vitro.
Résumé
L’émergence de la souche aviaire H5N1 hautement pathogénique et la récente pandémie de 2009 nous rappellent qu’il est nécessaire de trouver une solution alternative aux vaccins présentement utilisés. L’une d’elles est de diriger la réponse immunitaire contre des protéines internes du virus qui sont conservées entre les différentes souches d’influenza. Dans cette étude, nous avons utilisé une nucléoprotéine recombinante (rNP) comme base vaccinale et nous l’avons multimérisée grâce à un ARN synthétique afin d’augmenter sa faible immunogénicité. Cette présentation, sous forme de pseudo-nucléoparticule virale (NLP) combinée à un adjuvant (PAL), a augmenté la réponse humorale et cellulaire spécifique à NP chez la souris. Une dose de NLP-PAL ne fut pas suffisante pour protéger des souris contre une infection létale. Des expériences additionnelles sont nécessaires pour identifier la formulation et le nombre d’immunisations optimales nécessaires pour protéger des souris contre une infection létale par l’influenza
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Increase of the influenza nucleoprotein
immunogenicity through in-vitro multimerization
Alexis RUSSELL, Gervais RIOUX, Marilène BOLDUC, Pierre SAVARD, Denis LECLERC
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Abstract
The emergence of the highly pathogenic H5N1 avian influenza and the pandemic of 2009 have pushed scientists to find novel vaccination strategies such as directing the immune response towards the internal proteins of the influenza. In this study, we have used the recombinant nucleoprotein (rNP) of influenza as a basis for the development of a broad spectrum vaccine. This type of vaccine is possible because NP is well conserved among the influenza A strains and its potential to confer cross- protection has been showed on several occasions.
To enhance the immunogenicity of the rNP, we increased the complexity of this antigen through its multimerization using a synthetic RNA as a scaffold to generate nucleocapsid-like-particles (NLP). The self-assembly of this protein into NLP is a natural function of the protein and we have chosen to take advantage of this characteristic to improve its immunogenicity. The multimerization increased the cellular and humoral immunogenicity of NP and addition of the PAL adjuvant increased it even further. The NLP-PAL candidate vaccine was not able to fully protect mice from a lethal challenge after one immunization. A prime-boost immunization increased the humoral and cellular response but further experiments are needed to confirm that a prime-boost strategy can protect against a lethal influenza challenge.
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Introduction
Each year, influenza epidemics are responsible for the infection of 5 to 15% of the worldwide population which result in 3 to 5 million cases of severe illness and up to 500 000 deaths [1]. Trivalent inactivated vaccine (TIV), which results in a neutralising antibody-based response directed at the variable surface proteins hemagglutinin (HA) and neuraminidase (NA), is still the most efficient way to prevent infection against the virus and has been used for more than 60 years [2]. However, influenza viruses are subjected to minor (drift) and major (shift) antigenic change which means that new vaccines have to be made each year to match the dominating strains [3]. Furthermore, these vaccines will be ineffective in case of a pandemic caused by the apparition of novel influenza A strain that express new HA and NA proteins [4]. With the recent pandemic caused by a novel pH1N1 and the emergence of the highly pathogenic H5N1 as a potential pandemic strain [5], there is a growing concern to produce a vaccine that would induce broader protection against different serotypes [6].
Immunisation with conserved proteins of influenza, such as nucleoprotein (NP) or matrix protein (M1/M2), is known to induce a broad, heterotypic response against a multitude of strains. This response is associated with a more rapid viral clearance and a reduction in both morbidity and mortality [7–10]. NP is extremely well conserved among influenza A strains [11–13] and it contains dominant CTL epitopes for most human HLA-type [14,15] . Many studies have shown that NP immunisation leads to a protection mainly mediated by CD8+ T lymphocytes that recognize CTL epitopes presented by infected cells [16–18] and that this protection is heterosubtypic [19–21]. The humoral response against NP has long been disregarded but recent studies have shown that immunisation with NP results in antibodies that can also contribute to cross-protection [22,23] by a mechanism involving both FcRs and CD8+ cells [24]. Immunisations with soluble recombinant NP seems to be an efficient way to protect against infection but high doses, multiple immunisations or the combination with an
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adjuvant is required since soluble recombinant proteins generally have a low immunogenicity [20,21,25–28].
