Norovirus (NoV) specific protective immune responses induced by recombinant P dimer vaccine are enhanced by the mucosal adjuvant FlaB
© The Author(s). 2016
Received: 18 September 2015
Accepted: 7 May 2016
Published: 17 May 2016
Noroviruses (NoVs) are a major cause of childhood gastroenteritis and foodborne diseases worldwide. Lack of appropriate animal models or cell-based culture systems makes the development and evaluation of NoV-specific vaccines a daunting task. VP1 is the major capsid protein of the NoVs that acts as a binding motif to human histo-blood group antigens (HBGAs) through its protruding 2 (P2) domain and can serve as a protective antigen candidate for vaccine development.
Recombinantly produced NoV specific P domain (Pd) vaccine was inoculated into groups of mice either alone or in conjugation with mucosal adjuvant FlaB, the flagellar protein from Vibrio vulnificus. Antigen specific humoral and cell mediated immune responses were assessed by enzyme linked immunosorbent assay (ELISA) or fluorescent activated cell sorting (FACS). A comparative analysis of various routes of vaccination viz. intranasal, sublingual and subcutaneous, was also done.
In this study, we show that a recombinant Pd-vaccine administered through intranasal route induced a robust TH2-dependent humoral immune response and that the combination of vaccine with FlaB significantly enhanced the antibody response. Interestingly, FlaB induced a mixed TH1/TH2 type of immune response with a significant induction of IgG1 as well as IgG2a antibodies. FlaB also induced strong IgA responses in serum and feces. FlaB mediated antibody responses were toll like receptor 5 (TLR5) dependent, since the FlaB adjuvanticity was lost in TLR5−/− mice. Further, though the Pd-vaccine by itself failed to induce a cell mediated immune response, the Pd-FlaB combination stimulated a robust CD4+IFNγ+ and CD8+IFNγ+ T cell response in spleen and mesenteric lymph nodes. We also compared the adjuvant effects of FlaB with that of alum and complete Freund’s adjuvant (CFA). We found that subcutaneously inoculated FlaB induced more significant levels of IgG and IgA in both serum and feces compared to alum or CFA in respective samples.
We validate the use of TLR5 agonist as a strong mucosal adjuvant that would facilitate the development of NoV specific vaccines for humans and veterinary use. This study also highlights the importance of route of immunization in inducing the appropriate immune responses in mucosal compartments.
KeywordsNorovirus FlaB Adjuvant Antibody Intranasal Sublingual Subcutaneous Alum
Human noroviruses (NoVs) are the leading cause of childhood gastroenteritis and foodborne diseases worldwide . Human NoVs, belonging to the genus Norovirus within the family Caliciviridae, are a group of small, positive sense, single-stranded, non-enveloped viruses having 7–8 kb RNA genome with four open reading frames (ORFs) [2, 3]. Major and minor capsid proteins VP1 and VP2 are encoded by the ORF2 and 3, respectively . ORF1 encodes the nonstructural protein and ORF4 has been described recently to play a significant role in the murine NoV pathogenesis [3, 5]. The absence of efficient and reproducible cell culture systems and small-animal models has hindered the studies concerning the pathogenesis and molecular mechanisms of NoV life cycle as well as the development of effective vaccines and therapeutic agents [5, 6].
Antigen preparation and LPS removal
Norovirus VA387 strain (GII.4) P dimer-specific DNA fragments were cloned between NdeI-KpnI restriction sites and expressed in pET30a+ vector (Novagen) as a His-tagged protein. All proteins were expressed in Escherichia coli BL21 with an induction by 0.1 mM isopropyl-β-d-thiogalactopyranoside (IPTG) at 37 °C overnight. Bacterial pellets were dissolved in 8 M urea followed by sonication (2 s on to 3 s off cycles/5 min at 30 % of max. voltage) on ice. Following centrifugation (8000 rpm/15 min/4 °C), protein from cell free supernatant was purified by affinity purification using Ni–NTA Agarose beads (Qiagen) as per manufacturer’s instructions. Protein was dialyzed extensively against sterile phosphate buffered saline (PBS) followed by LPS removal by treatment with TritonX-114 (Sigma). Traces of Triton X-114 were removed by treatment with Bio-Beads™ SM-2 (Bio Rad) as per manufacturer’s instructions. For the production of FlaB protein, a 1.5-kb fragment containing the open reading frame of V. vulnificus flaB was cloned into pTYB12-yielding pCMM250 (New England Biolabs). Recombinant FlaB was purified as previously reported . Finally all proteins were suspended in sterile PBS at appropriate concentrations.
