The impact of size on particle drainage dynamics and antibody response

Vaccine-induced immune response can be greatly enhanced by mimicking pathogen properties. The size and the repetitive geometric shape of virus-like particles (VLPs) influence their immunogenicity by facilitating drainage to secondary lymphoid organs and enhancing interaction with and activation of B-cells and other innate humoral immune components. VLPs derived from the plant Bromovirus genus, specifically cowpea chlorotic mottle virus (CCMV), are T=3 icosahedron particles. They can be easily expressed in an E. coli host system and package ssRNA during the expression process. Recently, we have engineered CCMV-VLPs by incorporating the universal tetanus toxoid (TT) epitope at the N-terminus. The modified CCMVTT-VLPs successfully form icosahedral particles T=3, with a diameter of ∼30nm analogous to the parental VLPs. Interestingly, incorporating TT epitope at the C-terminus of CCMVTT-VLPs results in the formation of Rod-shaped VLPs, ∼1µm in length and ∼30nm in width. In this study, we have investigated the draining kinetics and immunogenicity of both engineered forms (termed as Round-shaped CCMVTT-VLPs and Rod-shaped CCMVTT-VLPs) as potential B cell immunogens using different in vitro and in vivo assays. Our results reveal that Round-shaped CCMVTT-VLPs are more efficient in draining to secondary lymphoid organs to charge antigen-presenting cells as well as B-cells. Furthermore, compared to Rod-shaped CCMVTT-VLPs, Round-shaped CCMVTT-VLPs led to more than 100-fold increased systemic IgG and IgA responses accompanied by prominent formation of splenic germinal centers. Round-shaped CCMVTT-VLPs could also polarize the induced immune response towards TH1. Up to our knowledge, this is the first study investigating and comparing the draining kinetics and immunogenicity of one and the same VLP monomer forming nano-sized icosahedrons or rods in the micrometer size.


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In 1956, Crick and Watson have stated that "it is a striking fact that almost all small 48 viruses are rods or spheres", "These shells are constructed from a large number of identical 49 protein molecules, of small or moderate size, packed together in a regular manner" (1). The 50 main reason for this arrangement is the small genome of viruses, especially RNA viruses. The 51 coat protein (CP) of many viruses is made up of multiple copies arranged in an icosahedron 52 or a helical-shaped geometry (2, 3). The icosahedral structure of viruses is more prevalent 53 than the helical-shaped one.

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Icosahedral VLPs can be manipulated by inserting few mutations to form rod-shaped VLP.

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For example, VLPs derived from the bacteriophage Qβ can assemble in a rod-shaped particle 73 following the mutation of five amino acid (a.a.) residues in the FG loop of its CP (17).

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It is known that the repetitive surface geometry of icosahedral VLPs enhances optimal

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consisting of 180 sequentially identical CPs. The coat protein can adopt multiple quasi-85 equivalent forms referred to as the coat subunits A, B and C forming either hexamers 86 (alternating subunits B and C) or pentamers (subunit A). The resulting virus particle consists 87 of 12 pentamers and 20 hexamers (29). Previously, it has been shown that icosahedral CCMV 88 can be converted into rod-shaped particles after a disassembly/reassembly process (30).

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In this study, we have demonstrated that CCMV-derived VLPs of different morphology 90 (icosahedral or rod-shaped structure) can be obtained directly from recombinant E.coli cells.

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We specifically manipulated CCMV-VLPs by inserting the universal tetanus toxoid (TT) 92 epitope at the C or N-terminus to form icosahedral VLPs in the nanometer scale or rod-shaped 93 VLPs with sizes in the micron range. Such intervention allowed us to study the impact of size 94 on particles in terms of drainage dynamic and magnitude of induced immune response using

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For the second round the PCR products from the first round were diluted 1:50 and re-amplified 114 with primers CC_N83_d24F and CC_AgeR (5' ACTTCGATACGCTGTAACCGGTCCA 3').

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The corresponding PCR fragments were analysed in 0.8% agarose gel and then purified using

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However, soluble proteins of CCMV-Ntt830-SS were precipitated using a mixture of PEG

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Serum was diluted 1/20 (total volume of 75µl) in PBS-Casein 0.15%. 25µl beads were used 233 per sample. Manufacturer's protocol was followed until step 3. of "Bind Antibody". Supernatant 234 was added to ELISA plates and a 1/3 serial dilution was performed. For IgA detection, goat 235 anti mouse IgA conjugated to HRP was used (ThermoFisher Scientific, Waltham,

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For OD50 calculations, if a sample did not reach the threshold a value of 1 was appointed.

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Therefore, the engineered VLPs in this study are salt-stable (SS). The shape and integrity of 303 the cloned VLPs were confirmed via electron microscopy which shows a size of ~30nm in 304 diameter for CCMV-Ntt-SS (Fig. 1a). The size of the CCMVTT-Ctt-SS greatly varies in length 305 but can reach up to more than 1µm, with a width of about 30nm. Figure 1b shows a magnified 306 image of CCMVTT-Ctt-SS VLPs for easy comparison of their width with the icosahedral 307 CCMVTT-Ntt-SS VLPs in figure 1a. To reach a rather homogenous population, we performed 308 sucrose gradient separation to focus on the long rods (Fig. 1c). For simplification we refer to 309 the two forms of engineered CCMVTT in this paper as Round-shaped CCMVTT-VLPs (CCMV-

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To further characterize the two forms of CCMVTT, we performed mass spectrometry

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Next, we studied which type of APCs are involved in interacting with the Round and

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shaped CCMVTT-VLPs after boosting (Fig. 3d and e). In a next step, we have produced Rod-

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<0.0001) isotype switching to IgA when compared to the Rod-shaped ones ( Fig. 5a and b).

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To rule out the role of TH-cells, we have also measured IFN-γ in the serum of vaccinated mice

Round and Rod-shaped CCMVTT-VLPs enhance neutrophil infiltration in lymph node
525 sinuses without causing any degenerative or vascular changes.

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To study the histopathological effect of Round and Rod-shaped CCMVTT-VLPs on LNs,

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we have performed histology of HE stained popliteal LNs collected 3h and 24h following 528 footpad injections. The main histopathological change of the examined LNs was restricted to 529 a mild to moderate increase of neutrophils within the sinuses, which was present in both 530 groups and absent or neglectable in control tissue (Table 1 and Fig. 7a-c). This neutrophil 531 infiltrate was more evident 3h than 24h after injection. No convincing histological difference 532 was observed between the two groups vaccinated with Round or Rod-shaped CCMVTT-VLPs.

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To achieve successful IgG class-switching and memory formation in B cells, co-

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The histologic analysis of LNs after immunization with either Round or Rod-shaped

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Taken together, our data demonstrate that antigen size is a key determinant of 659 immunogenicity and that icosahedral antigens of ~30nm in diameter are far more 660 immunogenic than essentially the same viral capsid protein assembled into µm sized rods.

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Trafficking from injection site to LNs as well as trafficking within LNs and direct interaction with 662 B cells explain the difference. 663 664