Intranasal c-di-GMP-adjuvanted plant-derived H5 influenza vaccine induces multifunctional Th1 CD4+ cells and strong mucosal and systemic antibody responses in mice. Vaccine

Influenza Centre, The Gade Institute, University of Bergen, N-5021 Bergen, Norway.
Vaccine (Impact Factor: 3.62). 05/2011; 29(31):4973-82. DOI: 10.1016/j.vaccine.2011.04.094
Source: PubMed


Vaccination is the best available measure of limiting the impact of the next influenza pandemic. Ideally, a candidate pandemic influenza vaccine should be easy to administer and should elicit strong mucosal and systemic immune responses. Production of influenza subunit antigen in transient plant expression systems is an alternative to overcome the bottleneck in vaccine supply during influenza pandemic. Furthermore, a needle-free intranasal influenza vaccine is an attractive approach, which may provide immunity at the portal of virus entry. The present study investigated the detailed humoral and cellular immune responses in mice vaccinated intranasally or intramuscularly with plant-derived influenza H5N1 (A/Anhui/1/05) antigen alone or formulated with bis-(3',5')-cyclic dimeric guanosine monophosphate (c-di-GMP) as adjuvant. The use of c-di-GMP as intramuscular adjuvant did not enhance the immune response to plant-derived influenza H5 antigen. However, intranasal c-di-GMP-adjuvanted vaccine induced strong mucosal and systemic humoral immune responses. Additionally, the intranasal vaccine elicited a balanced Th1/Th2 profile and, most importantly, high frequencies of multifunctional Th1 CD4(+) cells. Our results highlight that c-di-GMP is a promising mucosal adjuvant for pandemic influenza vaccine development.