Viral structural proteins expressed in an ordered and repetitive fashion, to form viral- like-particles (VLPs), are considerably more immunogenic than in their soluble form [29,30]. This technique can be applied to a multitude of nucleocapsids [31–34] to form nucleocapsid-like-particles (NLPs) which results in the enhancement of the antigen immunogenicity [35,36]. In this study, we have produced in E. coli and purified a monomeric recombinant influenza nucleoprotein (rNP) that we multimerized using a synthetic RNA as a scaffold to form a NLP. We evaluated the effect of the multimerization on both the humoral and cellular response against the nucleoprotein and we assessed the potential of the NLP combined with the PapMV (PAL) adjuvant as a one-shot candidate vaccine against a lethal influenza challenge in mice.
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Results
Production and purification of the monomeric NP recombinant protein
The sequence coding for the nucleoprotein (NP) of the pandemic strain A/California/04/2009 (H1N1) combined with a 6xHis tag at the C-terminal end (Fig. 2-1A) was cloned using an expression vector and a bacterial host (E. coli strain). After a 16h expression induced by IPTG, the recombinant NP (rNP) was purified via an affinity Nickel column. The eluted protein was diafiltrated with a 30 kDa MWCO membrane using Tangential Flow Filtration to further purify the rNP. The yield of production of rNP was 80-90 mg per litre of bacterial culture. The SDS-PAGE profile showed that the eluted rNP is highly purified (Fig. 2-1B, lane 3). The SDS-PAGE profile also showed minor degradation of the rNP after the diafiltration (Fig. 2-1B, lane 4). A western blot assay using a polyclonal antibody reacting specifically to the nucleoprotein of Influenza A viruses (data not shown) or using serum from mice immunized with NP-GST (Fig. 2-1C) confirmed that the purified protein was rNP although a little degradation can be seen.
Next, we confirmed that the recombinant NP was in a monomeric form and not associated with bacterial nucleic acids. Since it is suitable for proteins and nucleic acids analysis, Superdex 200 size-exclusion chromatography column and a FPLC system were used to reveal putative contamination with nucleic acids as a protein sample that is contaminated with nucleic acids will have a higher absorbance at 254 nm than at 280 nm. The FPLC profile (Fig. 2-1D) showed that the rNP, which is eluted between 15mL and 18mL, is highly purified and not contaminated with nucleic acids. The profile also showed a small fraction eluted between 19 and 22mL. This fraction is probably associated with degraded rNP as seen in the Anti-NP Western Blot (Fig. 2-1B, lane 4). The Superdex 200 column was also used to measure the molecular weight of the rNP. Using different proteins to calibrate the Superdex 200 (Fig. 2-1D, shown as arrows), the apparent molecular weight of the purified rNP was estimated between 29 000 and 76 000 kDa. Therefore, we concluded that the purified
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rNP was in a monomeric form since it is known that the apparent influenza nucleoprotein has a molecular weight of 56 kDa [37].