Animals, vaccination and sampling
Specific pathogen free (SPF) female Balb/c WT mice were purchased from Charles River Inc. while TLR5−/− mice on Balb/c background were bred and maintained under SPF conditions at the animal facility of Clinical Vaccine R&D Center of Chonnam National University. The mouse study protocol was approved by the Committee on Animal Welfare at Chonnam National University Medical School. Mice were immunized at an age of 6–7 weeks. The animals were housed in a temperature- and light-controlled environment and had free access to food and water. Various antigen combinations were used at equimolar concentrations. Vaccination groups included (1) P dimer (Pd), (2) Pd + FlaB, (3) FlaB, and (4) PBS. All antigens were inoculated through intranasal (i.n.) or sublingual (s.l.) routes into anaesthetized animals. Final volume for i.n. as well as s.l. vaccination was 10 μl/animal. In a separate experiment, groups of five mice were immunized subcutaneously with either alum precipitated Pd , Pd-CFA mixture, or Pd+FlaB mixture. In all the adjuvant groups, concentration of Pd antigen inoculated into mice was kept constant at 0.1 μM/dose. Animals were immunized thrice. In the CFA group, the first vaccination was done with CFA + Pd followed by two immunizations at one-week interval with incomplete Freund’s adjuvant along with Pd. In all immunization groups, before each respective vaccination, mouse serum as well as feces were collected and processed for antibody determination. One week after the third immunization, final blood and feces samples from mice were procured. Feces samples were made into a 20 % solution (w/v) in ice cold PBS containing 1 mM phenylmethylsulfonyl fluoride (PMSF). All clarified serum and feces samples were kept at −80 °C until used.
NoV specific enzyme linked immunosorbent assay (ELISA)
Antibody titers in serum and feces samples from individual animals were estimated by ELISA using Pd as the coating antigen. Samples were serially diluted in a dilution buffer (PBS + 1 % BSA + 0.05 %Tween20). Anti-mouse IgG, IgA, IgG1 and IgG2a specific Horse radish peroxidase (HRP) conjugated secondary antibodies (Southern Biotech) were used at a dilution of 1:2000. Finally HRP-specific optical reactions were developed using the BD OptEIA™ (BD Biosciences, San Diego, USA) for 15 min followed by stopping the reaction with 2 N H2SO4. Reactions were read at OD450 using Spectra Max 190 microplate reader (Molecular Devices). The cut-off value was decided as per formula: (Mean OD450 of negative control wells) + 3 (standard deviation of OD450 of negative control wells). Log2 of the reciprocal of highest dilution showing OD450 value equivalent to or higher than the cut off value was taken as the antibody titer.
Tissue processing, lymphocyte isolation, cell stimulation assay and flow cytometry
Spleen, mesenteric lymph nodes (MLNs) and Payer’s patches (PPs) were harvested from animals in various immunization groups 2 weeks after the final immunization and kept in ice-cold cell culture medium until processed. All tissues were mashed through 40 μm cell strainer (BD Falcon). In case of spleen, lymphocytes were purified by density centrifugation using the Lymphoprep™ (Life Technologies) followed by red cells lysis by ACK lysis buffer (Lonza Inc., USA) for 5 min at room temperature (RT). Cells were finally suspended in the cell culture medium (RPMI1640 + 10 % FBS + 1 % Penicillin/Streptomycin) and kept on ice until used. For in vitro stimulation assays, 1 × 106 cells in duplicate were plated in separate flat-bottom 96 wells and stimulated with 1 μg of Pd protein for 12 h. Golgistop™ (BD Biosciences, San Diego, CA) was added for the entire duration of incubation. Post-incubation cells were fixed and permeabilized using the Cytofix/Cytoperm kit (BD Biosciences) in accordance with the manufacturer’s instructions. The anti-mouse antibodies used for intracellular and surface staining (eBioscience or BD Pharmingen™) were CD4-APC (clone GK1.5), CD8-APC (clone 53–6.7) and IFNγ-PE (clone XMG1.2). Stained cells were acquired on FACS Accuri (BD Biosciences). Data analysis was performed using the FlowJo software (TreeStar, San Carlos, CA).