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    • "Madhun et al. showed that c-di-GMP improved the immunogenicity of an intranasally delivered subunit influenza vaccine , compared to antigen only, by inducing strong mucosal and systemic immune responses [8]. Additionally, the authors showed that intranasal administration of the c-di-GMP adjuvanted antigen induced protective antibody titers and cellular immune responses that far exceeded the responses induced by intramuscular administration of the same vaccine [8]. Moreover, Svindland et al. tested vaccination with c-di-GMP combined with a second adjuvant, Chitosan, and showed that vaccination with the combination of these molecules can further improve the humoral and cellular immune responses against target antigens [9]. "
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    ABSTRACT: Annually influenza virus infections are responsible for hospitalization and mortality, especially in high risk groups. Constant antigenic changes in seasonal influenza viruses resulted from antigenic shifts and antigenic drifts, enable emerging of novel virus subtypes that may reduce current vaccine efficacy and impose the continuous revision of vaccine component. Currently available vaccines are usually limited by their production processes in terms of rapid adaptation to new circulating subtypes in high quantities meeting the global demand. Thus, new approaches to rapidly manufacture high yields of influenza vaccines are required. New technologies to reach maximal protection with minimal vaccine doses also need to be developed. In this study, we evaluated the systemic and local immunogenicity of a new double-adjuvanted influenza vaccine administered at the site of infection, the respiratory tract. This vaccine combines a plant-produced H1N1 influenza hemagglutinin antigen (HAC1), a silica nanoparticle-based (SiO2) drug delivery system and the mucosal adjuvant candidate bis-(3′,5′)-cyclic dimeric guanosine monophosphate (c-di-GMP). Mice were vaccinated by intratracheal route with HAC1/SiO2 or HAC1/c-di-GMP (single-adjuvanted vaccine) or HAC1/SiO2/c-di-GMP (double-adjuvanted vaccine) and evaluated for target-specific immune responses, such as hemagglutination inhibition and hemagglutinin-specific IgG titers, as well as local antibody (IgG and IgA) titers in the bronchoalveolar lavage (BAL). Furthermore, the HAC1-specific T-cell re-stimulation potential was assessed using precision-cut lung slices (PCLS) of vaccinated mice. The double-adjuvanted vaccine induced high systemic antibody responses comparable to the systemic vaccination control. In addition, it induced local IgG and IgA responses in the BAL. Furthermore, HAC1 induced a local T-cell response demonstrated by elevated IL-2 and IFN-γ levels in PCLS of c-di-GMP-vaccinated mice upon re-stimulation. Overall, the present study showed the potential of the double-adjuvanted vaccine to induce systemic humoral immune responses in intratracheally vaccinated mice. Furthermore, it induced a strong mucosal immune response, with evidence of antigen-primed T-cells in the lung.
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    • "Contrary to that, local mucosal immunization with an adjuvant via the respiratory tract at the site of the virus entry may stimulate local humoral and cell-mediated immune responses [16], [17]. Consequently, mucosally administered adjuvanted influenza vaccines showed increased immunogenic effects with potential to reduce the required vaccine doses to be supplied to the global market [18], [19]. In context of local vaccination, a drug delivery system like micro- or nanoparticles (NP) might be of great benefit in terms of protein stabilization or controlled antigen release [20], [21]. "
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    ABSTRACT: Annual outbreaks of influenza infections, caused by new influenza virus subtypes and high incidences of zoonosis, make seasonal influenza one of the most unpredictable and serious health threats worldwide. Currently available vaccines, though the main prevention strategy, can neither efficiently be adapted to new circulating virus subtypes nor provide high amounts to meet the global demand fast enough. New influenza vaccines quickly adapted to current virus strains are needed. In the present study we investigated the local toxicity and capacity of a new inhalable influenza vaccine to induce an antigen-specific recall response at the site of virus entry in human precision-cut lung slices (PCLS). This new vaccine combines recombinant H1N1 influenza hemagglutinin (HAC1), produced in tobacco plants, and a silica nanoparticle (NP)-based drug delivery system. We found no local cellular toxicity of the vaccine within applicable concentrations. However higher concentrations of NP (≥10(3) µg/ml) dose-dependently decreased viability of human PCLS. Furthermore NP, not the protein, provoked a dose-dependent induction of TNF-α and IL-1β, indicating adjuvant properties of silica. In contrast, we found an antigen-specific induction of the T cell proliferation and differentiation cytokine, IL-2, compared to baseline level (152±49 pg/mg vs. 22±5 pg/mg), which could not be seen for the NP alone. Additionally, treatment with 10 µg/ml HAC1 caused a 6-times higher secretion of IFN-γ compared to baseline (602±307 pg/mg vs. 97±51 pg/mg). This antigen-induced IFN-γ secretion was further boosted by the adjuvant effect of silica NP for the formulated vaccine to a 12-fold increase (97±51 pg/mg vs. 1226±535 pg/mg). Thus we were able to show that the plant-produced vaccine induced an adequate innate immune response and re-activated an established antigen-specific T cell response within a non-toxic range in human PCLS at the site of virus entry.
    PLoS ONE 08/2013; 8(8):e71728. DOI:10.1371/journal.pone.0071728 · 3.23 Impact Factor
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    • "Such T cells have been strongly associated with immunity to HIV [1], [2]; influenza [3], [4]; leishmania [5] and tuberculosis (TB) [6], [7], [8], [9], [10], [11], [12]. Consequently, they are seen as a promising measure of vaccine efficacy [13] and are a commonly measured immunological parameter in clinical trials [14], [15], [16], [17], [18]. "
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    ABSTRACT: The assessment of antigen-specific T cell responses by intracellular cytokine staining (ICS) has become a routine technique in studies of vaccination and immunity. Here, we highlight how the duration of in vitro antigen pre-stimulation, combined with the cytokine accumulation period, are critical parameters of these methods. The effect of varying these parameters upon the diversity and frequency of multifunctional CD4 T cell subsets has been investigated using a murine model of TB vaccination and in cattle naturally infected with Mycobacterium bovis. We demonstrate a substantial influence of the duration of the antigen pre-stimulation period on the repertoire of the antigen-specific CD4 T cell responses. Increasing pre-stimulation from 2 to 6 hours amplified the diversity of the seven potential multifunctional CD4 T cell subsets that secreted any combination of IFN-γ, IL-2 and TNF-α. However, increasing pre-stimulation from 6 to 16 hours markedly altered the multifunctional CD4 T cell repertoire to a dominant IFN-γ+ only response. This was observed in both murine and cattle models. Whilst these data are of particular relevance to the measurement of vaccine and infection induced immunity in TB, more generally, they demonstrate the importance of the empirical determination of the optimum duration of the individual culture steps of ICS assays for any model. We highlight the potential significance of variations in these parameters, particularly when comparing data between studies and/or models including clinical trials.
    PLoS ONE 06/2012; 7(6):e38926. DOI:10.1371/journal.pone.0038926 · 3.23 Impact Factor
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