Mulitmerization of the monomeric NP recombinant protein using different RNA template
Since it is well known that viral structural proteins expressed in an ordered and repetitive fashion are considerably more immunogenic than in their soluble form [30], we multimerized the rNP into a NLP using two different single-stranded synthetic RNA as a scaffold: polycytidylic acid potassium salt (NLP-Poly-C) or polyuridylic acid single-stranded RNA polymer (NLP-Poly-U). Briefly, the monomeric rNP were mixed in a 10:1 ratio with either Poly-C or Poly-U, incubated for 2 hours, treated with RNase A for another 2 hours and diafiltrated on a 100 kDa MWCO membrane to remove the monomeric rNP that did not bind to the RNA. The NLPs were analyzed on a Superdex 200 to evaluate their molecular weight and to confirm that there was no monomeric rNP left after the diafiltration. Both NLPs elution profile (Fig. 2-2A) showed a heterogeneous population of NLPs with higher molecular weight than the monomeric rNP. The elution profile also showed that the diafiltration efficiently removed free monomeric rNP since there was no protein eluted between 15 and 18 mL. To complete the biochemical characterization of the NLPs, we used dynamic light scattering (DLS) to estimate the average length of the nanoparticles and their stability at various temperatures. Both NLPs had approximately the same average length: 22.1 nm for NLP-Poly-C and 23.9 for NLP-Poly-U (Fig. 2-2B, left panel). NLP-poly-C was stable to 50C whereas the NLP-Poly-U initiated aggregation at 37 °C (Fig. 2-2B, right panel). Transmission electron microscopy of both types of NLP showed a population of rod shape structures of various lengths (Fig. 2-2C, middle and right panel), while we could not see any visible monomeric rNP (Fig. 2-2C, left panel).
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Importance of the stability on the immune response
Since the two different NLP behaved differently under a temperature gradient, we wanted to see if there was a difference between them on the humoral immune response against NP. Mice were immunized twice at 14 days interval by the intramuscular route with two different doses of either monomeric rNP, NLP-Poly-U or NLP-Poly-C. Antibody levels against NP in the blood were measured by ELISA at day 14 and 28 post immunisation. Total IgG titers (Fig. 2-3A) and IgG2a titers (Fig. 2-3B) showed that NLP immunisation led to a humoral response against NP but there was no significant difference in the humoral response between the two NLPs, at day 14 and 28 post immunisation. While conducting this experiment, the stability of the NLPs was verified on the Superdex 200 column since both NLPs were stored at 4 °C for the last 3 months. The elution profile of the NLPs had changed (Fig. 2-4) when compared to the original elution profile (Fig. 2-2A). This change was especially noteworthy for NLP-Poly-C as we observed the apparition of an eluted peak between 19 and 23 ml. Aggregates were also seen in the NLPs solution stored at 4 °C. This result was surprising since NLP-Poly-U initiated aggregation at 37 and was less stable than NLP-Poly-C under a temperature gradient (Fig. 2-2B, right panel). Based on the fact that there were no differences in the humoral response between the two NLPs and that NLP-Poly-U was more stable than NLP-Poly-C after 3 months at 4 °C, we chose to continue the assessments with NLP-Poly-U only.
Updated production of the NLP vaccine
We made a few changes to the process for the production of both monomeric rNP and the NLP-Poly-U to improve their long term stability in solution. After capture and elution from the affinity column, the rNP solution was filtered through an anionic charged membrane for endotoxins removal. This operation decreased the LPS concentration from 50 EU/injection to below the 0,1 EU/mL detection limit of the
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Limulus test. As mentioned above, the monomeric rNP was then mixed with Poly-U for 2 hours at 22 °C but there was no RNase A treatment before the final diafiltration. A full biochemical characterization was made to see if the modifications to the production protocol affected the NLP. The monomeric rNP still eluted at the same volume on the Superdex 200 (data not shown) while the elution profile of the NLP- Poly-U was consistent with previous results shown in figure 2-2A. DLS analysis showed that NLPs had a mean length of 27 nm (Fig. 2-5B, left panel) and that they were stable throughout the temperature gradient (Fig. 2-5B, right panel). The stability of the NLP-Poly-U throughout the temperature gradient was improved when compared with the previous result (Fig. 2-2B, right panel). Transmission electron microscopy of the NLPs still showed rod shape structures of various lengths (Fig. 2- 5C).
