Infectious vaccine-derived rubella viruses emerge, persist, and evolve in cutaneous granulomas of children with primary immunodeficiencies.

Rubella viruses (RV) have been found in an association with granulomas in children with primary immune deficiencies (PID). Here, we report the recovery and characterization of infectious immunodeficiency-related vaccine-derived rubella viruses (iVDRV) from diagnostic skin biopsies of four patients. Sequence evolution within PID hosts was studied by comparison of the complete genomic sequences of the iVDRVs with the genome of the vaccine virus RA27/3. The degree of divergence of each iVDRV correlated with the duration of persistence indicating continuous intrahost evolution. The evolution rates for synonymous and nonsynonymous substitutions were estimated to be 5.7 x 10-3 subs/site/year and 8.9 x 10-4 subs/site/year, respectively. Mutational spectra and signatures indicated a major role for APOBEC cytidine deaminases and a secondary role for ADAR adenosine deaminases in generating diversity of iVDRVs. The distributions of mutations across the genes and 3D hotspots for amino acid substitutions in the E1 glycoprotein identified regions that may be under positive selective pressure. Quasispecies diversity was higher in granulomas than in recovered infectious iVDRVs. Growth properties of iVDRVs were assessed in WI-38 fibroblast cultures. None of the iVDRV isolates showed complete reversion to wild type phenotype but the replicative and persistence characteristics of iVDRVs were different from those of the RA27/3 vaccine strain, making predictions of iVDRV transmissibility and teratogenicity difficult. However, detection of iVDRV RNA in nasopharyngeal specimen and poor neutralization of some iVDRV strains by sera from vaccinated persons suggests possible public health risks associated with iVDRV carriers. Detection of IgM antibody to RV in sera of two out of three patients may be a marker of virus persistence, potentially useful for identifying patients with iVDRV before development of lesions. Studies of the evolutionary dynamics of iVDRV during persistence will contribute to development of infection control strategies and antiviral therapies.

Assessing Immunity to Rubella Virus: a Plea for Standardization of IgG (Immuno)assays.

Immunity to rubella virus (RV) is commonly determined by measuring specific immunoglobulin G (RV IgG). However, RV IgG results and their interpretation may vary, depending on the immunoassay, even though most commercial immunoassays (CIAs) have been calibrated against an international standard and results are reported in international units per milliliter. A panel of 322 sera collected from pregnant women that tested negative or equivocal for RV IgG in a prior test (routine screening) was selected. This panel was tested with two reference tests, immunoblotting (IB) and neutralization (Nt), and with 8 CIAs widely used in Europe. IB and Nt gave concordant results on 267/322 (82.9%) sera. Of these, 85 (26.4%) sera were negative and 182 (56.5%) sera were positive for both tests. All 85 IB/Nt-negative samples were classified as negative with all CIAs. Of the 182 IB/Nt-positive samples, 25.3 to 61.5% were classified as equivocal and 6 to 64.8% were classified as positive with the CIAs. Wide variations in titers in international units per milliliter were observed. In our series, more than half of the women considered susceptible to RV based on CIA results tested positive for RV antibodies by IB/Nt. Our data suggest that (i) sensitivity of CIAs could be increased by considering equivocal results as positive and (ii) the definition of immunity to RV as the 10-IU/ml usual cutoff as well as the use of quantitative results for clinical decisions may warrant reconsideration. A better standardization of CIAs for RV IgG determination is needed.

Rubella virus neutralizing antibody response after a third dose of measles-mumps-rubella vaccine in young adults.

