Identification of Immune and Viral Correlates of Norovirus Protective Immunity through Comparative Study of Intra-Cluster Norovirus Strains

<div><p>Whether or not primary norovirus infections induce protective immunity has become a controversial issue, potentially confounded by the comparison of data from genetically distinct norovirus strains. Early human volunteer studies performed with a norovirus-positive inoculum initially led to the conclusion that primary infection does not generate long-term, protective immunity. More recently though, the epidemiological pattern of norovirus pandemics has led to the extrapolation that primary norovirus infection induces herd immunity. While these are seemingly discordant observations, they may in fact reflect virus strain-, cluster-, or genogroup-specific differences in protective immunity induction. Here, we report that highly genetically related intra-cluster murine norovirus strains differ dramatically in their ability to induce a protective immune response: Primary MNV-3 infection induced robust and cross-reactive protection, whereas primary MNV-1 infection induced modest homotypic and no heterotypic protection. In addition to this fundamental observation that intra-cluster norovirus strains display remarkable differences in protective immunity induction, we report three additional important observations relevant to norovirus:host interactions. First, antibody and CD4⁺ T cells are essential to controlling secondary norovirus infections. Second, the viral minor structural protein VP2 regulates the maturation of antigen presenting cells and protective immunity induction in a virus strain-specific manner, pointing to a mechanism by which MNV-1 may prevent the stimulation of memory immune responses. Third, VF1-mediated regulation of cytokine induction also correlates with protective immunity induction. Thus, two highly genetically-related norovirus strains displayed striking differences in induction of protective immune responses, strongly suggesting that the interpretation of norovirus immunity and vaccine studies must consider potential virus strain-specific effects. Moreover, we have identified immune (antibody and CD4⁺ T cells) and viral (VP2 and possibly VF1) correlates of norovirus protective immunity. These findings have significant implications for our understanding of norovirus immunity during primary infections as well as the development of new norovirus vaccines.</p></div

B cells and CD4⁺ T cells, but not CD8⁺ T cells and IFN-γ, are essential for MNV-3 protective immunity.

<p><b>A</b>) Groups of mice of the indicated knockout strains (n = 7 mice per condition tested in at least two independent experiments) were inoculated with mock inoculum (black bars) or 10⁴ TCID₅₀ units of MNV-3 (grey bars). Six weeks later, all mice were challenged with 10⁷ TCID₅₀ units of MNV-3. One day later, animals were perfused, the indicated organs harvested, and viral burden determined by plaque assay. The data for all mice in each group were averaged. Limits of detection are indicated by dashed lines. The mock→MNV-3 infection group was compared to the MNV-3→ infection group for each mouse strain for statistical analysis. <b>B</b>) The mock→MNV-3 (1°) infection viral titers were divided by the MNV-3→ (2°) infection titers for the mice presented in panel A to determine the fold-reduction in titers as a quantitative measure of protective immunity. <b>C</b>) The same experiment was carried out for MNV-1; shown only are the fold-reductions in viral titers.</p

MNV-3 initiates replication faster than MNV-1.

<p><b>A</b>) RAW 264.7 cells were infected with MNV-1 or MNV-3 at MOI 5. Supernatant was collected from two independent wells at the indicated hours post-infection (hpi) and virus titers determined using TCID₅₀ assay. The entire experiment was repeated three times and data from all experiments are averaged. The limit of detection of the assay is indicated by a dashed line. <b>B</b>) Infected cells from the same cultures used for panel A were lysed and viral proteins were detected by western blotting using the indicated antibodies. These data are representative of duplicate samples tested from each of three independent experiments. <b>C</b>) 1.5×10⁵ HEK-293T cells were transfected with 0.4, 2, 10 or 50 ng of purified MNV-1 or MNV-3 genomic RNA. The virus titers at the indicated hpi were determined using TCID₅₀ assay. Data for all replicates are averaged. Data for MNV-1 and MNV-3 at each time point were compared for statistical purposes.</p

VP2 regulates antigen presenting cell maturation in vitro.