Importance of the multimerization on the nucleoprotein immunogenicity
After NLP-Poly-U was chosen as the candidate influenza vaccine, an immunisation program was set up to validate that the multimerization of the monomeric rNP into a NLP led to increase in the NP immunogenicity. Balb/c mice (10/group) were immunized once intramuscularly in the thigh with formulation buffer or 0.5 µg of either rNP or NLP-Poly-U. At day 14 post-immunisation, the humoral response against NP was assessed by ELISA. As seen in figure 2-3A and 2-3B, the multimerization of the monomeric rNP into a NLP led to a significant (p<0.001) increase in both total IgG (32-fold increase) (Fig. 2-6A, left panel) and IgG2a (128- fold increase) (Fig. 2-6A, left panel) titers.
It has been established that NP is one of the main targets of the cellular immune response against influenza and that it contains multiples conserved MHC class I and class II epitopes [38]. To evaluate if the multimerization of the rNP into a NLP led to a better cellular immune response, an ELISPOT assay on the mice immunized with either rNP or NLP-Poly-U was performed. Briefly, IFN-γ secretion of T cells was evaluated using the H-2KD Influenza NP peptide TYQRTRALV or NLP-Poly-U to
45 reactivate splenocytes that were harvested 2 weeks post-immunisation. The ELISPOT assay (Fig. 2-6B) showed that the multimerization did not improved the number of T cells secreting IFN-γ. Since there was no difference, even with the control group that received formulation buffer, we hypothesized that a single immunisation was not sufficient to induce a potent cellular response. A boost immunisation was made and the splenocytes were harvested 1 week later. This ELISPOT assay (Fig. 2-6C, left panel) showed that boost immunisation with the NLP-Poly-U led to a significant increase (p<0.01) in NP-specific CD8+ T-cells when compared with the group immunized with the monomeric rNP. The same increase was not seen for the splenocytes stimulated with the NLP-Poly-U (Fig. 2-6C, right panel). The results of the ELISPOT and ELISA assays confirmed that the multimerization of the monomeric rNP into a NLP significantly increased both the humoral and cellular response against the influenza nucleoprotein.
Multimerization is still beneficial with the addition of an adjuvant
Immunisation with recombinant purified antigens frequently results in the induction of a modest antibody response with little or no T cell response. To bypass the weak immunogenicity of the antigen, a high dose or an adjuvant is needed to enhance the efficacy and trigger the appropriate immune response [39]. In this experiment, the PapMV nanoparticles adjuvant, also known as PAL (PapMV-Adjuvant Long-lasting response), was used in combination with monomeric rNP or the NLP-Poly-U. We assessed whether there was still a benefit to the multimerization of NP or if similar results could be achieved with only adjuvanted monomeric rNP. Balb/c mice (10/group) were immunized once intramuscularly with 0.5 µg of either rNP or NLP- Poly-U adjuvanted with 5, 10, 20 or 40 µg of PapMV nanoparticles and the humoral response against NP was measured by ELISA 14 days post-immunisation. Total IgG and IgG2a titers (Fig. 2-7A) revealed that the multimerization of NP was still beneficial on the NP-specific immunity even with the addition of the PAL adjuvant. The addition of different concentrations of PAL also improved the humoral response against NP with 40 µg of PAL leading to the most substantial increase in mean titers
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of total IgG (12.4) and IgG2a (12.8) when compared to NLP alone (Fig. 2-6A, Total IgG: 8.8, IgG2a: 8.25). We also assessed if the addition of PAL increased the cellular response to NP and if the multimerization of the NP was still beneficial for the cellular response when compared to adjuvanted rNP. The ELISPOT assay, as mentioned above, was used and the results showed no difference in IFN-γ secretion between the adjuvanted monomeric rNP and the adjuvanted NLP groups after one immunisation (Fig. 2-7B). A boost immunisation was given but no significant differences were seen between the two groups (Fig. 2-7C).