BACKGROUND: Third doses of measles-mumps-rubella (MMR) vaccine have been administered during mumps outbreaks and in various non-outbreak settings. The immunogenicity of the rubella component has not been evaluated following receipt of a third dose of MMR vaccine. METHODS: Young adults aged 18-31 years with documented two doses of MMR vaccine received a third dose of MMR vaccine between July 2009 and October 2010. Rubella neutralizing antibody titers were assessed before, 1 month, and 1 year after receipt of a third dose of MMR vaccine. RESULTS: Among 679 participants, 1.8% had rubella antibody titers less than 10 U/ml, immediately before vaccination, approximately 15 years after receipt of a second dose of MMR vaccine. One month after receipt of a third dose of MMR vaccine, average titers were 4.5 times higher and >50% of participants had a 4-fold boost. Response was highest among those with titers less than 10 U/ml prior to vaccination (geometric mean titer ratio = 18.8; 92% seroconversion) and decreased with increasing pre-vaccination titers. Average titers declined 1 year postvaccination but remained significantly higher than pre-vaccination levels. The proportion classified as low-positive antibody levels increased from 3% 1 month postvaccination to 24% 1 year postvaccination. CONCLUSIONS: Vaccination with a third dose of MMR vaccine resulted in a robust boosting of rubella neutralizing antibody response that remained elevated 1 year later. Young adults with low rubella titers are more likely to benefit from a third dose of MMR vaccine.

Inhibition of rubella virus replication by the broad-spectrum drug nitazoxanide in cell culture and in a patient with a primary immune deficiency.

Persistent rubella virus (RV) infection has been associated with various pathologies such as congenital rubella syndrome, Fuchs's uveitis, and cutaneous granulomas in patients with primary immune deficiencies (PID). Currently there are no drugs to treat RV infections. Nitazoxanide (NTZ) is an FDA-approved drug for parasitic infections, and has been recently shown to have broad-spectrum antiviral activities. Here we found that empiric 2-month therapy with oral NTZ was associated in the decline/elimination of RV antigen from lesions in a PID patient with RV positive granulomas, while peginterferon treatment had no effect. In addition, we characterized the effects of NTZ on cell culture models of persistent RV infection. NTZ significantly inhibited RV replication in a primary culture of human umbilical vein endothelial cells (HUVEC) and Vero and A549 epithelial cell lines in a dose dependent manner with an average 50% inhibitory concentration of 0.35 µg/ml (1.1 µM). RV strains representing currently circulating genotypes were inhibited to a similar extent. NTZ affected early and late stages of infection by inhibiting synthesis of cellular and RV RNA and interfering with intracellular trafficking of the RV surface glycoproteins, E1 and E2. These results suggest a potential application of NTZ for the treatment of persistent rubella infections, but more studies are required.

Differences in Establishment of Persistence of Vaccine and Wild Type Rubella Viruses in Fetal Endothelial Cells.

Both wild type (WT) and vaccine rubella virus (RV) can pass through the placenta to infect a human fetus, but only wtRV routinely causes pathology. To investigate possible reasons for this, we compared establishment of persistence of wtRV and RA27/3 vaccine strains in fetal endothelial cells. We showed that yields of RA27/3 and wtRV were similar after the first round of replication, but then only vaccine-infected cultures went through a crisis characterized by partial cell loss and gradual decline of virus titer followed by recovery and establishment of persistent cultures with low levels of RA27/3 secretion. We compared various steps of virus replication, but we were unable to identify changes, which might explain the 2-log difference in RA27/3 and wtRV yields in persistently infected cultures. Whole genome sequencing did not reveal selection of virus variants in either the wtRV or RA27/3 cultures. Quantitative single-cell analysis of RV replication by in situ hybridization detected, on average, 1-4 copies of negative-strand RNA and ~50 copies of positive-strand genomic RNA in cells infected with both vaccine and WT viruses. The distinct characteristics of RA27/3 replication were the presence of large amounts of negative-strand RV RNA and RV dsRNA at the beginning of the crisis and the accumulation of high amounts of genomic RNA in a subpopulation of infected cells during crisis and persistence. These results suggest that RA27/3 can persist in fetal endothelial cells, but the characteristics of persistence and mechanisms for the establishment and maintenance of persistence are different from wtRV.