Neutralization escape in kidney transplant recipients with persistent BK polyomavirus replication

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HAL Id: inserm-02160211

https://www.hal.inserm.fr/inserm-02160211

Submitted on 19 Jun 2019

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Neutralization escape in kidney transplant recipients with persistent BK polyomavirus replication

Dorian Mcilroy, M. Hohnemann, Y. Verres, C. Peltier, F. Halary, A. Rodallec, E. Przyrowski, M. Hourmant, C. Bressollette-Bodin

To cite this version:

Dorian Mcilroy, M. Hohnemann, Y. Verres, C. Peltier, F. Halary, et al.. Neutralization escape in kidney transplant recipients with persistent BK polyomavirus replication. Réunion du Labex IGO, Apr 2018, Nantes, France. �inserm-02160211�

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D. McILROY

1,2, 3

_{, M. HOHNEMANN}

4 _{, Y. VERRES}

1,2

_{, C.PELTIER}

1,2

_{, F. HALARY}

1,2

_,

A. RODALLEC

5 _{, E.PRZYROWSKI}

5 _{, M. HOURMANT}

2,6

_{, C.BRESSOLLETTE-BODIN}

1,2,5,7

1. CRTI, UMR 1064, INSERM, Université de Nantes; 2. ITUN, CHU Nantes; 3. Faculté des Sciences et des

Techniques, Université de Nantes; 4. Institut für Virologie, Universität Leipzig; 5. Service de Virologie,

CHU Nantes; 6. Service de Néphrologie et Immunologie Clinique, CHU Nantes; 7. Faculté de Médecine,

Université de Nantes

Neutralization escape in kidney transplant recipients with

persistent BK polyomavirus replication

1. Introduction

The BK polyomavirus (BKPyV) infects more than 80% of the adult population in Europe. The virus persists in a latent state in the kidney and active replication is only seen in immunosuppressed individuals, such as kidney transplant (KTx) recipients, in whom BKPyV reactivation is associated with interstitial nephropathy and allograft dysfunction. Asymptomatic primary infection Lifelong persistence in renal epithelium Reactivation Allogeneic HSC Graft Kidney Transplant Disease Viruria (~50%) Viremia (10-20%) Hemorrhagic cystitis Polyomavirus Associated Nephropathy (2-5%)

No specific antiviral therapy

2. VP1 gene evolution in KTx patients

4. Effects of VP1 mutations on infectivity

6. Conclusions

Evolution of VP1 is consistently observed in KTx recipients with sustained viremia and viruria. Non-synonymous VP1 mutations reduce the efficacy of entry into 293TT cells mediated by both genotype I and genotype IV VP1 proteins.

In addition, clear neutralization escape from cognate serum was observed in one patient with gIb2 infection (3.4), and one patient with gIV infection (3.3). Both of these variants were neutralized as effectively as wild-type VP1 by serum from patient 3.1 who had successfully resolved post-KTx BKPyV viremia. These results indicate that the humoral response against BKPyV in KTx recipients is heterogeneous, since some patients develop a narrow neutralizing response that is vulnerable to neutralization escape mutants. They suggest that neutralization escape contributes to the persistence of BKPyV replication in patients with durably high viral loads.

Nevertheless data from patient 3.5 indicate that lower infectivity in 293TT cells is not always accompanied by neutralization escape. One possible explanation is that the VP1 variant observed in this patient was associated with a modification of viral tropism.

3. Location of VP1 mutations on BKPyV capsid

5. VP1 mutations and neutralization escape

No VP1 mutations found by Sanger sequencing in urine of patients who controlled BKPyV reactivation

Non-synonymous VP1 mutations accumulate over time in patients with persistent viremia despite the presence of a robust humoral response

Wt Wt

Wt

gIV gIb2 gIb2

Glc Gal NeuNAc Gal GalNAc NeuNAc GD1b Ganglioside

Capsid consists of 72 VP1 Pentamers

Yellow : a2-8 disialic acid

Blue : VP1 AA interacting with disialic acid

Green : VP1 AA interacting with Gal-GalNAc (predicted)

R69K E73A D77N E73Q E82Q A72V

VP1 mutations cluster on the external surface of the capsid

proximal to the receptor binding site

E82Q K69N

D60N

Pseudotype production of different VP1 variants: 1. Mutagenesis to generate panel of VP1 variants

2. Co-transfect into 293TT cells with VP2, VP3 and EGFP 3. Extract and purify on Optiprep gradient

4. Quantify by qPCR – number of pEGFP copies/µL 5. Test pseudotype infectivity in 293TT cells

Name Time Sequence

AEE M9 71SAENDFSSDSPERKM AQE M17 71SAQNDFSSDSPERKM AQQ M21 71SAQNDFSSDSPQRKM VQQ M24 71SVQNDFSSDSPQRKM 1.0×105 _1.0×106 _1.0×107 0.01 0.1 1 10 100 AEE AQE AQQ VQQ pEGFP copies/well % G F P + c e ll s gIb2 variant D60N K69N A72V E82Q from patient 3.4 not

neutralized by cognate serum

gIb2 variant A72V E73Q E82Q

from patient 3.5 is NOT a neutralization escape mutant

gIV gIb2 gIb2

VP1 mutations reduce the ability of genotype I and genotype IV

capsid proteins to mediate entry into 293TT cells

Wt EE QE QQ VQQ EE - pl t3 EQ -plt3 DNEQ_NNVEQ -4.5 -4.0 -3.5 -3.0 -2.5 L o g 1 0 R a ti o In fe c ti o u s p a rt ic le s t o E G F P c o p ie

s Genotype Ib2 variants Genotype IV variants

gIV variant

R69K E73A D77N

from patient 3.3 resists

neutralization by cognate serum

SDRED SDREN SD KA N DR ED - pl t 3 DRKD DRVKD -4.5 -4.0 -3.5 -3.0 -2.5 Wt E73Q E73Q E82Q 2 4 6 0 50 100 150 BARO M12 GRPI M0 GRPI M6 GRPI M12

Log10 Serum Dilution

% M a x im a l E G F P e x p re s s io n E73Q E82Q A72V 2 4 6 0 50 100

% M a x im a l E G F P e x p re ss io n 2 4 6 0 50 100 SDKAN B M12 SDKAN S M0 SDKAN S M6 SDKAN S M12

% M a x im a l E G F P e x p re ss io n Sérum Pt 3.1 M12 Pt 3.3 M0 Pt 3.3 M6 Pt 3.3 M12 Sérum Pt 3.1 M12 Pt 3.4 M0 Pt 3.4 M6 Pt 3.4 M12 Wt _{R69K E73A D77N} Wt _{D60N K69N A72V E82Q}

Wt _{A72V E73Q E82Q}

2 4 6

0 50

100 BAGA M12

BARO M12

% M a x im a l E G F P e x p re s s io n 2 4 6 0 50 100 150

% M a x im a l E G F P e x p re s s io n Sérum Pt 3.1 M12 Pt 3.5 M12 2 4 6 0 50 100

% M a x im a l E G F P e x p re s s io n