Boost-immunisation increases both humoral and cellular response
As shown in figure 2-6B and 2-6C, immunisation with 0.5 µg of NP only induced a weak cellular response after a single immunisation and a boost was needed to observe a significant increase in the T-cell response when comparing rNP and NLP. Therefore, we tested if the use of a higher quantity of antigen could improve the cellular response after a single dose and we assessed whether a prime-boost immunisation increased both humoral and cellular responses. Balb/c mice (5/group) were immunized once or twice at 14 days interval with 10 µg NLP alone or combined with 40 µg of PAL adjuvant. Blood was collected on day 13 (one day before the boost-immunisation) and day 21 (7 days after the boost) and the humoral response against NP was assessed by ELISA. The boost immunisation (Fig. 2-8A) led to a 15- fold increase for total IgG and 21-fold increase IgG2a titers for the NLP group while mice immunized with NLP + PAL had a 9-fold in their total IgG and 10-fold increase in their IgG2a titers (p <0.001). Even though the fold increase was lower in the group immunized with the PAL adjuvant after the boost immunization, the total IgG and IgG2a titers were higher than the titers of the group immunized with NLP alone.
Splenocytes were harvested 7 days after the last immunisation and reactivated with the NLP-Poly-U or the H-2KD peptide to evaluate the IFN-γ secretion of the T cells. The boost immunisation significantly increased (p<0.001) the number of T cells secreting IFN-γ (Fig. 2-8B) when stimulated with the NLP or the H-2KD peptide. A
47 similar increase after the boost immunisation was seen in the groups that received NLP with 40 µg of PAL adjuvant (Fig. 2-8C). Even when a more substantial quantity of antigen was used, the addition of PAL did not increase the cellular response against NP but it did increase the humoral response against NP. Based on the results mentioned in this experiment, we decided to use NLP-Poly-U combined with the PAL adjuvant as our candidate influenza vaccine.
A single immunisation with NLP-Poly-U + PAL does not provide protection from infection with a lethal dose of influenza H1N1
Even though an enhancement of the humoral and cellular response was observed with a boost immunisation, we assessed whether the candidate vaccine was able to protect mice from a lethal challenge with a H1N1 influenza strain after a single immunisation. Therefore, mice (10/group) were immunized with the vaccine formulations listed in Table 2.1. At day 14 post-immunisation, blood was collected to measure the IgG2a titers against NP. For this ELISA protocol, NP-GST was used instead of rNP as the capture antibody because rNP showed cross-reactivity with the PAL antibodies. This cross-reactivity may be explained by the presence of a His-tag in both rNP and PAL. NP-GST was also used to validate that the increase in the humoral response elicited by the PAL adjuvant was directed towards NP and not simply an addition of the anti-NP and the cross-reactive anti-PAL. IgG2a titers (Fig. 2-8) revealed that groups immunized with NLP + PAL had a significantly increased humoral response against NP (10-fold increase) when compared to the groups immunized with NLP only.
Mice were then challenged with 1 or 2 LD50 of the mouse-adapted strain A/WSN/33 (H1N1) and mice were monitored daily for weight loss, symptoms and survival. The results of the challenge showed that a single immunisation with NLP-Poly-U + PAL was not sufficient to provide a complete protection from a lethal challenge with the A/WSN/33 (H1N1) strain. There were no significant differences in the weight loss between the different groups challenged with 1 or 2 LD50 (Fig. 2-10A). No
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significant differences were also observed in the survival (Fig. 2-10B, left panel) and symptoms (Fig. 2-10C, left panel) of the groups infected with 1 LD50. However, in the challenge with 2 LD50, mice immunized with 20 µg NLP + 80 µg PAL had a significantly (p<0.001) higher survival percentage (Fig. 2-10B, right panel) and exhibited significantly (p<0.05) less symptoms (Fig. 2-10C, right panel) at the peak of infection when compared with mice that did not received the NLP vaccine. The significant difference (p<0.01) in survival between the mice immunized with 20 µg of NLP alone and the mice immunized with 20 µg of NLP combined with 80 µg of PAL also showed the importance of the adjuvant in the protection against an influenza