A Coxsackievirus B vaccine protects against virus-induced diabetes in an experimental mouse model of type 1 diabetes

Abstract

Aims/hypothesis: Epidemiological studies suggest a role for Coxsackievirus B (CVB) serotypes in the pathogenesis of type 1 diabetes, but their actual contribution remains elusive. In the present study, we have produced a CVB1 vaccine to test whether vaccination against CVBs can prevent virus-induced diabetes in an experimental model.

Methods: NOD and SOCS1-tg mice were vaccinated three times with either a formalin-fixed non-adjuvanted CVB1 vaccine or a buffer control. Serum was collected for measurement of neutralising antibodies using a virus neutralisation assay. Vaccinated and buffer-treated mice were infected with CVB1. Viraemia and viral replication in the pancreas were measured using standard plaque assay and PCR. The development of diabetes was monitored by blood glucose measurements. Histological analysis and immunostaining for viral capsid protein 1 (VP1), insulin and glucagon in formalin-fixed paraffin embedded pancreas was performed.

Results: The CVB1 vaccine induced strong neutralising antibody responses and protected against viraemia and the dissemination of virus to the pancreas in both NOD mice (n = 8) and SOCS1-tg mice (n = 7). Conversely, 100% of the buffer-treated NOD and SOCS1-tg mice were viraemic on day 3 post infection. Furthermore, half (3/6) of the buffer-treated SOCS1-tg mice developed diabetes upon infection with CVB1, with a loss of the insulin-positive beta cells and damage to the exocrine pancreas. In contrast, all (7/7) vaccinated SOCS1-tg mice were protected from virus-induced diabetes and showed no signs of beta cell loss or pancreas destruction (p < 0.05).

Conclusions/interpretation: CVB1 vaccine can efficiently protect against both CVB1 infection and CVB1-induced diabetes. This preclinical proof of concept study provides a base for further studies aimed at developing a vaccine for use in elucidating the role of enteroviruses in human type 1 diabetes.

Keywords: Antibody; Coxsackievirus; Enterovirus; Mouse model; NOD mice; Type 1 diabetes; Vaccine.

Conflict of interest statement

Duality of interest

HH is a minor (5%) shareholder and member of the board of Vactech Ltd., which develops vaccines against picornaviruses. The other authors declare that there is no duality of interest associated with their contribution to this manuscript.

Contribution statement

MFT and VPH are the guarantors of this work and, as such, had full access to all of the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis. VMS planned experiments, generated, analysed and interpreted data and wrote the manuscript. MMH planned experiments, generated, analysed and interpreted data and edited the manuscript. ES and SO generated and analysed data and edited the manuscript. ASK contributed to discussions, analysed data and edited the manuscript. HH, OHL and VPH contributed to planning and discussions and edited the manuscript. MFT planned the study and experiments, analysed and interpreted data and edited the manuscript. All authors approved the final version of the manuscript.

Figures

Fig. 1

A CVB1 vaccine protects NOD mice against viraemia and systemic viral spread following infection with CVB1. (a) Schematic illustrating the experimental timeline; NOD mice and SOCS1-tg mice were vaccinated or given buffer alone, followed by infection with CVB1. For NOD mice, the experiments were terminated on day 3 p.i. Mock vaccinations and CVB1 vaccinations are represented by the empty syringe; CVB1 challenge is shown with the black syringe. (b) Percentage of infected vaccinated (n = 8) or buffer-treated (n = 6) mice determined by the presence of CVB1 RNA in the blood of NOD mice on day 3 p.i. as detected by RT-PCR. ***p < 0.001, χ2 test. (c) Cytopathic virus in the blood and (d) in the pancreas on day 3 p.i. in NOD mice treated with buffer (n = 6) or CVB1 vaccine (n = 8) as measured by standard plaque assay. Mean values ± SD; **p < 0.01 and ***p < 0.001, Mann–Whitney U test. (e) Representative images of VP1 positivity (brown staining) in pancreas sections of buffer-treated and (f) CVB1-vaccinated NOD mice on day 3 p.i. (×16 magnification; scale bar, 50 μm) and (g) percentage of mice with VP1 positivity in the pancreas. ***p < 0.001, χ2 test

Fig. 2

SOCS1-tg mice are protected from virus-induced diabetes by the CVB1 vaccine. (a) Neutralising antibody titres in the serum of vaccinated mice (n = 7) sampled prior to vaccination on days 0, 14 and 28 and before infection on day 35. The dotted line illustrates the neutralising capacity threshold in the virus neutralisation assay. Each serum sample was analysed in two independent neutralisation assays and the mean neutralising antibody titre calculated. Mean values are indicated by the line ± SD; ***p < 0.001 compared with day 0 or indicated time point as determined by one-way ANOVA. (b, c) Weight changes of individual mice treated with vaccine buffer (n = 6) (b), or CVB1 vaccine (n = 7) (c) after infection with 106 PFU CVB1. Each individual animal is represented by a single line. Three of the buffer-treated animals developed diabetes and were removed prior to day 21. (d) Percentage of buffer-treated (n = 6) or vaccinated mice (n = 7) positive for CVB1 in the blood on day 3 p.i. as detected by RT-PCR. ***p < 0.001, χ2 test. (e) Cytopathic virus measured in the blood of buffer-treated (n = 6) or vaccinated (n = 7) mice on day 3 p.i. by standard plaque assay. Mean values ± SD; **p < 0.01, Mann–Whitney U test. (f) Cumulative diabetes incidence in buffer-treated (black line) and vaccinated (dotted line) SOCS1-tg mice after infection with CVB1, p < 0.05 comparing the two groups as determined by logrank Mantel–Cox test. Formalin-fixed, paraffin embedded SOCS1-tg mice pancreas sections stained with insulin or glucagon antibodies by immunohistochemistry. Shown are representative images from (g) buffer-treated and (h) CVB1-vaccinated mice. Images on the left of each panel are at ×16 magnification and the white box indicates the area of magnification shown in the right panels (at ×40 magnification). Scale bars, 50 μm. (g) Note the loss of acinar tissue and immune cell infiltration in tissue from buffer-treated animals