AAC Accepted Manuscript Posted Online 26 May 2015 Antimicrob. Agents Chemother. doi:10.1128/AAC.00878-15 Copyright © 2015, American Society for Microbiology. All Rights Reserved.

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Title: Characterization of poliovirus neutralization escape mutants of single-domain antibody

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fragments (VHHs).

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Running title: Poliovirus type 1 neutralization escape mutants against VHHs

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Lise Schotte1, Bert Thys1, Mike Strauss2, David J. Filman2, Bart Rombaut1,* and James M. Hogle2,#.

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Neurosciences, Vrije Universiteit Brussel, Laarbeeklaan 103, 1090 Brussels, Belgium

Department of Pharmaceutical Biotechnology and Molecular Biology, Center for

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240 Longwood Avenue, Boston 02115, Massachusetts, USA

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*

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# Corresponding author: [email protected]

Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School,

Deceased

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Corresponding author: James Hogle: [email protected], Phone: 617-432-3918,

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FAX: 617-432-4360

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Word count: summary: 216

main text (excl. acknowledgements, excl. legends): 6248

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Key words: poliovirus, VHH, nanobody, drug-resistance, escape-mutants

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1|Page

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Summary

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To complete the eradication of poliovirus and to protect unvaccinated people subsequently, the

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development of one or more antiviral drugs will be necessary. A set of five single-domain

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antibody fragments (VHHs) with an in vitro neutralizing activity against poliovirus type 1 was

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previously developed and their mechanisms of action have been studied. In this study,

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neutralization escape mutants were selected for each VHH. Sequencing of the P1-region of the

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genome showed that amino acid substitutions are found in the four viral proteins of the capsid

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and that they are located both in proximity to the binding sites of the VHHs and in regions

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further away from the canyon and hidden beneath the surface. Characterization of the mutants

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demonstrated that they have similar single cycle replication kinetics as compared to their

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parental strain and that they are all drug (VHH) independent. Their resistant phenotypes are

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stable, as they do not regain full susceptibility to the VHH after passage over HeLa cells in the

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absence of VHH. They are all at least as stable as the parental strain against heat inactivation at

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44°C and three of them are even significantly (P 6014-fold (6A-mutants) when compared to Mahoney. It can also be seen

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(Fig. 3) that the mutations are sufficiently stable to prevent reversion to their initial VHH-

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susceptible state. However, four mutants, 6A_1, 6A_2, 19B_3 and 21E_5 did show an increase in

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susceptibility to the VHHs, indicated by a decrease in their EC50 values.

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3.5 Thermal stability

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The thermal stability of the mutants was tested by heating of viral samples to 44°C. These

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experiments were done because our previous paper investigating the mechanism of

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neutralization showed that all five VHHs interfered with the uncoating of the virus and stabilized

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the capsid (19). As represented in Fig. 4, all of the mutants showed a very good resistance

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against heat inactivation. The titer at time 0 min was 4.2 x 107 ± 0.57 x 107 pfu/ml, and there

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were no significant (α=0.05) differences between the titers of the mutants and Mahoney at time

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0 (data not shown). After heating the virus samples at 44°C, the fraction of infectivity remaining

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seemed to be higher for most of the mutants as compared to Mahoney when looking at the

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graphs in Fig.4. However, when using an unpaired t-test to calculate the differences between

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the fractions of infectivity remaining after heating the virus 60 minutes at 44°C (Fig.4), only

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mutants 6A_4, 8A_1 and 19B_2 were shown to be significantly more stable than Mahoney to

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thermal inactivation at 44°C.