Statistical analysis of data was done using the GraphPad Prism software ver. 6.0 or Microsoft Excel as appropriate. P < 0.05 was taken as significant.
Cloning and expression of NoV P dimer (Pd)
Norovirus specific P dimer (Pd) containing two P1 domains and one central P2 domain (Fig. 1a) was expressed as a recombinant protein in Escherichia coli BL21. Homology modelling  suggested that though the complete viral protein had slightly dispersed structure (Fig. 1b), the P domain from ORF2 encompassing amino acid 222–539 had globular structure that would tend to have a compact three dimensional (3D) organization (Fig. 1b). This observation was substantiated by the non-denaturing native polyacrylamide gel electrophoresis (PAGE) of the cloned protein that showed that most of Pd protein remained in the stacking gel (Fig. 1c). The Western blot analysis from sodium dodecyl sulphate (SDS)-PAGE showed that the recombinantly produced protein had a major single monomeric unit of 37 kDa while minor fractions of ca. 80 kDa and over 120 kDa, representing dimer and multimeric proteins respectively, were also present (Fig. 1b).
FlaB enhances Pd-specific antibody responses in a TLR5-dependent manner
FlaB polarizes a mixed TH1/TH2 type humoral immune response against NoV Pd-vaccine
FlaB potentiates Pd-specific cell mediated immune responses in systemic and local immune compartments
FlaB induces similar systemic immune responses compared to alum and CFA, while inducing significantly higher secretory antibody responses in feces
In the present study, we have shown that NoV specific P domain (Pd) recombinant vaccine, produced in E. coli system, effectively induced substantial humoral immunity in mice. The combination of Pd with the mucosal adjuvant FlaB  enhanced the humoral immune responses in a TLR5 dependent manner and also induced significant cell mediated immunity in systemic and local immune compartments. In general, the route of vaccination is critical for successful immunization outcomes against specific infections . Most parenterally administered vaccines fail to induce local mucosal immune responses that act as a first line of defense against pathogens that invade through gastrointestinal surfaces. An important feature of present study is the information on the kinetics of antibody induction after vaccination through intranasal and sublingual routes. In agreement with previous reports, the immunization through intranasal as well as sublingual routes seems to induce considerable systemic as well as local antibody responses . However, the Pd vaccine given through i.n. route induced higher IgG antibody titers after two immunizations, which were significantly higher than the antibody titers induced after s.l. immunization. On the other hand, secretory IgA antibody levels in feces were higher after the s.l. immunization compared with the i.n. route. The difference in vaccine efficacy given through two routes can be attributed to the initial tissue types associated with antigen capture. Nasal associated lymphoid tissue (NALT) is a secondary lymphoid organ with organized cell clusters while the sublingual mucosa is a non-organized type II mucosal tissue with dispersed immune cells that lack an organized cell clustering. However, combination of Pd with the mucosal adjuvant FlaB seems to overcome this hurdle and induce high antibody titers after two immunizations that might be due to the induction of immune responses mediated through the epithelial cells of buccal mucosa  or indirect activation of innate immune cells .
Direct effects of flagellin on B cell activation and antibody production  have been refuted variously , while stimulatory effects of flagellin on dendritic cells and T cells have been reported to promote a dramatic increase in T cell dependent antibody production . The humoral responses enhanced by FlaB were characterized by high levels of IgG1 and IgG2a that are required to provide complete protection against the targeted pathogen . Like many other subunit vaccines, the Pd vaccine administered through either mucosal route (i.n. or s.l.) induced a TH2 skewed immune response resulting in higher IgG1 over IgG2a titers. However, addition of FlaB to the formulation resulted in the induction of TH1 type immune response providing a mixed TH1/TH2 antibody response. We envisage that the multifunctional immune response generated after immune-potentiation by FlaB will provide superior efficacy in preventing NoV infections in human and animals. Moreover, we have previously shown that FlaB is a very effective and safe mucosal adjuvant as after i.n. immunization it does not accumulate in olfactory tissues and the central nervous system [18, 28].
