T cell deficiency precipitates antibody evasion and emergence of neurovirulent polyomavirus

Decision letter after peer review:

[Editors’ note: the authors submitted for reconsideration following the decision after peer review. What follows is the decision letter after the first round of review.]

Thank you for submitting the paper "T Cell Deficiency Precipitates Antibody Evasion and Emergence of Neurovirulent Polyomavirus" for consideration by eLife. Your article has been reviewed by three peer reviewers, one of whom is a member of our Board of Reviewing Editors, and the evaluation has been overseen by a Senior Editor.

The reviewers all agreed that the results are fascinating and important, due to the role of compromised immune systems for polyoma and indeed other viruses. Nonetheless, as you will see below, the reviewers had detailed issues with experimental justification for some of the conclusions, overly complicated prose, and organization. In the cases of immune depletion, additional controls are requested.

The policy of eLife is not to accept papers that require more than a few months of work. Considering the requests for additional experimentation and, just as importantly, revision of the flow and logic of the manuscript, it is not accepted for publication or revision at this point. However, the Editors may be willing to consider a revised version if comments such as requests for genetic reconstructions, confirmations of antibody depletion, and careful editing interpretations can be fully addressed.

Below, the comments of the reviewers are combined, in general, and figure-by-figure. This organization was necessary because the writing made it difficult to distinguish which experiments supported which conclusion.

Of all manuscripts submitted to eLife, only a minority will ultimately be accepted for publication. The policy of eLife is to reject manuscripts that require major revision, to facilitate the authors' submitting the manuscript elsewhere. In the case of this interesting manuscript, we encourage resubmission provided you and your colleagues are willing to address the critiques below. Otherwise, we hope that the reviews and our feedback will help you in publishing your manuscript. Thank you for giving us the opportunity to review this work.

Summary

This is an interesting study of mouse polyomavirus (MuPyV) as a model for the human pathogen JCPyV. The latter can spread to the brain in immunocompromised individuals, where replication leads to progressive multifocal leukoencephalopathy (PML). This replication is often accompanied by mutations in the major viral capsid protein, VP1, that make it resistant to certain neutralizing antibodies. The selection for such resistance can be recapitulated in the mouse model. The authors examine the contributions of the T cell response to the appearance of these resistant viruses and examine the biology of an interesting mutant that they discovered. Thus, this study addresses an important question, especially given a resurgence in PML in recent years due to the increasing use of immunomodulatory monoclonal antibodies to treat various diseases. JC polyomavirus causes Progressive Multifocal Leukoencephalopathy (PML) in T-cell immunosuppressed patients. The host-pathogen interaction behind this condition is not fully understood. Using a complicated murine infection model, the authors address the particular role of T cells in this evolution. The model includes commercially available B-cell depleted mice, first infected with MuPyV, then treated with an anti-VP1mAb, and 16 days later with IgG control or anti-CD4 and/or anti-CD8 antibodies to induce T-cell depletion. Viral replication is monitored in the kidney and blood at different times post-infection in T-cell-depleted versus control mice and the emergence of resistant variants is characterized. Finally, the fitness of a resistant variant presenting two mutations is compared in cells, tissues, and mice. The authors show that CD4 T-cells play a critical role in limiting or delaying the emergence of resistant variants. In addition, they highlight that variants that can emerge in absence of T-cell control show fitness loss in kidney but increased neurovirulence. These observations shed some light on JC virus pathogenesis in T-cell suppressed patients. The conclusions are overall supported by the data and confirm the expected critical role of T-cells in controlling viral replication. However, concerns about the presentation will require additional editing and experimental work.

Comments concerning the presentation

1. An overall concern I have is that the first part of the introduction and the entire abstract sound as though JCPyV itself is under investigation. These possibly misleading sections need to be rewritten. There is no need to be concerned that the relevance of their model is going to be questioned.

2. The relevance of these mutations relative to existing JC mutants is not made clear. In fact, two out of three reviewers missed the point that the mouse model involved a different virus from the human one. Clarified prose is needed.

3. It would be good to give some background on JCPyV receptor usage and also if the receptor(s) is (are) conserved between mouse and human and in the different tissues.

