key: cord-0694532-0b7vv2qp
authors: Anderluzzi, Giulia; Lou, Gustavo; Woods, Stuart; Schmidt, Signe Tandrup; Gallorini, Simona; Brazzoli, Michela; Johnson, Russell; Roberts, Craig W.; O'Hagan, Derek T.; Baudner, Barbara C.; Perrie, Yvonne
title: The role of nanoparticle format and route of administration on self-amplifying mRNA vaccine potency
date: 2021-12-10
journal: J Control Release
DOI: 10.1016/j.jconrel.2021.12.008
sha: 4a28df46e5f9416f72ffcca1d343a5bce73eadc8
doc_id: 694532
cord_uid: 0b7vv2qp

The efficacy of RNA-based vaccines has been recently demonstrated, leading to the use of mRNA-based COVID-19 vaccines. The application of self-amplifying mRNA within these formulations may offer further enhancement to these vaccines, as self-amplifying mRNA replicons enable longer expression kinetics and more potent immune responses compared to non-amplifying mRNAs. To investigate the impact of administration route on RNA-vaccine potency, we investigated the immunogenicity of a self-amplifying mRNA encoding the rabies virus glycoprotein encapsulated in different nanoparticle platforms (solid lipid nanoparticles (SLNs), polymeric nanoparticles (PNPs) and lipid nanoparticles (LNPs)). These were administered via three different routes: intramuscular, intradermal and intranasal. Our studies in a mouse model show that the immunogenicity of our 4 different saRNA vaccine formulations after intramuscular or intradermal administration was initially comparable; however, ionizable LNPs gave higher long-term IgG responses. The clearance of all 4 of the nanoparticle formulations from the intramuscular or intradermal administration site was similar. In contrast, immune responses generated after intranasal was low and coupled with rapid clearance for the administration site, irrespective of the formulation. These results demonstrate that both the administration route and delivery system format dictate self-amplifying RNA vaccine efficacy.

The role of mRNA vaccines in global healthcare is now well established. mRNA vaccines can be classified into modified and non-modified mRNA and self-amplifying mRNA (saRNA) vaccines. saRNA are developed from the genome of positive-stranded RNA viruses (usually alphaviruses) in which the genes encoding the viral structural proteins are replaced by the gene(s) encoding the antigen(s) of interest.

They also contain the alphavirus-based open read frame that encodes four nonstructural proteins (nsP1-4). When expressed, nsP1-4 form RNA-dependent RNA polymerase (RDRP) complexes, which enables self-amplification [1] . As a consequence, saRNA replicons enable longer expression kinetics [2] and significantly more potent immune responses [3] than non-amplifying mRNAs. However, RNAs are polyanionic and susceptible to enzymatic degradation, limiting their entry into cells, therefore, delivery systems are needed. Incorporation of RNA vaccines into nanoparticles provides RNA protection and improved delivery into cells. To date, lipid nanoparticles (LNPs) based on ionizable amino-lipids are the most advanced RNA delivery systems [4] and this technology is deployed in COVID-19 vaccines [5, 6] .

Previous studies on saRNA-LNPs suggest that the route of administration strongly influences the kinetics and magnitude of antigen expression as well as the potency of the immune response, though most studies focus on intramuscular (IM) as the preferred way to deliver both mRNA and saRNA vaccines [7] [8] [9] [10] . For example, Geall and co-workers demonstrated that the intramuscular injection of a saRNA encoding respiratory syncytial virus fusion protein (RSV-F) either unformulated or formulated within lipid nanoparticles elicited neutralizing antibody titers in both mice and rats; however, saRNA-LNPs were significantly more potent than naked saRNA [11] . It has also been reported that LNPs based on either 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) or dimethyldioctadecylammonium (DDA) and coformulated with saRNA-HIV-1 Env gp140 induced equivalent IgG antibody responses against the target protein in mice when administered intramuscularly [12] . However, antigen-specific immunity with mRNA can be achieved via several other administration routes, e.g. intravenous, intradermal (ID), subcutaneous (SC), intranodal, and intrasplenic [13] . For example, the immunogenicity of a saRNA vaccine encoding the HIV gp140 surface glycoprotein, formulated in LNPs based on the ionizable lipid DLin-DMA, was tested after administration by a variety of routes and it was shown to be more effective when administered via the IM route compared with the ID and SC routes, though the differences between IM and ID groups was not significant [11] . Similarly, IM or ID vaccination with a hemagglutinin (HA)-encoded saRNA vaccine formulated in LNPs resulted in comparable antibody and HA inhibition titers [14] . In a third study, also with an HA-mRNA-LNP vaccine, HAI titers were significantly higher J o u r n a l P r e -p r o o f following ID vaccination compared to IM two weeks after the boost, but equivalent at later time points [15] .

