key: cord-0280477-vjr0pnw9 authors: Francis, David M.; Chen, Runqiang; Khorsandzadeh, Sahba; Hu, Qidong; Lyu, Xiaoxuan; Wang, Hua; Lim, Wan-lin; Sun, Haotian; Xie, Hui; Shaabani, Namir; Ross, Russell; Cooley, Brian; Ji, Henry title: Directing an mRNA-LNP vaccine toward lymph nodes improves humoral and cellular immunity against SARS-CoV-2 date: 2021-08-25 journal: bioRxiv DOI: 10.1101/2021.08.25.457699 sha: 0ca34045229ae7f2d169adea656fcc3a777878c1 doc_id: 280477 cord_uid: vjr0pnw9 The exploration and identification of safe and effective vaccines for the SARS-CoV-2 pandemic has captured the world’s attention and remains an ongoing issue in order to protect against emerging variants of concern (VoCs) while generating long lasting immunity. Here, we report the synthesis of a novel messenger ribonucleic acid (mRNA) encoding the spike protein in a lipid nanoparticle formulation (LNP) (STI-7264) that generates robust humoral and cellular immunity following immunization of C57Bl6 mice. In efforts to continually improve immunity, a lymphatic drug delivery device (MuVaxx) was engineered and tested to modulate immune cells at the injection site (epidermis and dermis) and draining lymph node (LN) to elicit adaptive immunity. Using MuVaxx, immune responses were elicited and maintained at a 10-fold dose reduction compared to traditional intramuscular (IM) administration as measured by anti-spike antibodies, cytokine producing CD8 T cells, and neutralizing antibodies against the Washington (Wild Type, WT) and South African (beta) variants. Remarkably, a 4-fold elevated T cell response was observed in MuVaxx administered vaccination as compared to that of IM administered vaccination. Thus, these data support further investigation into STI-7264 and lymphatic mediated delivery using MuVaxx for SARS-CoV-2 and VoCs vaccines. The SARS-CoV-2 virus has accounted for more than 210 million cases of the coronavirus disease 2019 (COVID-19) and over 4.4 million fatalities worldwide since its original outbreak in December 2019. This is the 3 rd outbreak of a Betacoronavirus since 2002, the SARS-CoV and Middle East respiratory syndrome coronavirus (MERS-CoV) being its predecessors, and is much more efficiently transmitted person to person. While vaccine efforts have greatly hampered the spread of disease, questions about their durability and protection against emerging VoCs remain. 1, 2 Thus, there is an urgent need to improve vaccine durability and efficacy to emerging VoCs, while balancing costs, stability, and manufacturing speed to scale up for world-wide vaccination efforts. There are currently two mRNA-based SARS-CoV-2 vaccines which have been authorized and widely disseminated. These vaccines encode for the spike (S) protein, which is the major surface protein on the coronavirus virion and are thus the primary target for neutralizing antibodies (nAb) as the S protein facilitates viral entry into host cells via interactions with the angiotensin-converting enzyme 2 (ACE2) receptor expressed in the upper and lower respiratory tract. The primary metric for SARS-CoV-2 vaccine efficacy has been on generating nAbs to prevent viral entry into host cells and promote viral clearance before replication. In addition to humoral immunity, an ideal vaccine would also generate cellular immunity as T cell immunity has been associated with less severe disease, 3, 4 faster recovery, 4 and memory persistence for decades. 5 To date, most vaccines are administered IM due to feasibility for healthcare workers, speed of injection, and immunological properties (i.e. muscle resident lymphocytes and antigen presenting cells). However, directing vaccines toward the dermis and draining LNs has been of interest for decades due to the high concentration of antigen presenting cells (APCs) including Langerhan cells that reside in the skin (epidermis and dermis) that are capable of taking up antigen and subsequently trafficking to draining LNs to elicit adaptive immunity. 6, 7 Moreover, the initial lymphatics are present at high concentrations just below the stratum corneum and provide direct access to draining LNs due to their high permeability and uni-directional flow towards draining LNs. 8, 9 Delivering vaccines directly to LNs provides a promising opportunity for improving vaccine efficacy as 1) lymphocytes reside in LNs at high concentrations, 10 2) are home to unique and strategically positions APCs that present incoming antigen to T and B cells rapidly to induce immunity, 11, 12 and 3) memory T and B cells reside in LNs during their lifespans. 