4. The last paragraph of the introduction somewhat exaggerates the findings, particularly the sentence beginning "In this study…." It is not clear that Ab-deficient mice are really the equivalent of PML patients since the patients DO produce antibodies against the virus. The model is not being questioned but it should not be oversold.

Comments concerning the figures

Figure 1: Shows that treatment with anti CD4⁺ T cell antibody, but not anti-CD8⁺ T cell antibody or IgG control antibody, reduces control of the virus in a mouse model that contains B cells.

1. The tissue should be clearly indicated in the legend and ideally on the panel.

2. The word 'resurgence' is not appropriate because the data in Figure 2B do not show early viremia.

3. The original comment from all three reviewers was that fluorescent cytometry is needed to confirm T-cell depletion, especially in the case of CD8⁺ T cells. However, it is stated somewhere that these are commercially obtained preparations. If this is the case and there is a validation of the depletions, that needs to be clearly stated. These controls are important, however they are obtained.

Supplemental Figure 1: Throughout the manuscript, the term 'blind spot' is used. This colloquial language implies a structural mechanism of neutralization that is often the case but not always.

1. Instead, more precise evolutionary language (especially for a general audience such as that of eLife) such as "anti-VP1 treatment allows the selection of VP1-antibody resistant viruses.

2. The data themselves show convincingly that the anti-VP1 antibody specifically neutralizes wild-type virus but not the A2Δ295 virus. At this point in the manuscript, the A2D295 virus has not been mentioned so it is not clear why it is S.Figure 1.

3. In (E), the y-axis of 'neutralizing titer' is not clear. Is it 'extent depletion'? Is it the

required serum dilution?

Figure 2 shows convincingly that the combination of anti-CD4⁺ and anti-CD8⁺ antibodies greatly reduce viral control in kidney, spleen, and brain.

1. Fluorescent cell quantitation is not provided to ensure the depletion of the relevant T cell populations,

2. The data in (B) cannot be clearly deciphered and the x-axis should be expanded.

However, the remaining panels in B, dissect the data in the time course. As in Figure 1, these data show that there is little control of the virus in the mice treated with anti-CD8⁺ antibodies.

Figure 3 is the strongest figure in the paper, showing that the sequences of viruses that grow upon anti-VP1 monoclonal antibody treatment of B-cell-deficient mice show a variety of sequence changes. Interestingly, a Δ295 and other amino acid 295 mutations are selected under all conditions and thus, as the authors conclude, confer resistance to the anti-VP1 antibody under all conditions. However, more mutations seem to be required in the absence of T cell depletion, and different spectra of mutations are found under each condition. Panel B shows the location in the VP1-antibody complex of several of the amino acids involved. Panel C shows a similar lack of neutralization for all selected viruses in cultured cells, and Panel D shows that there is different tropism and fitness for the various viruses in the kidney and spleen of infected B-cell-deficient mice in the presence of the anti-VP1 antibody.

1. It is not clear whether the mutations of interest have been installed in the otherwise wild-type virus free of other mutations that might have been selected; if it is not, this should be clearly stated.

Figure 4 shows explicit challenges with the 295 virus.

1. This virus is, presumably, reconstructed so that only this mutation is present. If it is not, it should be, and several points brought up by one of the reviewers is then relevant. Specifically, "The extensive analysis of the A2.D295N, A2.Δ297, and double mutant viruses is impressive. Given that the double mutant has enhanced replication properties, was the entire genome sequenced to confirm nothing has happened in the non-coding control region or the early region. It is not clear from the methods. Another sequencing-related question relates to the paragraph on lines 214-221: how many clones did they analyze, in order to rule out the presence of the D295N/A mutations alone? Could there have been a low-frequency present that was missed (and that might only be detected by NGS)?"

2. The legend does not describe this but the text (line 152) states that the mice used were B-cell deficient. If that is the case, this experiment with the B-cell escape variant is the same experiment done with wild-type virus previously and thus does not enhance the findings of the paper at this point, unless this reviewer is missing the point. Perhaps it was performed in the B-cell-deficient mouse supplemented with the specific antibody?

