Clinical and In Vitro Evidence Favoring Immunoglobulin Treatment of a Chronic Norovirus Infection in a Patient With Common Variable Immunodeficiency

Abstract

Background: Immunocompromised individuals can become chronically infected with norovirus, but effective antiviral therapies are not yet available.

Methods: Treatments with nitazoxanide, ribavirin, interferon alpha-2a, and nasoduodenally administered immunoglobulins were evaluated sequentially in an immunocompromised patient chronically infected with norovirus. In support, these components were also applied to measure norovirus inhibition in intestinal enteroid cultures in vitro. Viral RNA levels were determined in fecal and plasma samples during each treatment and viral genomes were sequenced.

Results: None of the antivirals resulted in a reduction of viral RNA levels in feces or plasma. However, during ribavirin treatment, there was an increased accumulation of virus genome mutations. In vitro, an effect of interferon alpha-2a on virus replication was observed and a genetically related strain was neutralized effectively in vitro using immunoglobulins and post-norovirus-infection antiserum. In agreement, after administration of immunoglobulins, the patient cleared the infection.

Conclusions: Intestinal enteroid cultures provide a relevant system to evaluate antivirals and the neutralizing potential of immunoglobulins. We successfully treated a chronically infected patient with immunoglobulins, despite varying results reported by others. This case study provides in-depth, multifaceted exploration of norovirus treatment that can be used as a guidance for further research towards norovirus treatments.

Keywords: VirCapSeq; enteroids; human intestinal organoids; immunocompromised; immunoglobulins; interferon alpha-2a; next-generation sequencing; nitazoxanide; norovirus; ribavirin.

Figure 1.

The effect of anti norovirus treatment on norovirus RNA levels. Norovirus RNA levels in feces (circles) and EDTA plasma (squares) during treatment are shown as cycle threshold (Ct) 40 (the lower limit of detection) minus the Ct value. The grey areas represent the duration of treatments. Treatment 1, 14 days of oral nitazoxanide, 500 mg twice daily. Treatment 2, 96 days of oral ribavirin, 400 mg twice daily. Treatment 3, 39 days of interferon alpha-2a, subcutaneous injections of 3 × 10⁶ units 3 times per week. Treatment 4, 2 days of oral immunoglobulin, 25 mg/kg 4 times daily.

Figure 2

Maximum likelihood tree of the GII.4 New Orleans 2009 VP1 sequences. The consensus sequences of the patient are colored orange. Selected bootstrap values > 70 are depicted.

Figure 3.

Accumulated number of nucleotide (A) and amino acid substitutions (B) in the viral genome compared to day 1 (March 2017), observed in the consensus sequences during treatment. The grey areas represent the duration of treatments. Treatment 1, 14 days of oral nitazoxanide, 500 mg twice daily. Treatment 2, 96 days of oral ribavirin, 400 mg twice daily. Treatment 3, 39 days of interferon alpha-2a, subcutaneous injections of 3 × 10⁶ units 3 times per week. Treatment 4, 2 days of oral Ig, 25 mg/kg 4 times daily.

Figure 4.

A, Effect of nitazoxanide and ribavirin on norovirus replication in human intestinal enteroid cultures compared to the dimethyl sulfoxide (DMSO) control. At a concentration of 100 µM nitazoxanide all cells were detached 72 hours after mock or virus inoculation. B, Effect of interferon alpha-2a on norovirus replication in human intestinal enteroid cultures. C, Virus neutralization with pre- and post-norovirus–infection serum from a patient with an acute GII.4 infection and 2 immunoglobulin (Ig) batches from Privigen. For each condition norovirus replication is shown as fold change of norovirus RNA equivalents at 72 hours postinoculation compared to 2 hours postinoculation. Error bars denote standard deviation, experiments were performed with two technical replicates.