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3.6 Cross-resistance

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To determine whether a mutant resistant against one VHH also shows resistance to other VHHs,

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we infected cells with a mutant and treated the cells with either a VHH different from the one

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against which original resistance was established, or with the antiviral compound pirodavir, or

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the poliovirus neutralizing antibody 35-1F4. The results of the mutants tested are represented in

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Fig. 5. In these graphs, the percentage of cell viability measured in the well after infection of the

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cells with the indicated mutant and treatment with the VHH or compound shown on the x-axis

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is given. This percentage was calculated using the following formula: (signal of sample – signal of

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0 % control)/(signal of 100 % control – signal of 0 % control) x 100, where the 0 % control is a

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control where cells were infected but not treated and where the 100 % control is a control in

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which cells were neither infected nor treated.

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As a first observation, we see that cross-resistance does occur for most of the variants, except

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for 6A_1, 6A_4, 21E_1 and both 29F_1 and 29F_5, which are still susceptible to neutralization by

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the four other VHHs. Reversely, all of the tested mutants could still be fully neutralized by at

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least two other VHHs at a concentration of 1 µM, e.g. the cells infected with variant 8A_1 and

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treated with VHH PVSP6A or PVSP19B retain full viability after three days of incubation at 37°C.

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Although all of the mutants showed cross-resistance to other VHHs, none of the aa-

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substitutions interfered with the binding and neutralizing activity of the antiviral compound

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pirodavir (except for 19B_1) or the monoclonal antibody 35-1F4.

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When looking at the mutants resistant to PVSP6A, 6A_1 and 6A_4 do not show cross-resistance

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to other VHHs. 6A_2 and 6A_3 are no longer susceptible to neutralization by PVSS8A and

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PVSS21E, probably due to the VP2 H142R and VP1 V61M substitutions also found in 21E_2 and

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21E_3, respectively. In the case of 6A_2 the resistance to neutralization by PVSS8A could also be

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due to the VP1 V47L substitution. 6A_4 also contains the VP2 H142R substitution, but the VP3

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D209V substitution might compensate for this and allow neutralization by PVSS21E.

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The mutants against PVSS8A have a single, dominant substitution (VP1 V47L) that induces cross-

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resistance against PVSS21E and PVSP29F.

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The mutants against PVSP19B are all resistant against PVSS21E. Interestingly, mutants 19B_1

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and 19B_3 only differ in the replacement of glutamic acid at position 168 of VP1 by either

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glycine or lysine, respectively. This seems to influence the cross-resistance pattern, as 19B_1

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shows resistance against pirodavir and 19B_3 does not. In contrast, 19B_3 is resistant to

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neutralization by PVSS8A and 19B_1 is not. The glutamic acid to glycine substitution somehow

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influences binding of pirodavir to 19B_1, while replacement of this same glutamic acid by lysine

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does not interfere with neutralization by pirodavir in 19B_3, but does disturb neutralization by

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PVSS8A.

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Of the five selected variants resistant against PVSS21E, four have only a single substitution of

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which one, in 21E_5, is located on VP4. 21E_1 (VP2 T139I) can still be neutralized by the other

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four VHHs, while an extra substitution (VP1 V61M) introduces resistance towards PVSS8A and

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PVSP19B.

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The mutants against PVSP29F have a rather limited variation in their aa-substitutions and these

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are not shared by any of the variants resistant against the other VHHs. They specifically

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introduce resistance against PVSP29F and do not seem to interfere with the binding of and

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neutralization by the other VHHs, pirodavir or the monoclonal antibody 35-1F4.

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Considering the frequency of development of cross-resistance against a certain VHH, PVSP6A is

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the best in class, with only one mutant out of ten selected by the other VHHs being cross-

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resistant against PVSP6A. PVSS21E is the weakest since 60 % of the mutants that were selected

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by other VHHs and that were tested for cross-resistance against PVSS21E were positive.

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PVSP29F (17 %), PVSP19B (18 %) and PVSS8A (46 %) have percentages between these two

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extremes.

