peptide against respiratory viruses including (2020)

Peer Review File - Reviewers' comments first round 
 
Reviewer #1 (Remarks to the Author): 
 
Zhao and co-workers show data on the alkaline peptide P9R with broad antiviral activity against 
the novel SARS-CoV-2, two other highly pathogenic CoV (MERS-CoV and SARS-CoV), influenza 
viruses and non-enveloped rhinovirus. Overall, the work is sound. Experiments are carefully 
conducted and results are clearly presented with reasonable discussion. The 2019 new coronavirus 
has spread to the world in many countries, and there is an urgent need to develop broad-spectrum 
antivirals for treating novel emerging viruses from now on. 
 
In this study, the authors are providing a novel concept of alkaline antiviral peptide. P9R could 
bind to different viruses and then inhibit host endosomal acidification. As far as I know, this is the 
first antiviral peptide which targets both of virus and host to exert very broad antiviral activity. The 
authors clearly showed that P9R could bind to different viruses and then could efficiently inhibit 
virus replication with low IC50. They used two control peptide PA1 and P9RS to indicate that the 
binding to virus only (PA1) or inhibiting host endosomal acidification only (P9RS) could not show 
antiviral activity. They used different methods to confirm the mechanism that the binding to 
viruses and the inhibition on host endosomal acidification are both needed for the antiviral activity 
of alkaline P9R. The results are solid supported by the experiments. The in vivo results indicated 
the efficiency of P9R for anti-influenza virus in mice. The low possibility of P9R inducing drug-
resistance virus indicated that this broad-spectrum antiviral peptide, in principle, may have some 
clinical advantages because of the quickly emergency of drug-resistance virus during treatment. 
 
Comments: 
 
- Why did the authors only study respiratory viruses? There are other non-respiratory viruses 
which are pH-dependent viruses. Dose P9R also have antiviral activity against other viruses? Have 
the authors tried other non-respiratory viruses? 
 
- Why can P9R bind to different viruses? It seems that cysteines are important for defensin 
peptide. The authors may discuss this a little bit more in discussion. 
 
- In line 121, authors made a peptide P9RS with multiple substitutions. As a result, P9RS lost the 
antiviral activity, but with the same positive charge as P9R. It is a good control for this study. How 
did the authors design this peptide P9RS? 
 
- In line 217, ‘enterovirus-A7’ should be ‘enterovirus-A71’ 
 
- Recently, the clinically approved drug Hydroxychloroquine was identified to be effective in 
inhibiting SARS-CoV-2. ‘Hydroxychloroquine, a less toxic derivative of chloroquine, is effective in 
inhibiting SARS-CoV-2 infection in vitro’. Please discuss this in the discussion related to the 
chloroquine part. 
 
- In line 559 of figure legend 4, ‘Zana’ is not necessary? 
 
- Figure 5a: It is suggested to show the concentration of both Zanamivir and P9R as nM. 
 
 
 
Reviewer #2 (Remarks to the Author): 
 
The manuscript from Yuen et al., entitled “A broad-spectrum virus- and host-targeting antiviral 
peptide against SARS-CoV-2 and other respiratory viruses” reports on a broadly antiviral peptide, 
which has been derived from mouse β-beta defensin 4. The authors performed an extensive study 
against several viral strains, which revealed that the previously reported lead peptide P9 (Yuen et 
al., 2016, Scientific reports) can be converted in a peptide (P9R) with higher antiviral activity, if 
four amino acids at the C-terminal region have been replaced by positively charged arginine. The 
authors claim that the peptide acts upon binding to the viral surface and by inhibiting endosomal 

