*2.4. VLP Vaccination Can Directly Activate B Cells*

B cells are an integral part of the adaptive immune response. The identification of the subtype of B cells that binds to VLP can help to understand the mechanisms of B cell activation, differentiation, gene regulation, cytokine, and antigen-specific antibody production. In an explicative study, the SIV, SHIV, and chimeric influenza HA/SIV (SIV gag plus influenza hemagglutinin) VLP, known to induce high titers of antibodies, were incubated with mouse splenocytes [72]. All three types of VLP bound directly to CD19<sup>+</sup> naive B cells but not to CD3<sup>+</sup> T cells, as expected by the fact that the mouse CD4 cannot be properly recognized by HIV envelope protein. Dose-dependent binding and unlabeled VLP competition assays showed that the VLP binding to naïve B cells is specific [72]. Indeed, B cells may be specific for VLP antigens, and binding occurs via their antigen receptor (BCR) rather than toll like receptors (TLR), and induces up-regulation of the activation markers CD69 and CD86 [72].

Among the population of naïve B cells (B220+IgM+), conventional B2 cells (CD43−CD5−) increased after treatment with VLP, compared with PBS control, while the B1 cell subpopulations B1a (B220+IgM+CD43+CD5+) and B1b (B220+IgM+CD43+CD5−) did not change after VLP exposure [72]. This data indicates that incubation of naïve B cells with VLP can stimulate B2 cells to expand in vitro.

Therefore, some VLP are capable of directly inducing the activation of the humoral response through specific binding to naïve B2 cells in vitro [72]. However, immunization with other VLPs may produce different results. A study with HPV16 vaccination, for instance, showed the ability of HPV16 VLP to activate naïve B cells, although, in this case, an increase in B1 cell subpopulation was reported [73]. Another study using Qβ and AP205 VLP induced the expansion of B1 cells from the marginal zone of the spleen [74]. The nature of the VLP structure and the type of infection are speculated to be involved in these differences.

VLP purification after expression in *Escherichia Coli* does not remove all the bacterial endotoxin, leaving traces of lipopolysaccharide (LPS) in the VLP formulation. Are the VLP alone responsible for inducing the expansion of naïve B cells, or is the residual endotoxin involved? Spleen cells incubated with VLP, LPS, or anti-CD40 antibody in the presence or absence of polymyxin B (PMB—an antibiotic that blocks LPS activity) helped to answer this question. The naïve B cell proliferation was reduced in the presence of LPS and PMB but was not affected when treated with VLP and anti-CD40 in the presence or absence of PMB, showing that activation of naïve B2 cells by VLP is not dependent upon the presence of endotoxin [72]. The same study showed that in the supernatant of naïve mouse splenocytes stimulated by treatment with VLP, the expression of IL-12, MIP-1α, and MIP-1β is elevated, while the expression of IL-4 and MCP-1, which favor IgG1 antibody production, was decreased. Therefore, VLP stimulation is conducive to IgG2a class-switch recombination (Figure 2) [72].

B cells can respond to antigen in a T-dependent or T-independent way. In both cases, besides antigen binding through the BCR, additional signals are required to induce B cells to proliferate and differentiate into plasma cells producing antibodies [75]. VLP bind and activate naive B cells, but can VLP induce B cells to differentiate into plasma cells? Splenocytes incubated for 48 h with VLP were transferred to a SIV VLP-coated polyvinylidene fluoride filter plate for 3 h at 37 ◦C. The ELISPOT assay showed that VLP treatment induces the differentiation of activated B cells into plasma cells, at least in vitro. These data were confirmed by real-time PCR analysis where the levels of Blimp-1 and XBP-1 increased after splenocytes incubation with VLP; these two proteins are essential for the differentiation of plasma cells. The level of antibodies produced after plasma cell differentiation was evaluated by ELISA, with a remarkable increase in both IgM and IgG2a, confirming that VLP stimulated a humoral

response in vitro [72]. VLP immunization can also stimulate B cell differentiation into a plasma cell and class-switch recombination in vivo [72].

**Figure 2.** Illustration of virus-like particles (VLP) triggering immune response. (A) The draining of nanoparticles to the lymphatic system is an essential property of nanoparticles. (B) VLP can directly activate naïve B cells and produce a long-lasting immune response. (C) VLPs processed by DC cells trigger immune response and development of effector mechanisms.