However, consideration of alternative routes for vaccination may offer opportunities. For example, the derma skin layer is abundant in professional antigen presenting cells e.g. dendritic dermal cells and Langerhans cells [16] which can enhance encoded antigen transportation to the lymph nodes and induce protective immune responses. Thus, intradermal administration may facilitate lower vaccine doses (dose-sparing) thereby reducing costs (including transport and storage) and expanding the supply chain.

Indeed, the potential of dermal non-viral delivery of saRNA vaccines was reported previously [17] ; the skin is extremely immune competent, easily accessible and drugs can be administered by means of needle-free devices, thus improving patient compliance, reducing the risk of needle-stick injures and reducing clinical waste. Intranasal (IN) vaccination is another needle-free, noninvasive administration route for vaccines. The nasal cavity is embedded with a high density of dendritic cells that can mediate strong systemic and local immune responses against pathogens [18] . The uptake of nasally administered vaccines is mediated by M cells, which can transport particulate antigens to the nasal lymphoid tissue by transcytosis. Nasal vaccination induces both systemic and mucosal immunity in the respiratory and genital tracts by the release of IgA into the nasal passage and intestinal tract. This administration route is adopted by AstraZeneca's FluMist (a live-attenuated influenza virus vaccine approved for human use) and has been investigated for the delivery of an mRNA-based HIV vaccine, with strong systemic and mucosal anti-HIV immune responses as well as cytokine productions being achieved [19] .

Whilst both intradermal and intranasal administration offers potential advantages, there is limited understanding on RNA vaccine efficacy when given via these routes compared with the conventional intramuscular route. Therefore, the aim of this study was to compare the efficacy of self-amplifying mRNA vaccines when delivered using 4 different delivery platforms and via the intramuscular, intradermal or intranasal route. Building on our pervious studies, where we show that lipid nanoparticles (LNPs), solid lipid nanoparticles (SLNs) and polymeric nanoparticles (PNPs) based on commercially available cationic lipids such as 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) efficiently deliver self-amplifying mRNA vaccines in mice [20, 21] , we investigate the role of administration route on the immunogenicity elicited. To compare their performance across different delivery routes, the same formulations were tested across the different routes. An saRNA encoding the rabies virus glycoprotein (RVG) was used, as commercial vaccines can be tested as benchmarks and immunological correlates of protection are well-established [22, 23] . Plasmid Maxi kits (Qiagen, Germantown, MD, USA). DNA was linearized following the 3' end of saRNA sequence by restriction digest. Linearized DNA templates were transcribed into RNA using a MEGAscript T7 kit (Life Technologies, Carlsbad, CA, MA, USA) and purified by LiCl precipitation. RNA was then capped using the Vaccinia Capping system (New England BioLabs, Ipswich, MA, USA) and purified by LiCl precipitation before formulation.

DOTAP-based formulations were prepared and characterized as previously described [20, 21] . In essence, 

Sera from individual mice were collected four weeks after first vaccination (day 28) and two weeks after second vaccination (day 42) and combined in five pools of two mice each. Total anti-RVG IgG titers were quantified with the PLATELIA RABIES II Kit Ad Usum Veterinarium [22] following manufacturer instructions.

Spleens from 3 randomly selected mice from each experimental group were collected on day 42 (two weeks after second vaccination). Single cell suspensions were obtained as described elsewhere [24] .

