Intranasal P particle vaccine provided partial cross-variant protection against human GII.4 norovirus diarrhea in gnotobiotic pigs

Abstract

Noroviruses (NoVs) are the leading cause of nonbacterial acute gastroenteritis worldwide in people of all ages. The P particle is a novel vaccine candidate derived from the protruding (P) domain of the NoV VP1 capsid protein. This study utilized the neonatal gnotobiotic pig model to evaluate the protective efficacies of primary infection, P particles, and virus-like particles (VLPs) against NoV infection and disease and the T cell responses to these treatments. Pigs either were vaccinated intranasally with GII.4/1997 NoV (VA387)-derived P particles or VLPs or were inoculated orally with a GII.4/2006b NoV variant. At postinoculation day (PID) 28, pigs either were euthanized or were challenged with the GII.4/2006b variant and monitored for diarrhea and virus shedding for 7 days. The T cell responses in intestinal and systemic lymphoid tissues were examined. Primary NoV infection provided 83% homologous protection against diarrhea and 49% homologous protection against virus shedding, while the P particle and VLP vaccines provided cross-variant protection (47% and 60%, respectively) against diarrhea. The protection rates against diarrhea are significantly inversely correlated with T cell expansion in the duodenum and are positively correlated with T cell expansion in the ileum and spleen. The P particle vaccine primed for stronger immune responses than VLPs, including significantly higher numbers of activated CD4+ T cells in all tissues, gamma interferon-producing (IFN-γ+) CD8+ T cells in the duodenum, regulatory T cells (Tregs) in the blood, and transforming growth factor β (TGF-β)-producing CD4+ CD25- FoxP3+ Tregs in the spleen postchallenge, indicating that P particles are more immunogenic than VLPs at the same dose. In conclusion, the P particle vaccine is a promising vaccine candidate worthy of further development.

Importance: The norovirus (NoV) P particle is a vaccine candidate derived from the protruding (P) domain of the NoV VP1 capsid protein. P particles can be easily produced in Escherichia coli at high yields and thus may be more economically viable than the virus-like particle (VLP) vaccine. This study demonstrated, for the first time, the cross-variant protection (46.7%) of the intranasal P particle vaccine against human NoV diarrhea and revealed in detail the intestinal and systemic T cell responses by using the gnotobiotic pig model. The cross-variant protective efficacy of the P particle vaccine was comparable to that of the VLP vaccine in pigs (60%) and to the homologous protective efficacy of the VLP vaccine in humans (47%). NoV is now the leading cause of pediatric dehydrating diarrhea, responsible for approximately 1 million hospital visits for U.S. children and 218,000 deaths in developing countries. The P particle vaccine holds promise for reducing the disease burden and mortality.

FIG 1

Th cells and CTLs induced by NoV infection or vaccination pre- and postchallenge. (A) Representative dot plots of frequencies of CD3⁺ CD4⁺ and CD3⁺ CD8⁺ T cells among lymphocytes from PBL of mock-vaccinated pigs postchallenge. FITC, fluorescein isothiocyanate; SPRD, Spectral Red; APC, allophycocyanin. (B) Mean total numbers plus standard errors of the means (n, 6 to 10) of CD3⁺ CD4⁺ and CD3⁺ CD8⁺ T cells in intestinal (duodenum, ileum) and systemic (spleen, PBL) tissues pre- and postchallenge. An asterisk above the error bars indicates a significant difference among groups for the same cell type and tissue at the same time point (P, <0.05 by Kruskal-Wallis one-way ANOVA). A section sign indicates that the numbers increased significantly following challenge in the same group. A number sign indicates that the numbers decreased significantly following challenge in the same group. IN, intranasal.

FIG 2

Activated nonregulatory CD4⁺ and CD8⁺ T cells pre- and postchallenge. (A) Representative dot plots of frequencies of CD4⁺ CD25⁺ FoxP3⁻ and CD8⁺ CD25⁺ FoxP3⁻ activated T cells in PBL from P-particle-vaccinated pigs prechallenge. PE, phycoerythrin. (B) Mean total numbers plus standard errors of the means (n, 6 to 10) of CD4⁺ CD25⁺ FoxP3⁻ and CD8⁺ CD25⁺ FoxP3⁻ activated T cells prechallenge and postchallenge in intestinal (duodenum, ileum) and systemic (spleen, PBL) tissues. See the legend to Fig. 1 for an explanation of the symbols indicating statistical significance.