3. Why are infections done in the hind footpad in the initial protocol and then by IV for the challenge or to test the fitness of the different mutants? The infection route should be clearly described and justified in the result section.

Figure 5 switches up the viruses under study. It shows a very good experiment in which the viruses have now clearly been reconstructed in isolation. However, several inconsistencies in the data likely make it necessary to repeat several aspects of these experiments.

1. 297, D295N, and double mutant viruses were measured by somewhat indirect means such as fold increase over input and fixed time points at two different MOIs, rather than growth curves at a fixed MOI so that any differences in total yield can be clearly assessed.

2. In Panel I, the frequencies of the D295N/Δ297 double mutation in the original mice in which they were isolated, as well as a different combination of D295A/297 from another mouse, are shown. These data do not contribute mechanistically to Figure 5 and should be moved to Panel 3 where the mutations are first discussed. They do, however, provide a basis for discussion of the potential order of mutational events, which should be in the discussion.

3. The double mutant virus seems fitter than the wt. However, the abundance of this variant is very low in Fig5I, how do the authors explain that?

4. Figure 5 D to F and Figure 5 sup 1 are showing very close assays. In addition, the results are not always consistent depending on the readout: in Figure 5 D to F D295N is consistently less fit than A2 while in the supplementary figure, in panel A it looks fitter. Is it a transfected virus in the sup panel A? Why is A2 del 297 not shown in figure 5 panel A? Again, in panel E of Figure 5, this variant is as fit as wt but not in the other assay.

5. Sensitivity to neuraminidase: based on Figure 5 G and 5 sup D, del295 seems insensitive to sialidase pre-treatment, then why in panels E and F of Figure 5 sup it is sensitive?

6. With the way results are presented, it is not clear that panel H represents kidney viral loads after mice infection.

7. In figure 5H, the double mutant is as fit as the wt in the kidney. This is not reproduced in Figure 6.

Figure 6.

1. Why did the authors perform IC inoculation rather than IV or inoculation in the hind footpad as before? There are likely reasons for this, but they need to be explained.

2. The authors show in several figures that the double mutant has an advantage in cells, and this is shown here in the mouse brain. If such variants present increased neurovirulence, the authors should try to understand by what mechanism. Is binding to the receptor changed in the brain? Binding assay in brain tissues should be done in the same way as with kidney tissues.

Supplement to Figure 6 presents further exploration into the comparative phenotypes of the four viruses presented in Figure 5.

Reviewer #1 (Recommendations for the authors):

– In the introduction, it would be good to give some background on JCPyV receptor usage and also if the receptor(s) is(are) conserved between mouse and human and in the different tissues.

– Figures 1 and 2 should be grouped in a single and simplified figure. The experimental setup is the same and only the site of genome replication and the time of analysis differ. In figure 1, the tissue should be clearly indicated in the legend and ideally on the panel.

– Figure 4: Viral loads are not different than in absence of virus challenge in figure 3, the authors should comment.

– Why are infections done in the hind footpad in the initial protocol and then by IV for the challenge or to test the fitness of the different mutants? The infection route should be clearly described and justified in the result section.

– Figure 5 and the supplementary figure 5 are confusing and several points should be clarified in the result section:

o the double mutant virus seems fitter than the wt. However, the abundance of this variant is very low in Fig5I, how do the authors explain that?

o Figure 5 D to F and Figure 5 sup 1 are showing very close assays. In addition, the results are not always consistent depending on the readout: in Figure 5 D to F D295N is consistently less fit than A2 while in the supplementary figure, in panel A it looks fitter. Is it a transfected virus in the sup panel A? Why is A2 del 297 not shown in figure 5 panel A? Again, in panel E of Figure 5, this variant is as fit as wt but not in the other assay.

o Sensitivity to neuraminidase: based on Figure 5 G and 5 sup D, del295 seems unsensitive to sialidase pre-treatment, then why in panels E and F of Figure 5 sup it is sensitive?

o With the way results are presented, it is not clear that panel H represents kidney viral loads after mice infection.

o In figure 5H, the double mutant is as fit as the wt in the kidney. This is not reproduced in Figure 6

– Figure 6: Why did the authors perform IC inoculation rather than IV or inoculation in the hind footpad as before? Also, the authors show in several figures that the double mutant is an advantage, in cells already and here in mice brain. If such variants present increased neurovirulence, the authors should try to understand by what mechanism. Is binding to the receptor changed in the brain? Binding assay in brain tissues should be done in the same way as with kidney tissues.

