*2.3. VLP and the Lymphatic System*

The draining of nanoparticles to LN is an essential property of VLP vaccines. As mentioned above, most VLP platforms have diameters ranging from 25 to 100 nm, making them small enough to be drained into the lymphatic vessels, whose diameters vary from 10–60 μm up to as much as 2 mm. Ultra-small nanoparticles such as Pluronic-stabilized polypropylene sulfide (PPS) (~25–100 nm) injected intradermally are transported highly efficiently into the lymphatic capillaries and their draining LN [63]. EαRFP, a recombinant protein injected subcutaneously (s.c.), is transported to the LN in 18 h [64]. In one lymphatic trafficking study, VLP of bacteriophage Qβ (30 nm of diameter) labeled with Alexa-488 were injected in C57BL/6 mice. Fluorescent polystyrene nanoparticles with diameters of 20, 500, and 1000 nm were also injected. Within two hours, VLP and 20 nm beads were detected in the subscapular sinus of the popliteal LN, where they were associated with LN-resident DCs, B cells and macrophages. This pattern is compatible with drainage of afferent lymphatic capillaries. By 48 h, they were localized at the subcapsular, paracortex, and cortex areas of the LN in the vicinity with B cell follicles [36]. Larger 500 and 1000 nm beads needed 24 to 48 h to reach the popliteal LN and colocalized to areas where DCs reside. They did not efficiently enter lymphatic vessels and were possibly carried into the LN by specialized cells immigrating from the skin. One week after injection, 20, 500 and 1000 nm particles were still present at the injection sites and popliteal LN. At this time, the presence of VLP was reduced, which may be due to its degradation. The transport differences of these nanoparticles indicate size-dependent entry and distribution into the lymphatic system [36].

By freely entering the afferent lymphatic vessels and the LN subcapsular sinus where they encounter B cells and resident DCs, the VLP can efficiently initiate a humoral immune response. The VLP then spreads through the small gaps (0.1–1 μm) of the sinus floor to the follicular area and interacts with naive B cells [65]. The VLP can be kept for an extended period in the germinal center by follicular DCs, leading to the clonal expansion and the development of a long-lived antibody response. Alternatively, the VLP can be processed by DCs in the paracortex region, where a substantial fraction of the DC population is immature and able to process new antigens [66,67], initiating an immune response and the development of effector mechanisms [37]. DCs are also involved in the transport of large particles from the interstitial space to the lymphatic vessels through an active mechanism involving cell adhesion molecules [38]. Larger particles confined to the interstitial space before entering the LN are susceptible to the action of phagocytic cells [36], reducing their ability to be drained or transported.

VLP represent a reliable antigen delivery system whose route of administration determines the strength of the immune response [37]. Studies of different immune routes using simian human immunodeficiency SHIV VLP (approximately 90 nm in diameter) and near-infrared (NIR) fluorescent dyes in SKH-1 hairless immunocompetent mice show significant differences in particles trafficked into LN. Five minutes after intradermal injection, the VLP can be detected in the inguinal, popliteal, lumber, and sciatic LN, remaining detectable for up to six days [37]. When the VLP reach the subcapsular sinus, a larger population of B cells is activated and migrate from the border between the T cell zone and follicle to receive proliferation signals from antigen-specific T helper cells [68]. The increase in B cell proliferation can lead to increased levels of antibody-secreting plasma cells. Hence, an increase in VLP uptake in the LN can result in a more robust immune response. Further studies using SHIV intradermal (i.d.) immunization in C57Bl/6 mice showed a healthy antibody level production, possibly due to the increase in LN involvement leading to a better overall immune response [37].

Through effective drainage, the VLP can generate antibodies with higher affinity for specific epitopes through somatic hypermutation (SHM) at the germinal center. VLP-stimulation of gene regulation, cytokine production, and antigen-specific antibody production provide important information for the development of VLP-based vaccine [69]. Intradermal vaccination seems to produce a higher activated germinal center B cells [37].

Spleen cells from C57BL/6 mice i.d. immunized with SHIV VLP were analyzed by flow cytometry for the expression of various activation markers. The results demonstrated that most B cells (B220+) were also positive for Fas and GL7 proteins, typically expressed on activated germinal center B cells. The population of B cells was also highly positive for the expression of CD80, a co-stimulatory molecule required for T cell activation and survival. When up-regulated on activated B cell, CD80 provides T cell co-stimulation through CD28 signaling. In the same experiment, the B220+PD1<sup>+</sup> population increased after VLP immunization. PD-1 is a member of the CD28/CTLA4 family that is up-regulated on activated macrophages, T cells, and B cells. This increase in the population of activated and germinal center B cells, as evidenced by the expression of specific surface markers, might explain why i.d. vaccination leads to significantly higher affinity levels of antibody production [37]. VLP immunization was also effective in inducing an expansion of CTLs, as demonstrated by a cytotoxicity assay using a simian immunodeficiency virus (SIV) gag peptide pool. CTLs are an essential component of the cellular immune response, where they induce the death of virally infected cells and tumor cells. The i.d. SHIV VLP immunization produced higher numbers of CTLs than other vaccination pathways [37].

Humoral and cell-mediated responses require the activation of multiple effector mechanisms to identify virus infected cells and tumor cells, which may not be achieved by all vaccines. Cancer vaccines rely mostly on the development of antigen-specific antibodies and on the generation of antigen-specific CTLs to recognize distinct antigens present on the cancer cell surface to eliminate them. It is well established that through cross-linking B cell receptors (BCRs), VLP activate the B cells, originating a strong activation response [70]. VLP can be modified to present a variety of epitopes, and to display B cell epitopes in a rigid, organized, and repetitive manner to effectively induce the production of neutralizing antibodies [71].