acidification. Although there is currently an emerging need for new antivirals especially against 
CoVid-19 (SARS-CoV-2), and the principle strategy to develop naturally inspired peptides with 
antiviral potency is a promising approach I noticed several significant weaknesses in the paper. 
Meaning more control experiments and clarifications are needed to provide more evidence on the 
mechanism of action of the peptide, and to show that the peptide is applicable in a therapeutic 
setup. 
Major concerns: 
1. Peptides: 
A) The solvent of the peptide needs to be stated. As the water solubility of the peptides is very low 
I was wondering which vehicle the authors used in their experiments, especially in vivo. This is 
also relevant in terms of the applicability of the peptide P9R. 
B) In Figure 1a the authors mentioned “positive charges” of the peptides. To which pH the given 
values for the “net charge” are related to. How does it change during endosomal acidification? 
C) The manuscript gives the impression that the positively charged (basic) peptides act buffering 
within endosomes. See page 6, line 115: “the degree of positive charge was correlated with the 
degree of inhibition of endosomal acidification”. However, I cannot follow the hypothesis of P9R as 
a buffering peptide from a theoretical point of few. The pKa value of the guanidium group of 
arginine is around 12, which means guanidinium is already protonated at physiological pH 7.4. 
These residues cannot buffer more protons. Histidines instead would act buffering at pH 4.5-6.5 in 
the endosomes. Can the authors comment on that. I suspect the sequence feature of P9R, 
provides the peptide another functionality, which brings me to the next point. 
D) With the introduction of positive charges at the C-terminus of the peptide the authors created 
an amphiphilic peptide. It is highly probable that peptide P9R now disturbs the integrity of lipid 
membranes. Thus, it is possible that P9R acts virucidal, which need to be shown (see below) and 
discussed in the manuscript. In addition, the peptide could lead to proton leakage at the 
endosomal membrane and thus lead to a delay or even inhibition of acidification. However, I see 
that this point would need more extensive cell culture studies. However, virus fusion inhibition 
assays (e.g. virus induced hemolysis inhibition or fluorescence dequenching assays) with H1N1 
virus are simple to setup and could give a first indication of the mode of action of the peptides 
(e.g. pH buffering). The same holds true for a potential virucidal inhibition: It would be highly 
relevant to show if peptide P9R is rupturing the virus integrity. Either by electron microscopy of 
virus particles treated with P9R or biochemically by virus incubation with peptide followed by 
dialysis. If the binding is reversible the virus titer should not decrease. 
E) In all in vitro assays with virus, the authors first incubated virus with peptides and then treated 
cells with the virus-peptide mix. However, this approach reflects only a prophylactically antiviral 
treatment. It would be necessary to show at least with one virus a therapeutic setup in which cells 
get first infected before the treatment takes place. In the best case with a virus growth curve. If 
the peptides do not bind unspecifically to lipid membranes, virus inhibition should also take place 
on virus progeny. This would also support and rationalize the in vivo studies, in which a 
therapeutic setup seems to work. 
F) For the in vivo studies no virus titers are given. It is not clear how the peptide treatments work, 
even in the therapeutic setup. Otherwise no direct correlation between peptide treatment and 
antiviral effect (virus titer reduction) can be drawn. 
G) Although the authors showed cytotoxicity data I think it is important to show the interaction of 
the peptides with lipid membranes. As the peptides act broadly antiviral it needs to be shown that 
the peptides do not bind unspecifically to hydrophobic surfaces in general. I suggest simple 
hemolysis assays with red blood cells at 37°C with the peptides, but other experiments (e.g. with 
liposomes) could also be implemented. Further the authors used a fluorescent peptide version. Can 
the authors provide images of cells treated with the fluorescent peptide only to show that there is 
no strong cell membrane binding? 
 
Minor concerns: 
- In several figures the authors did not provide the meaning of the error bars. Is it SEM or SD? 
- No NMR spectra or data are given for figure 3g. 
- In the abstract, the authors write that P9R exhibited antiviral activity against pH-dependent 
viruses for membrane fusion. However, non-enveloped viruses do not fuse with the endosomal 
membrane although a relevance of the pH reduction in the endosomes might play a role for virus 
uncoating. This needs to be rephrased. 
- Figure 3h should be Figure 3g in the article 

 
 
 
Reviewer #3 (Remarks to the Author): 
 
In this study, Zhao et al. analyze the broad-spectrum activity of peptides against various 
respiratory viruses including SARS-CoV-2 and H7N9. The rationale of the antiviral peptides is that 
it interacts with the virus particle and inhibits endosomal acidification. This is a very elegant way 
to inhibit replication of viruses with an endosomal stage and thus provides –at least theoretically- 
a target for a broad range of viruses. The peptides are then evaluated in cell culture and later the 
mouse model for influenza was used. In general, the study is interesting and has promising 
components. However, as it stands, the study has some major weaknesses that should be 
addressed. 
 
Major points: 
 
#1. Figure 1-3: Antiviral assessment was performed in cell culture. However, antiviral effects are 
shown in percentage, which is not very significant given that any reduction below 1log with 
influenza viruses is not considered of biological relevance. The authors should show the data on 
“infectious particles” (e.g. p.f.u.) rather than copy number that is not very robust. 
 
#2: Best hit peptide P9R should be tested on primary airway epithelia with all viruses. Peptide 
should be tested at various concentrations to evaluate its impact on virus replication kinetics. The 
results should be shown as p.f.u.. This is particularly important since the mouse model is only 
used to assess the antiviral activity of the peptide P9R against H1N1 influenza. 
 
#3: Figure 4: The mouse data are not convincing. The authors used in some groups only 5 mice. 
The challenge dose used with 3xLD50 is very low for H1N1. Given the low animal number, even 
variations among biological replicates could alter survival rates. The authors should use n=10-20 
per peptid/concentration. For a solid and convincing challenge 10xLD50 should be used. 
Additionally, virus titers in the lungs of treated and non-treated mice should be measured. 
 
#4: Figure 4: In the absence of animal data on coronaviruses, the authors should at least repeat 
the mouse experiments with H7N9 to prove their claim of having an in vitro and in vivo potent 
antiviral peptide against various respiratory viruses. 
 
Minor point: 
 
The manuscript requires amendments in language and grammar.