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Given the similar binding sites of the different VHHs, we were concerned that the presence of

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the original selecting VHH could interfere with the binding of other VHHs in the cross-

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neutralization experiments. Thus, the cross-resistance test was done in the absence of the first

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‘selector’ VHH. To test whether the variant virus, which formed in the wells where cross-

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resistance was seen, was still the original mutant and not a newly selected genotype, these

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variant ‘multiresistant’ viruses were once more tested against the original VHH. The

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percentages of viable cells infected with the multiresistant virus and treated with its original

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VHH are shown in Fig. 6. For example, 6A_2/8A is the virus that replicated in the well infected

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with 6A_2 and treated with VHH PVSS8A. As controls, the mutant virus from the same cross-

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resistance experiment and treated with its original selector VHH (e.g. 6A_2/6A) was also

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measured in this experiment. No significant difference (α=0.05) was seen between any of the

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variants which replicated in the absence of the original selector VHH as compared to the one

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which replicated in the presence of the same VHH.

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4. Discussion

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Genotypes of the neutralization escape mutants

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The sequencing of the P1 genome region of five plaque purified neutralization escape variants

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of poliovirus type 1 for each of five in vitro neutralizing VHHs (PVSP6A, PVSS8A, PVSP19B,

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PVSS21E and PVSP29F) lead to some interesting observations. First, aa-substitutions in all of the

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four viral proteins were found. Even though most of the substitutions are located nearby the

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binding sites of the VHHs on the virion (which was shown to be the canyon), six different

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sustitutions (VP1 A43T, VP1 V47L, VP1 V61M, VP2 A121T, VP3 D209V and VP4 I62T) were found

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to be located in regions of the capsid that are further away from the canyon and are more

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internal in the capsid. The VP1 V47L and the VP4 I62T substitutions even occur as single aa-

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substitutions and thus are fully responsible for the resistant phenotype of variants 8A_1 and

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21E_5, respectively. Mutants resistant against conventional antibodies tend to mutate in

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epitopes that are more exposed onto the surface of the capsid, e.g. in the loop regions at the

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five-fold axis of symmetry (BC- and EF-loop of VP1) or the loops surrounding the canyon (EF-

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loop of VP2 and GH-loop of VP1) (28-36). The dimensions of conventional antibodies

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(approximately 10 times those of VHHs) usually allow them to recognize only these epitopes on

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the capsid that are easily accessible and escape mutations are therefore clustered in defined

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regions that are consistent with the area of the antigen binding site at the tip of each Fab. In

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contrast, the aa-substitutions selected by the VHHs that are in exposed residues are at the

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periphery of the footprint of the VHH rather than in the center of the site. Note that the GH-

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loop of VP1 and the EF-loop of VP2 are within the VHH footprint, but are also highly exposed

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components that belong to major antigenic sites, and are therefore the locations of escape

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substitutions from both conventional antibodies and VHHs. The conformation and dimensions

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of VHHs make it advantageous for them to interact inside the canyon in order to maximize the

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contact area between the VHH and the virus. Since the binding sites of the VHHs and that of the

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natural poliovirus receptor (CD155) (19) (Strauss et al, in preparation) are so similar, the virus is

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now more limited in the residues it can mutate without affecting its infectivity. Thus, changing

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the residues at the center of the VHHs’ binding sites would be likely to also have a deleterious

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effect on receptor binding. The occurrence of aa-substitutions in internal and non-accessible

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regions of the capsid also contrasts with the almost exclusive surface localization of escape

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mutations selected by monoclonal antibodies. It should be noted, however, that the presence of

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aa-substitutions outside the immediate binding site is not unique to the VHHs. Thus, Blondel

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and colleagues have previously shown that mutations affecting neutralization by antibodies

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recognizing the N2 antigenic site can be located both outside and inside the binding site (37)

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and must therefore act by a mechanism other than simple steric interference with antibody

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binding. In our data, some substitutions (VP1 A43T, VP1 V47L, VP1 V61M, VP4 I62T) are located

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remarkably far from the binding site on the inner surface of the virion, and several others, that

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may map closer to the VHH binding site (including VP1 A150V, VP2 A121T, and VP3 D209V) are

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in residues in intersubunit interfaces that are not exposed on the surface, and are therefore also