In a few NoV vaccine studies done in humans  and chimpanzees  the correlates of protection indicate that both antibody and cell mediated immune responses are necessary to clear NoVs. In particular, in a mouse model, CD4 and CD8 T cells were required for clearance of virus from the intestine [31, 32]. Immune responses in mucosal tissues are governed by the nature of the antigen, the type of APCs involved, and the local microenvironment. With most types of non-adjuvanted peptide/protein antigens, the ‘default’ immune response seems to be the TH2 type response that may cause active suppression of systemic immunity . However, antigens and adjuvants, including most PAMPs sensed by mucosal APCs as ‘danger signals’ (e.g. TLR ligands), favor the development of stronger and broader immune responses engaging both the humoral-secretory and cellular immunity effector arms. In the present study we found that P domain based vaccine, given alone, did not induce a substantial cell mediated immunity. However, as shown previously for other antigens , combination of Pd vaccine with flagellin resulted in induction of significantly higher IFNγ secreting CMI. Interestingly, i.n. immunization using Pd+FlaB tended to induce much higher levels of IFNγ secreting CD8+ and CD4+ lymphocytes than the same antigen combination given through s.l. route. Though there is a consensus regarding flagellin’s ability to induce CD4+ T cell-mediated immune responses, the TLR5-dependent CD8+ immune response is less defined. Data in the present study support previous reports that testified the significant activation of CD8+ lymphocytes through TLR5 signaling [34, 35]. However, there are studies that did not note the induction of cytotoxic T cell responses after immunization with flagellin adjuvant  highlighting the fact that the nature and characteristics of co-administered antigen, and route of immunization should determine the type and magnitude of the resulting immune response. Hence, every candidate antigen should be tested with adjuvant rather than predicting effects based upon published results.
Alum is the most common adjuvant used in approved prophylactic vaccines . However, the propensity of alum based adjuvants to induce a TH2-skewed immune response and their inability to induce cell mediated immune responses , limits wider application against diseases where TH1 or cytotoxic T cell responses are critical for immune protection. Hence, we compared the adjuvant effects of FlaB with alum and CFA. CFA is an experimental adjuvant composed of oil-in-water emulsion incorporated with killed mycobacteria and is not recommended for human usage because of severe adverse inflammatory reactions at the site of inoculation . Nonetheless, it is one of the strongest inducers of antibody and cell mediated immune responses. We found that FlaB induced high levels of serum IgG that were comparable to IgG levels induced by CFA but were significantly higher than that induced by alum. We further found that the levels of IgG2a induced after alum vaccination were significantly less than that induced by FlaB or CFA, though the levels of IgG1 were comparable among three groups. Moreover, induction of high levels of secretory IgA antibodies in feces of FlaB immunized animals but not in other two groups established the superiority of FlaB as an adjuvant given through mucosal (i.n. or s.l.) or non-parenteral routes. Taken together, these results indicate the importance of use of an appropriate adjuvant such as FlaB for induction of protective immune reactions in local settings. These results, in conjugation with our previously published reports, further support the superiority of FlaB as a mucosal adjuvant in inducing the antigen specific antibody as well as cell mediated immune reactions in the local mucosal settings.
We show that NoV specific immune responses could be significantly fortified by the use of TLR5 agonist when given through mucosal route. The data in the present manuscript also conclusively demonstrate that for induction of relevant immune responses in appropriate immune compartments, route of immunization is a decisive factor. These results pave a way for the development of a relevant vaccine for human as well as veterinary use, in which NoV induced diarrhea and foodborne infections are a major cause of distress and financial loss.
VV performed the experiments with inputs from WT and SP. VV, SEL and JHR conceived of the study, developed the idea, analyzed the results and wrote the manuscript. JHR, SEL and K-OC contributed the reagents. All authors read and approved the final manuscript.
This study was supported by a grant from the Korean Health Technology R&D Project, Ministry of Health & Welfare, Republic of Korea (No. A110983) and Chonnam National University Hospital Biomedical Research Institute (HCRI 14004-1).
The authors declare that they have no competing interests.
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