<p>5.0×10⁵ RAW 264.7 cells were plated overnight in 24-well plates and then exposed to mock inoculum or recombinant MNV-1, MNV-3, MNV-1.3 VP2, or MNV-3.1 VP2 at MOI 5 for 24 h. In certain experiments, a well was also co-infected with equivalent titers of parental MNV-1 and MNV-3 such that cells were infected at MOI 5. Cells were stained with antibodies to MHC class I and MHC class II molecules, co-stimulatory molecules CD40 and CD80, or CD103. Flow cytometry was carried out as described in the <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003592#s4" target="_blank">Methods</a>. Six independent experiments were performed for single infections, and three experiments included co-infections. <b>A</b>) Representative data from one experiment are presented in the histograms. In these graphs, surface expression of the indicated marker is shown for mock-inoculated cells (light grey histogram filled with dashed lines); cells infected with parental MNV-1 or MNV-3 (black histogram); and cells infected with chimeric MNV-1.3VP2 or MNV-3.1VP2 (dark grey histogram). The parental and chimeric virus pair is shown to the left of the histograms. The dashed vertical lines indicate the mean basal expression levels in mock-inoculated cells. <b>B</b>) In the bar graphs, the percentage of cells displaying upregulated surface expression of the indicated marker is presented. Data from all experimental replicates are averaged. Cells inoculated with UV-inactivated MNV-1 or MNV-3 displayed statistically similar levels of each marker compared to mock-inoculated cells (data not shown). Statistical comparisons were made between mock and each parental MNV; MNV-1 and MNV-3; each parental and chimeric pair; and each parental MNV and the co-infected cells.</p

Recombinant ORF3-swap viruses replicate comparably to parental viruses.

<p><b>A</b>) RAW 264.7 cells were infected with recombinant MNV-1, MNV-3, MNV-1.3VP2, or MNV3.1VP2 at MOI 0.05 or MOI 5 and growth curves were carried out as described in the legend of <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003592#ppat-1003592-g006" target="_blank">Figure 6A</a>. Data from four independent experiments, with duplicate wells per condition tested in each experiment, are averaged. <b>B</b>) RAW 264.7 cells were infected with the indicated virus at MOI 5 for 0 or 24 hpi and cell lysates analyzed for viral proteins using western blotting. Data are representative of duplicate samples tested from each of three independent experiments.</p

A relatively attenuated MNV strain induces more robust homotypic and heterotypic immunity than a virulent strain.

<p><b>A</b>) Groups of wild-type B6 mice (a minimum of 5 mice per group tested in two independent experiments) were inoculated with mock inoculum, 10⁴ TCID₅₀ units of MNV-1 (1), 10⁴ TCID₅₀ units of MNV-3 (3), or 5×10³ TCID₅₀ units of each MNV-1 and MNV-3 (1+3) by the peroral route; this is referred to as challenge 1. Six weeks later, mice were infected with 10⁷ TCID₅₀ units of MNV-1 or MNV-3; this is referred to challenge 2. One day following challenge 2, animals were perfused, the indicated organs harvested, and viral burden determined by plaque assay. The data for all mice in each group were averaged. Mice receiving mock inoculum at challenge 1 and virus infection at challenge 2 (the primary infection groups) are indicated by black bars; mice receiving homologous virus at challenges 1 and 2 are indicated by grey bars; mice receiving heterologous virus at challenges 1 and 2 are indicated by white bars; and mice co-infected with MNV-1 and MNV-3 at challenge 1 and either MNV-1 or MNV-3 at challenge 2 are indicated by black striped bars. Limits of detection are indicated by dashed lines. <b>B</b>) Homotypic challenge studies were performed on groups of B6 mice (n = 4) as described in panel A, with the exception that challenge 2 was administered to the mice six months (instead of six weeks) following challenge 1. The parallel groups of mice challenged after six weeks from panel A are included here for clarity. In all experiments, the indicated groups were compared in statistical analysis as described in the <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003592#s4" target="_blank">Methods</a>. MLNs = mesenteric lymph nodes.</p

MNV-3 elicits protection from severe disease in the absence of type I interferon signaling.

