Traditional vaccine platforms such as the flu vaccine is primarily based on the growth of viruses in chicken eggs, which is time consuming (takes more than 6 months) and the process is tedious. Furthermore, it is no more than 50% effective due to the ability of influenza viruses to mutate rapidly  . The emergency use of mRNA-based vaccines to stem the spread of COVID-19 has been a huge game changer in vaccine production. In an effort to discover a cost-effective vaccine that also offers broad and long-lasting protection against the influenza virus, there has been a focus on an alternative vaccine platform based on the creation of a rod-shaped nanoparticle composed of the coat protein of a plant virus called the papaya mosaic virus (PapMV) that self-assembles around an ssRNA, which is then fused with a peptide from the influenza virus . This strategy has been shown to trigger specific immune responses , resulting in 100% of survival rate in mice studies, along with decreased weight loss, viral titers in lungs and nasal turbinate, and flu symptoms compared to a control group .
Surface plasmon resonance (SPR) is a real-time, label-free optical technique to detect and observe biomolecular interactions. SPR is mostly accessible to large pharma and CRO facilities which can be an obstacle for research facilities to quickly obtain valuable data. Portable SPR has shown to remove such bottleneck and improve SPR access to scientists everywhere. For example, Affinité's portable P4SPR™ has recently been deployed in an university hospital to detect antibodies specific to the SARS-CoV-2 nucleocapsid and spike proteins, and to determine the cross-reactivity of antibodies towards the viral proteins from other variants of concern. The portable SPR can similarly evaluate the efficacy of a novel vaccine platform such as the PapMV nanoparticle that is fused with a peptide specific to a flu virus strain, a project that has been actively engaged in Prof. Denis Leclerc’s lab at Centre Hospitalier de l’Université Laval (CHUL). This blog details how sera from pre-immunized and immunized mice with the PapMV-peptide fusion nanoparticle were analyzed by our P4SPR to compare the levels of antibodies induced by this novel vaccine platform.
SPR Experimental Details
Two batches of serum samples were prepared for this experiment from the same group of mice. The serum samples were drawn from mice before immunization (pre-immunized) and after immunization (immunized). The mice were immunized with the PapMV nanoparticle that was fused with the m2 peptide, a peptide that is part of a conserved protein of the influenza virus. Pre-immunized and immunized serum samples were collected and were diluted in the running buffer (1x PBS buffer with 0.005% Tween20 and 0.1% BSA) by using the following dilution factors: 1:500, 1:250, 1:150, 1:100, 1:50, 1:25. The SPR data were collected by Affinité's quad inlet P4SPR™ (4 channels).
First, the P4SPR gold sensor chips were functionalized with Afficoat™, a self-assembled monolayer that serves both as a covalent linker and a coating that minimizes non-specific protein adsorption, which is especially crucial for complex samples such as serum. The PapMV nanoparticles were immobilized onto Afficoat-functionalized chips by using NHS and EDC chemistries in 10 mM acetate buffer (pH 5.5) for 20 min. Figure 1 depicts the general immobilization scheme. The chip surfaces were further passivated with 1 M ethanolamine (pH 8.5) for 10 min. After the baseline was stabilized with the running buffer, the sensorgram data of the binding interaction between the antibodies in mice serum and immobilized PapMV were collected by the P4SPR from the most to least diluted without any regeneration steps. Two pre-immunized serum samples and subsequent dilutions were injected into the first 2 microfluidic channels and the same was repeated for immunized serum samples for the last 2 channels of the P4SPR. The average SPR signals were obtained from each set of the 2 measurement channels and they were used to plot the final sensorgram data (Figure 2).
Figure 1. SPR setup of the experiment. PapMV nanoparticles were immobilized onto Afficoat-modified gold sensor chips.
Comparison of Pre-Immunized and Immunized Mice Sera
Figure 2 shows the difference in average SPR signal between the mouse serum that came from pre-immunized mice and immunized mice from the most to least diluted. Immunized mice had serum that contained higher levels of antibodies, demonstrating that the use of the PapMV-m2 peptide vaccine platform was effective in eliciting a heightened immune response. These data also show that the P4SPR can differentiate the level of antibodies between the two types of samples while immersed in a complex matrix, i.e. mouse serum. Besides preparing dilutions of the samples, they did not require any other processing steps to enable analysis by the P4SPR.
Figure 2. Average sensorgram data obtained from P4SPR of pre-immunized (blue) and immunized (orange) mouse sera samples (starting from most diluted to least diluted).
The entire set of sensorgrams took about 40 min to obtain once the baseline was stabilized following the PapMV immobilization and surface passivation steps. With these data on hand, it is also possible to estimate the KD for the PapMV and antibody interactions by using the SPR shift at equilibrium from each dilution and its corresponding dilution factor. The P4SPR control software allows determination of KD easily when these values are input into the data processing table.
P4SPR is a simple SPR system that can be used to evaluate the humoral effects of a novel vaccine platform in mice which is based on a PapMV nanoparticle that is fused with an influenza-specific antigenic peptide. The immobilization steps were straightforward and the generation of SPR data could be obtained quickly even for complex biological samples such as mouse serum.
The Affinité Advantage
Affinité Instruments’ P4SPR™ is a very user-friendly, compact, and portable instrument. In addition, samples do not need much preparation and can be manually injected into the instrument. The P4SPR™, compared to a traditional immunoassay such as ELISA, provides fast, real-time affinity and/or kinetic data.
Simplicity - Fast training, fast results
Versatility - Pharmaceutical, biosensing, assay development applications
Economy - Affordable, accessible
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 Marilène Bolduc, Mariana Baz, Marie-Ève Laliberté-Gagné, Damien Carignan, Caroline Garneau, Alexis Russel, Guy Boivin, Pierre Savard, Denis Leclerc. The quest for a nanoparticle-based vaccine inducing broad protection to influenza viruses. Nanomed.: Nanotechnol., Biol., and Med., 2018, 14, 2563–257.
 Marie-Eve Laliberté-Gagné, Marilène Bolduc, Caroline Garneau, Santa-Mariela Olivera-Ugarte, Pierre Savard and Denis Leclerc. Modulation of Antigen Display on PapMV Nanoparticles Influences Its Immunogenicity. MDPI Vaccines 2021, 9, 33.