13, 14 To address the SARS-CoV-2 pandemic and need for effective vaccines, we engineered a novel mRNA construct encoding the S protein in a lipid nanoparticle formulation (LNP) referred to as STI-7264 and interrogated the immunological response using a traditional intramuscular (IM) injection compared to our proprietary Sofusa MuVaxx Lymphatic Drug Delivery Platform (MuVaxx). We first explored the ability of MuVaxx to deliver antigen to LNs to induce an improved immunological response by vaccinating against a model antigen, Ovalbumin (OVA) in a preclinical mouse model compared to an IM injection. We next compared STI-7264 against a marketed reference mRNA-LNP (Reference) vaccine for comparison and characterized the humoral and cellular response in a preclinical mouse model measuring circulating anti-S antibodies and peripheral cytokine producing T cells. Our STI-7264 formulation led to similar antibody production compared to the Reference vaccine, however the CD8 T cell response was dramatically improved. MuVaxx administration of STI-7264 enabled an approximately 10-fold reduction in dose needed compared to an IM injection while maintaining similar B cell immunity and an elevated T cell immunity. Thus, STI-7264 and MuVaxx represent an intriguing approach for vaccinating against SARS-CoV-2 and its VoCs as well as moving forward to clinical applications. The vaccine is comprised of an active drug substance, a single-stranded mRNA encoding for the full-length SARS-CoV-2 S glycoprotein encapsulated in lipid nanoparticles (LNPs). The sequence was derived from the strain "Severe acute respiratory syndrome coronavirus 2 isolate Wuhan-Hu-1". Mutations were introduced into S protein to substitute residues 986 and 987 to produce prefusion-stabilized SARS-CoV-2 S(2P) protein. 15 To achieve optimal expression in humans, the sequence was further codon-optimized and cloned into a pVAX1-based backbone that contains T7 promoter, 5′-UTR, 3′-UTR and optimized Poly-A tail with minimal overhang. The template was then linearized immediately downstream of the Poly-A tail and used for in vitro transcription (IVT) ( Figure 1A ). To facilitate mRNA expression and reduce innate immune response, during the IVT, Cap 1 structure was added to the 5' terminus of the RNA co-transcriptionally by CleanCap ® AG, and UTP completely replaced by N1-methylpseudo-UTP. This process can be readily scaled up to produce desired amounts of capped mRNA. To confirm the expression, the IVT mRNA was introduced into monocyte-derived dendritic cells (DC) by electroporation. Twenty-four hours post-transfection, the cells were collected and stained with anti-Spike antibody STI-2020 and detected with allophycocyanin-conjugated anti-human Fc antibody. The flow cytometry showed that the codon-optimized Cap 1 mRNA can be efficiently translated into prefusion-stabilized spike protein in the primary DC ( Figure 1B ). To explore the potential of delivering vaccine constituents to draining LNs, we utilized MuVaxx, our lymphatic drug delivery device, which is connectable to any luer lock syringe ( Figure 2A) , and which consists of microneedles that puncture the stratum corneum and release drug at the epidermal/dermal boundary. To evaluate LN delivery using MuVaxx, we used Indocyanine Green (ICG) which can be visualized in vivo with near-infrared fluorescence (NIRF). Following injection, we observed ICG accumulation within minutes in the draining brachial LN ( Figure 2B ). We then explored the potential of MuVaxx to augment the immunogenicity of a model antigen, specifically Ovalbumin (OVA) in mice. Mice were injected with OVA and an oligonucleotide adjuvant (CpG) on days 0 and 14 using an IM or MuVaxx administration. All mice treated with MuVaxx generated anti-OVA IgG by day 13 compared to 4 of 8 in the IM cohort. Additionally, following the booster shot (day 14), anti-OVA IgG was measured on day 34 and mice treated with MuVaxx displayed significantly higher titers compared to mice treated IM resulting in over a 60-fold increase in titers ( Figure 2C ). The cellular immune response was additionally measured in both cohorts of mice looking at cytokine production in CD8 T cells following the booster shot on days 20 and 28. Interferon-Gamma (IFNγ) and Tumor Necrosis Factor-Alpha (TNFα) were assessed following ex vivo stimulation with SIINFEKL, an OVA derived class I peptide. Mice treated with MuVaxx displayed higher proportions of cytokine producing CD8 T cells compared to naïve mice on both days 20 and 28 ( Figure 2D ). Taken together, these results highlight the potential of MuVaxx to deliver cargo to draining LNs to improve immunity of vaccines. To evaluate the potential of MuVaxx to enhance immunity, we compared the humoral To assess the Th1/Th2 bias elicited after immunization, whole blood was collected to measure cytokine producing CD4 T cells 6 days after the booster shot via intracellular cytokine staining (ICS) and IgG subclass titers from day 49 serum ( Figure 4A ). The ratio of Th1 (CD3 + CD4 + IFNγ + ) to Th2 (CD3 + CD4 + IL4 + ) T cells favored