Effects of Rotavirus NSP4 on the Immune Response and Protection of Rotavirus-Norovirus Recombinant Subunit Vaccines in Different Immune Pathways

Abstract

Diarrheal disease continues to be a major cause of global morbidity and mortality among children under 5 years of age. To address the current issues associated with oral attenuated rotavirus vaccines, the study of parenteral rotavirus vaccines has promising prospects. In our previous study, we reported that rotavirus nonstructural protein 4 (NSP4) did not increase the IgG antibody titer of co-immune antigen but did have a protective effect against diarrhea via the intramuscular injection method. Here, we explored whether NSP4 can exert adjuvant effects on mucosal immune pathways. In this study, we immunized mice via muscle and nasal routes, gavaged them with the rotavirus Wa strain or the rotavirus SA11 strain, and then tested the protective effects of immune sera against both viruses. The results revealed that the serum-specific VP8* IgG antibody titers of the mice immunized via the nasal route were much lower than those of the mice immunized by intramuscular injection, and the specific IgA antibodies were almost undetectable in the bronchoalveolar lavage fluid (BALF). NSP4 did not increase the titer of specific VP8* antibodies in either immune pathway. Therefore, in the two vaccines (PP-NSP4-VP8* and PP-VP8*+NSP4) used in this study, NSP4 was unable to perform its potential adjuvant role through the mucosal immune pathway. Instead, NSP4 was used as a co-immunized antigen to stimulate the mice to produce specific binding antibodies that play a protective role against diarrhea.

Keywords: immune method; nonstructural protein 4; norovirus P particle; recombinant subunit vaccine; rotavirus.

Figure 1

Production and characterization of the antigens. (A) SDS-PAGE result of PP-NSP4-VP8*, PP-VP8*, and PP-V; M represents the prestained protein markers with the indicated molecular sizes in kDa. (B) Western blot results for the three antigens. (C) Western blot results for NSP4; the black arrow represents the size of NSP4. Lane 1: Nickel column elution concentrate; lane 2: size exclusion column elution concentrate. (D–F) Representative EM micrographs of nanoparticles and aggregates assembled from PP-NSP4-VP8* (D), PP-VP8* (E), and PP-V (F). The typical subviral particles are indicated by black arrows.

Figure 1

Production and characterization of the antigens. (A) SDS-PAGE result of PP-NSP4-VP8*, PP-VP8*, and PP-V; M represents the prestained protein markers with the indicated molecular sizes in kDa. (B) Western blot results for the three antigens. (C) Western blot results for NSP4; the black arrow represents the size of NSP4. Lane 1: Nickel column elution concentrate; lane 2: size exclusion column elution concentrate. (D–F) Representative EM micrographs of nanoparticles and aggregates assembled from PP-NSP4-VP8* (D), PP-VP8* (E), and PP-V (F). The typical subviral particles are indicated by black arrows.

Figure 2

VP8*- and NSP4-specific IgG titers detected by enzyme-linked immunosorbent assay (ELISA). (A) Specific IgG responses of VP8*. (B) Specific IgG responses of NSP4. The differences between groups are indicated as follows: *, significant differences with p ≤ 0.05; ***, significant differences with p ≤ 0.001; ns, nonsignificant differences with p > 0.05.

Figure 3

VP8*- and NSP4-specific IgA titers detected by enzyme-linked immunosorbent assay (ELISA). (A) VP8*-specific IgA responses. (B) NSP4-specific IgA responses.

Figure 4

Neutralization of various vaccine-immunized mouse sera against the rotavirus Wa strain. (A) The 50% neutralization titers (y-axis) of mouse sera after administration with different immunogens (x-axis) against rotavirus (Wa strain) in cell culture were measured by fluorescence-based plaque reduction assays. (B) Representative images of the tested sera. The green spots represent the location of the rotavirus.

Figure 5

Binding curves and blocking effects of norovirus P particles in response to HBGA with various antigen−immunized sera and BALFs. (A) The binding curve of PP-V protein to type A saliva sample was used to select the appropriate saliva sample and PP-V subviral particle concentration for blocking experiments. The absorbance at 450 nm (OD450) reflects the binding ability. (B) Inhibitory effects on the 50% blocking titer (BT50) of various antigen−immunized mouse sera and BALFs. BT50 was tested with PP-V at 5 μg/mL and saliva sample 2. ns: non-significant.

Figure 6

Four representative images of a suckling mice with diarrhea following a challenge with rotavirus. (A) Severely watery stool image, representing a score of 4. (B) Moderate watery stool image, representing a score of 3. (C) Mildly watery stool image, representing a score of 3. (D) Normal diarrhea image, representing a score of 2.

Figure 7

Passive protection of mouse sera immunized with various vaccine candidates against murine diarrhea caused by rotavirus Wa strain challenge. (A) Diarrhea rates (y-axis) of suckling mice after a challenge with the rotavirus Wa strain that was treated with mouse sera after immunization with various immunogens (x-axis). (B) Diarrhea scores of suckling mice after a rotavirus Wa strain challenge.

Figure 8

In vivo effects of VP8* and NSP4 on the rotavirus Wa strain. The immunofluorescence results revealed differences in virus loading in the intestinal tissues. The blue fluorescence represents the position of the cell nucleus, whereas the green fluorescence represents rotavirus. RV+: positive control group; RV−: negative control group.

Figure 9

Passive protection of mouse sera immunized with various vaccine candidates against murine diarrhea caused by rotavirus SA11 strain challenge. (A) Diarrhea rates (y-axis) of suckling mice after being challenged with a rotavirus SA11 strain that was treated with mouse sera after immunization with various immunogens (x-axis). (B) Diarrhea scores of suckling mice after rotavirus SA11 strain challenge.

Figure 10

In vivo effects of VP8* and NSP4 on the rotavirus SA11 strain. The immunofluorescence results revealed differences in virus loading in the intestinal tissues. The blue fluorescence represents the position of the cell nucleus, whereas the green fluorescence represents rotavirus. RV+: positive control group; RV−: negative control group.