Cells were then incubated with RBC lysis buffer (2 mL) at 4 °C for 2 minutes, resuspended in complete RPMI (cRPMI) and passed again through cell strainers. Cells were counted in a Vi-CELL XR cell counter 

Statistical analysis of T cell responses and biodistribution experiments was performed by one-way analysis of variance (ANOVA) followed Tukey's honest significance test. Statistical analysis of IgG titers was performed by Kruskal-Wallis followed by Dunn's test. P values below 0.05 (*) were considered significant. All analyses were done in GraphPad Prism 7.0.

We have previously reported the microfluidic production of several nanoparticles based on the commercially available cationic lipid DOTAP [20, 21] . The use of microfluidics in the manufacturing process supports process driven size control and scale-independent production [26, 27] . Within this study, we selected three different nanoparticle formats (LNPs, PNPs and SLNs) to further investigate the role of administration route on self-amplifying RNA vaccine performance (Figure 1 ). Whilst cationic LNPs tend to display bilayer-like structures [28] , PNPs consisting of a polymer core and SLNs have a lipid monolayer surrounding the polymer core [29] . These formulations were selected based on previous studies which demonstrated these formulations were capable of associating with cells, inducing antigen expression in vitro and protecting SaRNA against enzymatic degradation [20, 21] . The same formulations were used across the different delivery routes to allow direct comparison. Our particles were from 65 to 135 nm in size, with low PDI (<0.2), near neutral zeta potential, except for the PNPs which were cationic in nature, and high saRNA encapsulation efficiency (>95%) (Figure 1 B-D) . Particle size has been suggested to play a role in the immunogenicity of mRNA vaccines in mice [30] . However more recent studies suggest this may only be a feature of small animal studies [31] ; a retrospective analysis of mRNA LNP vaccine in vivo studies revealed a relationship between LNP particle size and immunogenicity in mice using LNPs of various compositions. Nevertheless, whilst small diameter LNPs were substantially less immunogenic in mice, all particle sizes tested yielded a robust immune response in non-human primates [31] .

J o u r n a l P r e -p r o o f Therefore, using the formulations outlined in Figure 1 , we assessed the impact of administration route on saRNA vaccine efficacy when delivered using the different nanoparticle formats. Mice were vaccinated twice, four weeks apart, with RVG-saRNA formulated in either SLNs, PNPs, cLNPs or benchmark iLNPs [33] and delivered intramuscularly (IM), intradermally (ID) or intranasally (IN). Control groups were vaccinated with or Rabipur, an inactivated rabies virus vaccine. The selected doses were based on our previous findings with these delivery systems [20, 21] (Table 1) . 5% HD 50 µL IgG responses were measured, prior to immunization, 4 weeks post first injection (day 28), 2 weeks after the second injection (day 42) and 10 weeks after the second injection (day 98). No anti-RVG IgGs were detected in mice sera prior to immunization (data not shown). Four weeks after the first injection, there was no significant difference between the IgG responses promoted by the 4 different nanoparticle formulations (SLNs, PNPs, cLNPs, iLNPs) when administered IM. All 4 nanoparticle formulations induced strong antigen-specific IgG titers above the correlate of protection of 0.5 EU/mL and these responses were significantly (p<0.05) higher than the control vaccine (Rabipur) (Fig. 2A) . When the mice were dosed ID, generally a similar response profile was shown with antigen-specific IgG titers above the correlate of protection of 0.5 EU/mL. However, PNPs promoted significantly lower responses compared to the SLNs, cLNPs and the iLNPs benchmark ( Fig. 2A) . After IN administration there was no notable IgG responses measured, with IgG titers below the limit of quantification in all but three samples, despite mice receiving a 10 folder higher dose via this route ( Fig. 2A) (Fig.2B) . Comparing between the nanoparticle formulations, with an IM booster injection, iLNPs produced significantly (p<0.05) higher IgG responses compared to the three DOTAP formulations (SLNs, PNPs, cLNPs). When a second dose was administered ID, there is no difference between SLNs, cLNPs and iLNPs. However, PNPs promoted significantly (p<0.05) lower IgG responses compared to iLNPs (Fig. 2B) . Again, the immune responses induced upon IN immunization were significantly weaker compared to IM or ID immunization for all of the formulations tested with J o u r n a l P r e -p r o o f only the iLNPs promoting an average response above the correlate of protection (Fig. 2B) . Overall, after the second immunization, iLNPs administered IM promoted the strongest IgG responses (Fig. 2B ).

