Rotavirus VP6 protein mucosally delivered by cell wall-derived particles from Lactococcus lactis induces protection against infection in a murine model

Abstract

Rotaviruses are the primary cause of acute gastroenteritis in children worldwide. Although the implementation of live attenuated vaccines has reduced the number of rotavirus-associated deaths, variance in their effectiveness has been reported in different countries. This fact, among other concerns, leads to continuous efforts for the development of new generation of vaccines against rotavirus.In this work, we describe the obtention of cell wall-derived particles from a recombinant Lactococcus lactis expressing a cell wall-anchored version of the rotavirus VP6 protein. After confirming by SDS-PAGE, Western blot, flow cytometry and electronic immunomicroscopy that these particles were carrying the VP6 protein, their immunogenic potential was evaluated in adult BALB/c mice. For that, mucosal immunizations (oral or intranasal), with or without the dmLT [(double mutant Escherichia coli heat labile toxin LT(R192G/L211A)] adjuvant were performed. The results showed that these cell wall-derived particles were able to generate anti-rotavirus IgG and IgA antibodies only when administered intranasally, whether the adjuvant was present or not. However, the presence of dmLT was necessary to confer protection against rotavirus infection, which was evidenced by a 79.5 percent viral shedding reduction.In summary, this work describes the production of cell wall-derived particles which were able to induce a protective immune response after intranasal immunization. Further studies are needed to characterize the immune response elicited by these particles as well as to determine their potential as an alternative to the use of live L. lactis for mucosal antigen delivery.

Fig 1. Analysis of the protein content of the CWDP.

A—Coomassie-blue stained SDS-PAGE corresponding to the total protein extract from: lane 1, non-induced L. lactis carrying RV VP6 protein; lanes 2 and 3, nisin-induced L. lactis expressing RV VP6 protein; lane 4, protein Molecular Weight Marker. B—Coomassie-blue stained SDS-PAGE corresponding to the CWDP obtained from: lane 1, untransformed L. lactis NZ9000 (NZ-CWDP); lane 2, recombinant L. lactis expressing RV VP6 protein (VP6-CWDP); lane 3, protein Molecular Weight Marker. C—Western blot analysis of the CWDP obtained from: lane 1, untransformed L. lactis NZ9000 (NZ-CWDP); lane 2, recombinant L. lactis expressing RV VP6 protein (VP6-CWDP); lane 3, positive control of RV particles. D—Histogram plot corresponding to flow cytometry analysis of CWDP obtained from untransformed L. lactis NZ9000, used to set negative FITC fluorescence (filled orange curve) or recombinant L. lactis expressing RV VP6 protein (unfilled green curve). VP6 protein was stained with a polyclonal anti-RV serum and a FITC-conjugated secondary antibody. Protein molecular weight markers (MWM) sizes as well as expected sizes of VP6 and VP6 fusion to cell wall anchor domain (VP6-CWA) bands are depicted in kiloDaltons (kDa). White arrows indicate bands corresponding to VP6-CWA protein.

Fig 2. CWDP structure analysis by TEM.

A—Entire recombinant L. lactis at 20,000 X magnification. B—CWDP obtained from recombinant L. lactis expressing RV VP6 protein at 20,000 X magnification. C—CWDP obtained from recombinant L. lactis expressing RV VP6 protein at 40,000 X magnification. D—CWDP obtained from recombinant L. lactis expressing RV VP6 protein at 60,000 X magnification. VP6 protein was detected with a polyclonal anti-RV serum and a secondary antibody labeled with 10 nm colloidal gold. Arrows indicate the electron-dense gold particles which are indicative of VP6 protein presence.

Fig 3. Anti-rotavirus IgG and IgA detection in serum at day 42 after the first immunization doseot. y with or without dmLT presented both IgG and IgA specific antibodies against RV. described in th.

A—Anti-rotavirus IgG titer determined for each mouse in three independent assays and B—OD_{490 nm} obtained for a 1/20 dilution of each mouse serum in the IgA determination ELISA. Vi and ViA denote groups administered intranasally with VP6-CWDP without or with dmLT, respectively. Lines or bars represent the mean and error bars represent standard error for each group. Statistical analysis was performed by Kruskal-Wallis test using Dunn’s Multiple Comparison post-test. **<0.01, ****< 0.0001.

Fig 4. Rotavirus challengeot. y with or without dmLT presented both IgG and IgA specific antibodies against RV. described in th.

A—Mean shedding curves obtained for mice groups immunized with PBS, VP6-CWDP intranasally (Vi), VP6-CWDP intragastrically (Vig), VP6-CWDP + dmLT intranasally (ViA) or VP6-CWDP + dmLT intragastrically (VigA). Each symbol represents the mean, and the error bars represent the standard error for each day. B—Percentage of viral shedding for each mouse within different groups respect to the 100% calculated for PBS. Bars represent the mean and error bars represent the standard error for each group immunized with: PBS, VP6-CWDP intranasally (Vi), VP6-CWDP intragastrically (Vig), VP6-CWDP + dmLT intranasally (ViA), VP6-CWDP + dmLT intragastrically (VigA). Statistical analysis was performed by Kruskal-Wallis test using Dunn’s Multiple Comparison post-test. **<0.01.

Fig 5. Anti-rotavirus IgG and IgA titration in serum at day 10 after infection with rotavirusot. y with or without dmLT presented both IgG and IgA specific antibodies against RV. described in th.

A—Anti-rotavirus IgG titer determined for each mouse in three independent assays and B—Anti-rotavirus serum IgA titer determined for each mouse in three independent assays 10 days after infection with RV for groups administered intranasally with VP6-CWDP with or without dmLT (ViA or Vi, respectively) or PBS. Lines represent the mean and error bars represent the standard error for each group. Statistical analysis was performed by Kruskal-Wallis test using Dunn’s Multiple Comparison post-test. **<0.01, ***< 0.001, ****< 0.0001.