a Th1 response and was similar between all cohorts, although the 10 μg STI-7264 IM group and 1 μg STI-7264 MuVaxx groups had slightly higher ratios favoring an enhanced anti-viral immune response ( Figure 4B ). Similarly, the ratio of IgG2c to IgG1 was skewed towards IgG2c for the 10 μg STI-7264 IM group and 1 μg STI-7264 MuVaxx cohorts suggesting bias towards a Th1 response ( Figure 4C ) in line with the CD4 T cell cytokine phenotypes. MuVaxx. In addition to CD4 T cells, the responses in the CD8 T cell compartment were evaluated on day 49 following incubation with spike associated peptides overnight via ICS ( Figure 5A ). Mice vaccinated with the Reference vaccine displayed a minor increase in cytokine producing CD8 T cells ( Figure 5B and C), in line with previous literature 16 . However, the STI-7264 10 µg formulation when administered IM led to a robust antigen specific CD8 T cell response as measured by IFNγ and TNFα. The response was dose dependent as IM administration of 1 µg of STI-7264 led to a minimal CD8 T cell response. Interestingly, when 1 µg of STI-7264 was administered via MuVaxx toward draining LNs, the CD8 T cell response was restored to similar levels to that of a 10 µg IM dose ( Figure 5B and C). Overall, these results highlight the improved CD8 T cell response observed with this STI-7264 formulation along with the benefit of directing spike encoding mRNA towards draining LNs to generate CD8 T cell immunity. To asses nAb generation, a Plaque Reduction Neutralization Test (PRNT) was performed in vitro, where VeroE6 cells were exposed to the live virus in the absence or presence of diluted mouse serum. PRNT detects plaque formation and is indication of cell infection by the SARS-CoV-2 virus whereas the absence of plaque formation represents nAb presence. Each cohort of treatments led to nAb generation by d49 against the WT strain ( Figure 6A ). To investigate protection against the Beta variant, VeroE6 cells were incubated with this strain of the virus. The Reference vaccine, STI-7264 10 µg IM, and STI-7264 1 µg MuVaxx cohorts displayed robust protection against this strain up to a 1:120 dilution whereas the lower 1 µg STI-7264 IM cohort of mice displayed much lower protection ( Figure 6B ). Taken together, these results show that STI-7264 generates nAbs and that lymphatic mediated delivery via MuVaxx can broaden protection at 1/10 th the IM dose. Here, we report a novel mRNA-based SARS-CoV-2 vaccine, STI-7264, that induces similar humoral immunity with elevated cellular immunity compared to a Reference vaccine formulation when administered IM. Immunity generated with the STI-7264 formulation was dose dependent as immunity was reduced when going from 10 to 1 µg IM. Interestingly, when administering the same 1 µg STI-7264 formulation via MuVaxx, dose sparing effects were observed where both humoral and cellular immunity were similar to that of a 10 µg IM dose highlighting the improved immunogenicity when directing vaccines towards LNs. Previous mRNA-based vaccines have reported dose-dependent side effects with higher doses linked to systemic and local adverse events 17, 18 underscoring an additional advantage of lower dose formulations; lessening side effects while expanding vaccine access to large populations. A vaccine that generates durable immunity is another hallmark of an effective vaccine and is a metric we investigated. The serum concentrations of both anti-S1 and anti-RBD IgG waned to a lesser degree in mice treated with MuVaxx relative to those treated with the same dose IM highlighting improved durability. This is of interest as recent reports prevention. 3, 4 Additionally, T cell responses against previous betacoronaviruses can persist for decades 5, 24 and display cross-reactivity against other betacoronaviruses [25] [26] [27] highlighting potential for long term protection and coverage against variants. In this work, we show STI-7264 delivered via MuVaxx elicits a strong CD8 T cell response towards SARS-CoV-2 peptides which may be advantageous for preventing COVID-19 and providing protection against re-infection. The CD4 Th2 phenotype has previously been associated with vaccine-associated enhanced respiratory disease (VAERD) in those vaccinated against the measles-and respiratory syncytial-virus. 28, 29 We therefore explored the CD4 Th1 vs Th2 response and Statistics. Statistical significance of differences between experimental groups was determined with Prism software (Graphpad). All data are expressed as standard error mean (SEM). ****P < 0.0001, ***P < 0.001, **P < 0.01, and *P < 0.05 by unpaired twotailed t tests or one-or two-way analysis of variance (ANOVA). Anti-S1 IgG (ng/mL) ** ** Durability of mRNA-1273-induced antibodies against SARS-CoV-2 variants. 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