This pattern of immune response was also seen 10 weeks post second immunization, demonstrating the ability of these nanoparticle formulations to induce persistent humoral immunity above the correlate of protection (Fig.2C) . When administered IM, iLNPs continued to promote significantly (p < 0.05) higher

IgG titers compared to the SLNs, PNPs and cLNP formulations. When administered ID, there was no significant different between the 4 different nanoparticle formulations but a similar trend of higher responses from iLNPs was seen (Fig. 2C) . Comparing between the routes of administration at this timepoint, IM and ID gave similar response profiles yet when administered IN, only the iLNPs promoted a notable IgG response with all responses above the correlate of protection (Fig. 2C ).

The results in Fig. 2 are in line with recent studies of Blakney and co-workers, who reported equivalent antibody production in mice vaccinated either IM or ID with saRNA formulated within poly(CBA-co-4amino-1-butanol) (ABOL)-based nanoparticles at different doses [34] . Although all formulations elicited antibodies titers above the level of protective response to rabies vaccination reported by WHO [35] , LNPs and SLNs were generally more potent than PNPs two weeks after the second vaccination, and overall iLNPs gave the highest long term response via both the IM and ID routes. In our previous studies [20, 21] To study the immune response profiles further, cytokine responses were also measured. The saRNAnanoparticles formulations induced multifunctional RVG-specific cellular immune responses two weeks after the second vaccination (Fig. 3) . Generally, LNPs injected either IM or ID induced the highest frequencies of cytokines-producing RVG-specific splenic CD4+ and CD8+ T cells (Fig. 3) . Similar to the IgG profiles, the frequencies of cytokine-producing CD8+ T cells in mice which received iLNPs were greater than the other formulations after IM (Fig. 3A) . When administered ID, there profiles are similar for the SLNs, cLNPs and iLNPs whilst the responses induced by the PNPs are low (Fig. 3A) . The majority of RVGspecific CD8+ T cells expressed IFN-γ in combination with TNF-α and/or IL-2, irrespective of the route of administration, and this is generally associated with a mature effector phenotype. The strong proliferation of CD8+ T cells triggered by saRNA vaccines is consistent with previous studies which J o u r n a l P r e -p r o o f demonstrated that saRNA formulated with LNPs injected IM induced antigen expression within muscle cells and its consequent presentation to APCs, suggesting cross-priming as the prevalent mechanism for CD8+ T-cell response activation by saRNA vaccines [37] . Similar to the IgG profiles, the frequencies of cytokine expression were low in mice vaccinated IN (Fig. 3A) . A similar trend was observed in the expression of the degranulation marker CD107a (Fig. 3B) , whose expression correlates with the cytotoxic activity of CD8+ T cells in vivo [38, 39] . In mice vaccinated IM, the frequencies of CD107a+ CD8+ T cells were highest with the iLNPs, whilst after ID, the responses induced by iLNPs reduced and were comparable with the cLNPs and SLNs (Fig. 3B) . After IN administration, only negligible percentages of CD107a+ CD8+ T cells were quantified (<0.1%, Fig. 3B ). With respect to the CD4+ T cell responses, again a similar profile of responses is seen (Fig. 3C) ; after IM injection iLNPs promote the highest responses in mice, whilst after ID these responses reduce and are similar to SLNs and cLNPs (Fig. 3C) . However, SLNs administered via the IN route, promoted responses in line with the responses promoted by SLNs given IM and ID (Fig. 3C) .