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unlikely to seriously impact binding of the VHHs to the capsid. This type of mutation seems to

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be sufficient to permit resistance against the VHHs with the lowest poliovirus neutralizing

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capacity (and highest EC50 values: PVSS8A and PVSS21E). For the other three VHHs, which have

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lower EC50 values, the substitution of surface-exposed residues at the periphery of the binding

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site seems to be essential to escape neutralization. Although most of these aa-substitutions are

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in very close proximity to the VHH binding site and may actually directly contact the selective

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VHH (19) (Strauss et al., in preparation), we would argue that the location at the periphery of

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the binding site would be expected to produce at most marginal effects on VHH binding affinity.

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Interestingly, in every case the substitutions occur in residues where the structure of the

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particle is known to change upon the receptor-induced conformational changes associated with

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cell entry. Based on the observation that in all cases binding VHHs stabilizes the virion, and that

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all of the VHHs neutralize at copy levels below those required to prevent receptor binding, we

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have previously argued that the VHHs neutralize virions by preventing conformational changes

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required for cell entry (19). Taken together the data presented here, it is suggested that both

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the internal and surface aa-substitutions may confer resistance to the VHHs at least in part by

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interfering with the ability of the VHH to prevent conformational changes needed for cell entry.

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It is interesting that when primate antibodies were identified that cross-neutralize both

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poliovirus serotypes 1 and 2 (and thus avoid the usual highly exposed antigenic sites), the

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antibody binding sites overlapped that of the receptor, and the escape substitutions included

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residues 65, 109 and 166 of VP1 (38, 39).Those findings are clearly very similar to our results

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using neutralizing VHHs, and suggest that the mechanisms of neutralization may be similar.

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Characteristics of the selected mutants

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When testing antiviral compounds, the possibility of developing therapy resistant variants

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should always be considered and knowledge of the implications of these resistant variants is

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very important. The single cycle replication kinetics of the mutants are very similar to those of

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their parental Mahoney strain. This shows that the aa-substitutions found in the variants offer

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little advantage or disadvantage for the variants, and that the variants will replicate to the same

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extent as the parental strain in the absence of the VHH. This has also been observed for other

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neutralization escape variants against poliovirus type 1, 2 and 3 (40, 41).

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Previously, a number of mutations in poliovirus type 2 (41) and type 3 (12) selected for

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resistance to neutralization by an antiviral drug were found to be dependent on the drug against

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which resistance was developed. They were called drug-dependent variants. The similarity

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between the single cycle replication curves of Mahoney and the mutants in the absence of

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compound provides evidence that none of the variants depends on the presence of its

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corresponding VHH in order to induce infection. However, this is not conclusive, as a drug-

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dependent mutant against poliovirus type 3 showed similar single cycle replication

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characteristics in the presence and absence of a drug (12), due to problems at the level of virus-

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release from the cells in the absence of compound. A more specific method to determine drug

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dependency is to calculate the drug plating index (DPI). This is the ratio between the titers of

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the variant measured in the presence and the absence of the drug. Variants with a DPI between

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0.001 and 0.01 (41) are only slightly resistant to the drug, those with a DPI around 1 are non-

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dependent on the drug for infectivity and those with a DPI of 100 or higher are drug-dependent.

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All of the variants characterized in this study have DPIs between 0.23 (21E_5) and 1.7 (8A_1)

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and have therefore no aa-substitutions that destabilize the capsid to such an extent that the

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VHH is essential for infection. The DPIs of the wild type Mahoney strain are all under 0.0048.

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We have shown that binding of the VHHs stabilizes the virions against heat degradation (19).

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We therefore hypothesized that the VHH-resistant escape substitutions would be destabilizing,

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thereby counterbalancing the effect of the VHHs. On the contrary, all of the tested variants

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were at least as stable against heat inactivation as the parental strain and most of them

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appeared to be even more stable at 44°C. The test was performed three times for each variant,

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but due to rather large variations, the difference was significant (P