Reviewer #2 (Recommendations for the Authors):

1. One overall concern I have is that in the first part of the introduction and the entire abstract, they make it sound as if they are studying JCPyV. These sections need to be rewritten because I think they are misleading. I don't think they need to be concerned that the relevance of their model is going to be questioned, at least not by this reviewer.

2. I found the middle of the last paragraph of the introduction to be another example of "exaggeration," particularly the sentence beginning "In this study…." I'm not sure Ab-deficient mice are really the equivalent of PML patients since the patients do produce antibodies against the virus. Again, I am not questioning their model but they should not oversell it.

3. What does "un-neutralized virus" on line 121 mean?

4. Do the authors have any sense of when during the persistent phase of the infection the mutations are arising? Is it before or after reactivation of all-out replication, for example?

5. The extensive analysis of the A2.D295N, A2.Δ297, and double mutant viruses is impressive. Given that the double mutant has enhanced replication properties, I am wondering whether they sequenced the entire genome to confirm nothing has happened in the non-coding control region or the early region. It is not clear from the methods. Another sequencing-related question relates to the paragraph on lines 214-221: how many clones did they analyze, in order to rule out the presence of the D295N/A mutations alone? Could there have been a low-frequency present that was missed (and that might only be detected by NGS)?

Reviewer #3 (Recommendations for the authors):

The narrative of this manuscript is difficult to follow although it contains much interesting biology. It begins by showing that T-cell control of JC polyomavirus in mice is important in the presence and absence of a B-cell response. Then, the authors show that the relevant T cells for viral control are CD4⁺, although controls for specific cell depletion are not provided. These data are presented in Figures 1, S.Figures 1, Figure 2, and one panel of Figure 3. The antibody-escape mutations in VP1 described in Figure 3 are very interesting and form the heart of this paper.

In Figure 5, four viruses are defined genetically and their properties compared in antibody-binding studies, antibody-neutralization studies, and in B-cell depleted mice. The viruses are A2 wild-type, Δ297, D295N, and double mutant. The authors show that the D295N virus (as does the Δ295 virus they have been studying up until this point) confers resistance to a particular neutralizing antibody. However, the Δ297 mutation is required to increase its fitness. Thus, Figure 5 is where all the pieces are together to ask about the properties of these viruses in the presence and absence of the neutralizing antibody and CD4⁺ T cells.

By stopping where they do in the narrative, the message of the manuscript is that compensatory mutations are often needed to increase the fitness of antibody- or drug-resistant mutations. This has been shown many times, but, as such, remains of interest to a specialized audience. ,

The writing is so confusing that suggestions, below, are to highlight the findings and make suggestions to increase their clarity and persuasiveness.

Figure 1: Shows that treatment with anti- depletion of CD4⁺ T cell antibody, but not anti-CD8⁺ T cell antibody or IgG control antibody, reduces control of the virus in mouse model that contains B cells.

1. The word 'resurgence' is not appropriate because data in Figure 2B do not show early viremia.

2. Fluorescent cytometry is needed to confirm T-cell depletion, especially in the case of CD8⁺ T cells.

Supplemental Figure 1: Throughout the manuscript, the term 'blind spot' is used. This colloquial language implies a structural mechanism of neutralization that is often the case but not always.

1. Instead, more precise evolutionary language (especially for a general audience such as that of eLife) such as "anti-VP1 treatment allows the selection of VP1-antibody resistant viruses.

2. The data themselves show convincingly that the anti-VP1 antibody specifically neutralizes wild-type virus but not the A2Δ295 virus. At this point in the manuscript, the A2D295 virus has not been mentioned so it is not clear why it is S.Figure 1.

3. In (E), the y-axis of 'neutralizing titer' is not clear. Is it 'extent depletion'? Is it the

required serum dilution?