<p><b>A</b>) Groups of mice lacking the type I interferon receptor (IFNAR^−/−; a minimum of 5 mice per condition) were inoculated with mock inoculum, 10⁴ or 10⁷ TCID₅₀ units of MNV-1 or MNV-3 by the peroral route. The percentage of mice surviving infection was calculated daily. All surviving mice were weighed daily and the weights compared to day 0 weights to calculate a relative weight. <b>B</b>) Groups of IFNAR^−/− mice (n = 3–5) were inoculated with either mock inoculum or 5×10³ TCID₅₀ units of MNV-3. Six weeks later, mice were challenged with 10⁷ TCID₅₀ units of MNV-3 or 10⁶ TCID₅₀ units of MNV-1 and monitored for survival and weight loss, as described above. <b>C</b>) Groups of IFNAR^−/− mice (n = 3) were inoculated with mock inoculum (1° mock; grey bars) or 5×10³ TCID₅₀ units of MNV-3 (1° MNV-3; black bars) by the peroral route. Six weeks later, mice were infected with 10⁷ TCID₅₀ units of MNV-1 or MNV-3 (2° challenge virus displayed on the x-axis). One day following 2° challenge, animals were perfused, the indicated organs harvested, and viral burden determined by plaque assay. Limits of detection are indicated by dashed lines. MLNs = mesenteric lymph nodes. Groups of mice receiving mock versus MNV-3-1° infection and the same 2° challenge virus were compared for statistical purposes.</p

VF1 regulates cytokine induction in vitro.

<p><b>A</b>) RAW 264.7 cells were infected with recombinant MNV-1, MNV-3, MNV-1.3VP2, or MNV3.1VP2 at MOI 5, or a mock inoculum. Total RNA was extracted from cells at 0, 12, and 24 hpi and cytokine expression determined through qRT-PCR, as described in the <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003592#s4" target="_blank">Methods</a>. Data from duplicate wells per condition, and two independent experimental replicates, were averaged. <b>B</b>) HEK-293T cells were transfected with pβLUX, pRL-SV40, pN-RIGI and one of the following test plasmids – pTriEx-1ORF4 (VF1-1), pTriEx-3ORF4 (VF1-3), pTriEx-1ORF2 (VP1-1), pTriEx-3ORF2 (VP1-3), pTriEx-1ORF3 (VP2-1), or pTriEx-3ORF3 (VP2-3). In the negative control (−), only pβLUX, pRL-SV40, and filler DNA were transfected. In the positive control (+), the test plasmid was replaced with filler DNA. Each condition was tested in duplicate and the entire experiment repeated three times. Data represent the averaged readings from all experiments. Each group was compared to the positive control for statistical purposes.</p

MNV-3 induces a more robust systemic and mucosal antibody response than MNV-1.

<p><b>A</b>) Clarified supernatants of Sf9 cells infected with recombinant baculoviruses expressing MNV-1 or MNV-3 rVP1/2 were visualized by electron microscopy. The insets show representative VLPs stained with a polyclonal antibody to the MNV-1 VP1 protein using immunogold labeling. <b>B–F</b>) In panels B, C, E and F, groups of mice were inoculated with mock inoculum, 10⁴ TCID₅₀ units of MNV-1, or 10⁴ TCID₅₀ units of MNV-3. In panel D, only mice infected with MNV-3 were tested. In all cases, mice were challenged with homologous inoculum six weeks later. One day later, serum and intestinal contents were collected as described in the <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003592#s4" target="_blank">Methods</a>. <b>B</b>) Serum samples from wild-type B6 mice (a minimum of 4 mice per condition tested in at least two independent experiments) were tested in ELISA on plates coated with the indicated rVP1/2. <b>C</b>) Serum was collected from groups of B6 mice (n = 3) inoculated with either mock inoculum or 10⁴ TCID₅₀ units of the indicated MNV strain for six weeks. The 50% neutralization titer was determined for each serum sample. The entire experiment was repeated three times. Data from all samples per condition were averaged and compared for statistical purposes. <b>D</b>) Serum samples from groups of mice of the indicated knockout strain (a minimum of 4 mice per strain tested in at least two independent experiments) were tested in ELISA on plates coated with either MNV-1 (grey) or MNV-3 (black) rVP1/2. Intestinal contents from the indicated small intestinal piece of wild-type B6 mice (a minimum of 6 mice per condition tested in at least two independent experiments) were tested in ELISA on plates coated with MNV-1 rVP1/2 (<b>E</b>) or MNV-3 rVP1/2 (<b>F</b>). For all panels, the data for all mice in each group were averaged. Statistical analyses were carried out on the indicated groups as described in the <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003592#s4" target="_blank">Methods</a>.</p