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

Animal husbandry and monitoring of animal health

NOD mice and SOCS1-tg mice on a NOD background (both from in-house breeding) were housed in specific pathogen-free conditions at Karolinska Institutet, Stockholm, Sweden. Ethics approval was granted for all experiments by the local ethics committee and were conducted in accordance with the NIH Principles of Laboratory Animal Care and national laws in Sweden. Extended health monitoring of mice was performed. Additional information is provided in the ESM Methods: Animals, together with details of primers used for SOCS1-tg mouse genotyping (ESM Table 1).

Virus and vaccine production

A CVB1 field isolate (CVB1-V200; [3]), was propagated in Vero cells (ECACC no. 84113001; mycoplasma negative), purified and then formalin inactivated for 3 days at 37°C to produce CVB1 vaccine. See ESM Methods: Vaccine production and Hankaniemi et al [7] for more details. Mice were infected with CVB1-10796 (propagated in HeLa cells, originally obtained from R Glas, Karolinska Institutet, Stockholm, Sweden, mycoplasma negative).

Vaccinations

Male and female age-matched NOD and SOCS1-tg mice (4–7 weeks old) were randomly assigned to groups and vaccinated on days 0, 14 and 28 with non-adjuvanted vaccine containing 1.8 μg protein (n = 8 and n = 7, respectively) or mock-vaccinated with vaccine buffer (M199-0.1% Tween80 (vol./vol.), 150 μl, n = 6 for NOD and for SOCS1-tg mice) by interscapular injection [6, 7]. Serum was collected before vaccinations and infection (day 35) by tail bleeding (experimental timeline displayed in Fig. 1a). The study was not blinded to the experimenter.

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Infections

Mice were challenged with 10⁶ plaque forming units (PFU) CVB1-10796 (diluted in serum-free RPMI to a final volume of 200 μl, administered by intraperitoneal injection, with the dose carefully optimised, data not shown) on day 35. Blood samples were collected on day 3 post infection (p.i.; 1:1 ratio with 12 mmol EDTA). NOD mice were killed on day 3 p.i. and pancreases saved for virus quantification and histological analysis. SOCS1-tg mice were monitored until diabetes development or day 21 p.i. and pancreases saved for histological analysis.

Monitoring of blood glucose and diabetes development

Blood glucose was measured in blood obtained from the tail-vein with a Bayer Contour XT blood glucose meter (Bayer, Basel, Switzerland) and a glucose concentration >18 mmol/l, or two consecutive measurements between 13 and 18 mmol/l were used to define diabetes and diabetic mice were killed.

Neutralising antibody detection

Neutralising antibody titres were determined by standard virus plaque reduction assay in green monkey kidney (GMK) cells (National Institute for Health and Welfare, Finland; mycoplasma negative) [3]. Plaque number reduction ≥80% compared with untreated virus suspension was considered positive. The detection limit of the assay was a fourfold dilution (1:4), and positivity cut-off for serum samples was set to ≥1:16.

Virus titration

Pancreases were homogenised and lytic virus quantified in either blood or pancreas by standard plaque assay in GMK cells. Viral titres are expressed as PFU/g of tissue or ml of blood. See ESM Methods: Virus titration and tissue homogenisation for more details.

PCR analysis

Enterovirus specific real-time PCR was performed using RNA extracted from blood samples; see ESM Methods: PCR analysis and Honkanen et al [8] for further details. Primers are shown in ESM Table 2.

Histological analysis and immunohistochemistry

Histological analysis and immunohistochemistry of pancreas samples with viral capsid protein 1 (VP1), insulin and glucagon were carried out as in Flodström et al and Larsson et al ([4, 6]; ESM Methods: Immunohistochemistry and antibodies).

Statistical analysis

Statistical analyses were executed using Prism 5 software (GraphPad, La Jolla, CA, USA). PCR and VP1 immunohistochemistry data were analysed by χ ² tests. Plaque assay virus titrations were analysed by Mann–Whitney U test. Neutralising antibody data was analysed by one-way ANOVA. Diabetes incidence was analysed by log rank Mantel–Cox test. Data are expressed as the mean ± SD. A p value ≤0.05 was regarded statistically significant.

CVB1 vaccine is well tolerated and is highly immunogenic

The newly produced CVB1 vaccine was well tolerated by NOD mice with no adverse effects on weight or blood glucose (ESM Fig. 1a–c). Furthermore, the vaccine was highly immunogenic and vaccinated mice produced CVB1 neutralising antibodies after the primary immunisation, which was augmented after the second immunisation (ESM Fig. 1d). Serum with a neutralising capacity was not detected in buffer-treated mice (data not shown).

CVB1 vaccine protects against CVB1 infection in NOD mice

We next examined whether the vaccine protects against viraemia caused by CVB1 infection and prevents virus replication in the pancreas on day 3 p.i. All vaccinated mice (8/8) were protected from viraemia, as determined by RT-PCR and plaque assay (Fig. 1b, c). Conversely, all buffer-treated mice were identified viraemic by plaque assay (Fig. 1c) and 5/6 were positive for CVB1 RNA (Fig. 1b). Similarly, replicating virus in the pancreas was measured in buffer-treated mice but not in vaccinated mice (Fig. 1d). Immunohistochemical analysis using the VP1 antibody further confirmed viral dissemination to the pancreas in all buffer-treated mice (Fig. 1e, g) but not vaccinated mice (Fig. 1f–g).

CVB1 vaccine protects against virus-induced diabetes

SOCS1-tg mice, which are susceptible to virus-induced diabetes [4], showed no adverse changes in weight and blood glucose after CVB1 vaccination (data not shown). Additionally, vaccinated SOCS1-tg mice developed a robust antibody response (Fig. 2a) similar to that observed in NOD mice (ESM Fig. 1d). Buffer-treated mice remained consistently negative for neutralising antibodies (data not shown).

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We next monitored SOCS1-tg mice after CVB1 challenge. No obvious differences were found in the weight of vaccinated and buffer-treated mice (Fig. 2b, c). Furthermore, viraemia measurements on day 3 p.i. revealed no signs of infection in the vaccinated animals (0/7; Fig. 2d, e). In contrast, all (6/6) buffer-treated mice were infected as indicated by the detection of both viral RNA (Fig. 2d) and infective virus by plaque assay (Fig. 2e).

We also tracked diabetes development in the infected SOCS1-tg mice until day 21 p.i. As expected, diabetes occurred in the buffer-treated SOCS1-tg mice with 50% (3/6) developing hyperglycaemia (p < 0.05; Fig. 2f). Pancreatic exocrine damage was notable in 4/6 mice (Fig. 2g), which corresponded with diabetes development. Moreover, mice that developed hyperglycaemia showed glucagon positivity but a loss of insulin positivity in a number of islets, indicating destruction of the insulin-producing beta cells (Fig. 2g). In contrast, all seven vaccinated SOCS1-tg mice were protected from diabetes (Fig. 2h) and showed normal pancreas morphology on day 21 p.i. with healthy exocrine tissue and intense insulin and glucagon staining in the islets of Langerhans (Fig. 2h).