The CD4+ T cells proliferation induced by RNA vaccines is likely to be related to the rapid activation of lymphatic cells. For example, Liang and colleagues [15] showed that mRNA-LNPs administered either intradermal or intramuscular in rhesus macaques specifically targeted APCs located both at the injection site and in draining lymph nodes, leading to antigen translation and upregulation of type I IFN-inducible genes. This rapid innate immunity induced priming of antigen-specific CD4+ T cells and generation of vaccine-specific immunity solely in the draining lymph nodes. Similar observations were also reported elsewhere [40] . The relative frequency of CD8+ and CD4+ T cells quantified for each formulation and route of administration (Fig. 3 ) was also consistent with the production of antibodies reported in Fig. 2 .

A combination of Th0 (IL-2+/TNF-α+, TNF-α+, or IL-2+) and Th1 (IFN-γ+ alone or in combination with IL-2+ and/or TNF-α+) phenotypes was observed in CD4+ T-cells 2 weeks after the second immunization in all groups (Fig. 3C) . Interestingly, ID injection of SLNs resulted in the highest frequencies of polyfunctional antigen-specific CD4+ T cells. The potential of ID vaccination has been widely established in many clinical trials, although results are not always consistent among different vaccines. For example, dermal injection of lower doses of a virus-inactivated influenza vaccine resulted in equivalent immunogenicity to the standard dose delivered intramuscularly [41] . With respect to the rabies virus, post-exposure IM or ID vaccination with Rabipur resulted in similar neutralizing antibody titers in humans but ID was slightly lower compared to IM in a pre-exposure prophylaxis regime [42] . Conversely, with hepatitis B vaccine, the benefit of dose-sparing was not fully evident [43] . Figure S1 in the supplemental material for the gating strategy.

Intranasally administered vaccines have the potential to induce persistent lung effector T cells, which could significantly benefit host immunity against respiratory pathogens [24] . Therefore, to further investigate this, we performed a T cell assay in lung cells from mice immunized IN. iLNPs and SLNs elicited higher frequency of RVG-specific CD8+ T cells compared to LNPs, PNPs and Rabipur when administered IN. Furthermore, both formulations gave comparable responses to Rabipur administered IM (Fig. 4A) . Interestingly, the quality of CD8+ T cell responses in the lungs varied among tested formulations: SLNs and PNPs induced polyfunctional CD8+ IFN-γ+ and TNF-α+/IL-2+ cells, while those elicited by cLNPs were IFN-γ/TNF-α+ and IFN-γ+/IL-2+ and those elicited by iLNPs were γ/TNF-α+, IFN-γ+/IL-2+ and IFN-γ+ (Fig. 4A) . However, the majority of RVG-specific CD8+ T-cells were CD107a- (Fig. 4B) irrespective of the nanoparticle formulation used, which correspond to a non-cytotoxic profile.

Regarding CD4+ T cells, the frequencies of RVG-specific cells were comparable between SLNs, cLNPs and iLNPs groups (around 0.2%); however, again the profiles were different with the iLNPs promoting more J o u r n a l P r e -p r o o f Journal Pre-proof TNF-α+ cells (Fig. 4C) . As observed in splenic CD4+ T-cells, cell profile was a combination of Th0/Th1 phenotypes, with SLNs inducing a higher frequency of Th1 cells than LNPs and PNPs respectively (Fig.   4C ). These differences in T cell responses may be attributed to differences in the nanoparticle chemical composition and/or mRNA delivery profile. For example, fatty acids are known to modulate cytokines secretion from activated T cells and the effect is dependent on both the saturation degree and length of fatty acid [44, 45] . In particular, it was reported that saturated fatty acids induced significantly higher release of pro-inflammatory cytokines in T cells than their unsaturated counterparts, possibly due to increased formation of free radicals, diacyl glycerol and activation of protein kinase C [46] . Figure S2 in the supplemental material for the gating strategy.

Several studies have suggested that the administration route of mRNA vaccines strongly influences the kinetics of antigen expression [47] . For example, in a study conducted with mRNA encoding luciferase formulated in LNPs, the half-life of antigen expression in mice was ranked in the order of intradermal >> intramuscular > intraperitoneal and subcutaneous >> intratracheal > intravenous [47] . Although antigen expression, biodistribution and immunogenicity are expected to be closely related, a defined correlation J o u r n a l P r e -p r o o f remains unclear. Indeed, we have previously shown that both cLNPs and iLNPs are retained at the injection site following intramuscular injection for up to 10 days [21] . Here, we compared the pharmacokinetics of saRNA-SLNs, PNPs and LNPs administered via IM, ID or IN in an effort to further understand the importance of the delivery route for effective mRNA vaccines.