Figure 2: (A) Shows a time course of viremia in B cell-deficient mice after treatment with IgG, anti-CD4⁺ , anti-CD8⁺, and a combination of anti-CD4⁺ and anti-CD8⁺ T cell antibodies. The finding that CD4⁺ , but not CD8⁺ T cells can control viremia in the absence of B cells is interesting but not pursued mechanistically.

1. Fluorescent cell quantitation is provided to ensure the depletion of the relevant T cell populations,

2. The data in (B) cannot be clearly deciphered and the x-axis should be expanded.

However, the remaining panels in B, dissect the data in the time course. As in Figure 1, these data show that there is little control of the virus in the mice treated with anti-CD8⁺ antibodies. The remaining panels show convincingly that the combination of anti-CD4⁺ and anti-CD8⁺ antibodies greatly reduce viral control in kidney, spleen, and brain.

Figure 3 is the strongest figure in the paper, showing that the sequences of viruses that grow upon anti-VP1 monoclonal antibody treatment of B-cell-deficient mice show a variety of sequence changes. Interestingly, a Δ295 and other amino acid 295 mutations are selected under all conditions and thus, as the authors conclude, confer resistance to the anti-VP1 antibody under all conditions. However, more mutations seem to be required in the absence of T cell depletion, and different spectra of mutations are found under each condition. Panel B shows the location in the VP1-antibody complex of several of the amino acids involved. Panel C shows a similar lack of neutralization for all selected viruses in cultured cells, and Panel D shows that there is different tropism and fitness for the various viruses in the kidney and spleen of infected B-cell-deficient mice in the presence of the anti-VP1 antibody.

1. It is not clear whether the mutations of interest have been installed in the otherwise wild-type virus free of other mutations that might have been selected; if it is not, this should be clearly stated.

Figure 4 shows an explicit challenge with the Δ295 virus, presumably specifically reconstructed so that only this mutation is present. Panel (B) shows viral growth following T cell depletion. The legend does not describe this but the text (line 152) that the mice used were B-cell deficient.

1. Therefore this experiment with the B-cell escape variant is the same experiment done with wild-type virus previously and thus does not enhance the findings of the paper at this point.

Figure 5 switches up the viruses under study. It shows a very good experiment in which the viruses have now clearly been reconstructed in isolation. A substitution at residue 295 (D295N), rather than the deletion that has been previously investigated, is reconstructed individually and together with a different single-amino acid deletion, Δ297, which was found in one of the natural isolates presented in Figure 3. They find (A) that only the Δ297 mutation confers neutralizing antibody resistance. This can be explained by the observed failure of either the Δ297 mutant virus or the double mutant virus to bind to the neutralizing antibody. In the other figures, the relative fitness of the wild-type A2, Δ297, D295N, and double mutant viruses were measured by somewhat indirect means such as fold increase over input and fixed time points at two different MOIs, rather than growth curves at a fixed MOI. However, the case is made convincingly that the double mutant virus grows better under all conditions in cultured cells. The authors show that these data can clearly be modified in animals, because, in the mouse kidney (H), a double mutant is present at equivalent abundance to wild-type.

1. In Panel I, the frequencies of the D295N/Δ297 double mutation in the original mice in which they were isolated, as well as a different combination of D295A/Δ297 from another mouse, are shown These data do not contribute mechanistically to Figure 5 and should be moved to Panel 3 where the mutations are first discussed. They do, however, provide a basis for discussion of the potential order of mutational events, which should be in the discussion.

Supplement to Figure 6 presents further exploration into the comparative phenotypes of the four viruses presented in Figure 5.

1. Could the authors discuss why the double-mutant virus generates the most virus but shows considerably less CPE than the D295N mutation?

Figure 6 shows pathogenesis experiments that compare wild-type and the D295N/Δ297 double mutant in B cell-deficient mice in the absence of T cell depletion. The double mutant shows similar viremia but increased growth in brain tissues. It is part of the interesting biology of this paper, that the mutations were isolated for their antibody-escape properties, but show differences in mice with no B cells. Thus, this provides the perfect jumping-off point to compare the properties of the viruses in the presence and absence of neutralizaing antiboy and T cells.