FIG 3

NoV-specific IFN-γ-producing CD4⁺ and CD8⁺ T cell responses pre- and postchallenge. (A) Representative dot plots of frequencies of NoV-specific CD3⁺ CD4⁺ IFN-γ⁺ and CD3⁺ CD8⁺ IFN-γ⁺ effector T cells in PBMCs isolated from the NoVPO group postchallenge and stimulated with P particles (17 h at 37°C). IFN-γ was detected using intracellular staining and flow cytometry. (B) Numbers of IFN-γ-producing CD4⁺ and CD8⁺ T cells following subtraction of isotype control and mock-stimulated background numbers. Data presented are mean total numbers plus standard errors of the means (n, 6 to 10) prechallenge and postchallenge in intestinal (duodenum, ileum) and systemic (spleen, PBL) tissues. See the legend to Fig. 1 for an explanation of the symbols indicating statistical significance.

FIG 4

Treg responses induced by NoV infection or vaccination pre- and postchallenge. (A) Representative dot plots of frequencies of CD4⁺ CD25⁻ FoxP3⁺ and CD4⁺ CD25⁺ FoxP3⁺ Tregs from PBL of P-particle-vaccinated pigs prechallenge. (B) Mean total numbers of CD25⁻ and CD25⁺ Tregs plus standard errors of the means (n, 6 to 10) among total MNCs prechallenge and postchallenge in intestinal (duodenum, ileum) and systemic (spleen, PBL) tissues. See the legend to Fig. 1 for an explanation of the symbols indicating statistical significance.

FIG 5

TGF-β-producing Treg responses induced by NoV infection or vaccination pre- and postchallenge. (A) Representative dot plots of frequencies of cytokine-secreting Tregs from unstimulated MNCs from PBL of P-particle-vaccinated pigs prechallenge. TGF-β production was detected using intracellular staining and flow cytometry. (B) Mean numbers plus standard errors of the means (n, 6 to 10) of cytokine-producing Tregs among Tregs prechallenge and postchallenge. See the legend to Fig. 1 for an explanation of the symbols indicating statistical significance.

FIG 6

IL-10-producing Treg responses induced by NoV infection or vaccination pre- and postchallenge. (A) Representative dot plots of frequencies of cytokine-secreting Tregs from unstimulated MNCs from PBL of P particle-vaccinated pigs prechallenge. IL-10 production was detected using intracellular staining and flow cytometry. (B) Mean numbers plus standard errors of the means (n, 6 to 10) of cytokine-producing Tregs among Tregs prechallenge (A) and postchallenge. See the legend to Fig. 1 for an explanation of the symbols indicating statistical significance.

FIG 7

Expansion of T cell subsets following NoV challenge. Expansion was determined by dividing the postchallenge numbers of each T cell subset by the prechallenge numbers. Shown is the expansion of Th cells and CTLs (A), activated nonregulatory CD25⁺ FoxP3⁻ T cells (B), IFN-γ-producing T cells (C), CD25⁻ and CD25⁺ Tregs (D), and TGF-β (E)- and IL-10 (F)-producing Tregs among NoVPO, P-particle-vaccinated, and control pigs. Correlations between T cell expansion and the protection rate against diarrhea were determined with Spearman's rank correlation coefficient (R, +1 or −1; P, <0.0001) in SAS. NC indicates a significant negative correlation with the protection rate against diarrhea (R, −1; P, <0.0001); PC indicates a significant positive correlation with the protection rate against diarrhea (R, 1; P, <0.0001). See Fig. 1 to 6 for the analysis of cell types.

FIG 8

Comparisons of P-particle- and VLP-induced T cell responses following NoV challenge. Shown are total numbers of activated nonregulatory CD25⁺ FoxP3⁻ T cells (A), IFN-γ-producing T cells (B), FoxP3⁺ Tregs (C), and TGF-β-producing Tregs (D) among P-particle-vaccinated or VLP-vaccinated pigs postchallenge. Error bars indicate the standard errors of the means. See Fig. 2 to 5 for the analysis of cell types. See the legend to Fig. 1 for an explanation of the symbols indicating statistical significance.