When considering the biodistribution of the different nanoparticle formulations (Fig. 5 and 6) , full body images of mice which received saRNA-nanoparticles via intramuscular or intradermal injection showed that the signal was mainly concentrated at the site of injection (Fig. 5 ). Long-term retention of all four nanoparticle formulations at the injection site was also observed after both IM (Fig. 6A) and ID (Fig. 6B) administration, with the area under the curve (AUC; calculated using the trapezoidal method) confirming that the drainage profile of the nanoparticles was comparable (Fig. 6D) . With respect to IN vaccinated groups, whole body images showed poor retention of all nanoparticles (Fig. 5) ; most of the administered dose was detected in the throat and stomach at 4 hours post administration (Fig. 5) suggesting that part of the vaccine dose had been rapidly swallowed and cleared a few hours after administration, irrespective of the nanoparticle format ( Fig. 6C and 6D) . The rapid clearance of the nanoparticles from the administration site after IN vaccination correlated with the weaker humoral and cellular immune response observed. This may result from ineffective interactions between the nanoparticles and mucosal tissue upon administration due to a lack of muco-adhesive/ mucopenetrating excipients within the nanoparticle formulations. The presence of muco-adhesive or mucopenetrating polymers (e.g. poly(acrylic acid) (PAA), alginate, cellulose derivatives, chitosan, poloxamers and poly(ethylene glycol) (PEG)) on the surface of particles can enhance the concentration of therapeutics delivered to the mucus mesh [48] . Furthermore, the weak potency of vaccines administered IN may also be linked to the unavoidable limitation of the animal model used; intranasal vaccination in small animals may trigger inhalation and ingestion of vaccine antigens, which consequently affects vaccine dosage [49] .

By comparing the retention of formulations at the injection site, we did not observe notable differences in clearance between the four saRNA-nanoparticle formulations from either the IM or ID administration, despite the formulations inducing different humoral and cellular responses ( Fig. 2 and 3 ). This suggests that other factors may contribute to the immunogenicity of SaRNA vaccines. These findings are in agreement with previous investigations which showed poor correlation between pharmacokinetics and immunogenicity [30] . Accumulation and trafficking of immune cells transporting the encoded antigen to J o u r n a l P r e -p r o o f the draining lymph nodes as well as the mode of antigen delivery to lymphoid tissue might also be involved in the immunostimulatory mechanism of mRNA and saRNA vaccines [40] . The slow clearance of the nanoparticles from the injection site could be due to active uptake by host cells via association with endogenous ligands (e.g. ApoE) and recognition by scavenger receptors and the low-density lipoprotein receptor [50] . ApoE easily associates with the surface of neutral lipid-based particles, resulting in enhanced ApoE-mediated cellular uptake [51] . As these receptors are ubiquitously expressed in all nucleated cells [52] , this active targeting could augment nanoparticle retention at the injection site. 

In this study, we demonstrate that the immunogenicity of our saRNA vaccines for a given delivery route was affected by the format of the nanoparticles. saRNA encapsulated within SLNs and LNPs tending to be more potent than PNPs after administration via the intramuscular or intradermal route and immune responses from these routes were similar. The clearance of all four saRNA nanoparticle formulations from either the IM or ID administration site was also similar. In contrast, immune responses generated after intranasal administration was low (despite receiving a 10-fold higher dose) and coupled with rapid clearance for the administration site irrespective of the formulation, suggesting that further optimization of these systems for this route is required. 

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We wish to thank the saRNA Vaccine Platform Team at GSK Rockville. We also thank the staff at the Animal Research Center and at the Flow-Cy-TOF Core Facility at GSK Siena and the Biological Procedures Unit at University of Strathclyde for technical assistance.