Development of an mRNA-lipid nanoparticle vaccine against Lyme disease.

Lyme disease is the most common vector-borne infectious disease in the United States, in part because a vaccine against it is not currently available for humans. We propose utilizing the lipid nanoparticle-encapsulated nucleoside-modified mRNA (mRNA-LNP) platform to generate a Lyme disease vaccine like the successful clinical vaccines against SARS-CoV-2. Of the antigens expressed by Borrelia burgdorferi, the causative agent of Lyme disease, outer surface protein A (OspA) is the most promising candidate for vaccine development. We have designed and synthesized an OspA-encoding mRNA-LNP vaccine and compared its immunogenicity and protective efficacy to an alum-adjuvanted OspA protein subunit vaccine. OspA mRNA-LNP induced superior humoral and cell-mediated immune responses in mice after a single immunization. These potent immune responses resulted in protection against bacterial infection. Our study demonstrates that highly efficient mRNA vaccines can be developed against bacterial targets.

Single immunizations of self-amplifying or non-replicating mRNA-LNP vaccines control HPV-associated tumors in mice.

As mRNA vaccines have proved to be very successful in battling the coronavirus disease 2019 (COVID-19) pandemic, this new modality has attracted widespread interest for the development of potent vaccines against other infectious diseases and cancer. Cervical cancer caused by persistent human papillomavirus (HPV) infection is a major cause of cancer-related deaths in women, and the development of safe and effective therapeutic strategies is urgently needed. In the present study, we compared the performance of three different mRNA vaccine modalities to target tumors associated with HPV-16 infection in mice. We generated lipid nanoparticle (LNP)-encapsulated self-amplifying mRNA as well as unmodified and nucleoside-modified non-replicating mRNA vaccines encoding a chimeric protein derived from the fusion of the HPV-16 E7 oncoprotein and the herpes simplex virus type 1 glycoprotein D (gDE7). We demonstrated that single low-dose immunizations with any of the three gDE7 mRNA vaccines induced activation of E7-specific CD8+ T cells, generated memory T cell responses capable of preventing tumor relapses, and eradicated subcutaneous tumors at different growth stages. In addition, the gDE7 mRNA-LNP vaccines induced potent tumor protection in two different orthotopic mouse tumor models after administration of a single vaccine dose. Last, comparative studies demonstrated that all three gDE7 mRNA-LNP vaccines proved to be superior to gDE7 DNA and gDE7 recombinant protein vaccines. Collectively, we demonstrated the immunogenicity and therapeutic efficacy of three different mRNA vaccines in extensive comparative experiments. Our data support further evaluation of these mRNA vaccines in clinical trials.

CAR T cells produced in vivo to treat cardiac injury.

Fibrosis affects millions of people with cardiac disease. We developed a therapeutic approach to generate transient antifibrotic chimeric antigen receptor (CAR) T cells in vivo by delivering modified messenger RNA (mRNA) in T cell­targeted lipid nanoparticles (LNPs). The efficacy of these in vivo­reprogrammed CAR T cells was evaluated by injecting CD5-targeted LNPs into a mouse model of heart failure. Efficient delivery of modified mRNA encoding the CAR to T lymphocytes was observed, which produced transient, effective CAR T cells in vivo. Antifibrotic CAR T cells exhibited trogocytosis and retained the target antigen as they accumulated in the spleen. Treatment with modified mRNA-targeted LNPs reduced fibrosis and restored cardiac function after injury. In vivo generation of CAR T cells may hold promise as a therapeutic platform to treat various diseases.

Lipid nanoparticles enhance the efficacy of mRNA and protein subunit vaccines by inducing robust T follicular helper cell and humoral responses.

Adjuvants are critical for improving the quality and magnitude of adaptive immune responses to vaccination. Lipid nanoparticle (LNP)-encapsulated nucleoside-modified mRNA vaccines have shown great efficacy against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), but the mechanism of action of this vaccine platform is not well-characterized. Using influenza virus and SARS-CoV-2 mRNA and protein subunit vaccines, we demonstrated that our LNP formulation has intrinsic adjuvant activity that promotes induction of strong T follicular helper cell, germinal center B cell, long-lived plasma cell, and memory B cell responses that are associated with durable and protective antibodies in mice. Comparative experiments demonstrated that this LNP formulation outperformed a widely used MF59-like adjuvant, AddaVax. The adjuvant activity of the LNP relies on the ionizable lipid component and on IL-6 cytokine induction but not on MyD88- or MAVS-dependent sensing of LNPs. Our study identified LNPs as a versatile adjuvant that enhances the efficacy of traditional and next-generation vaccine platforms.

Highly efficient CD4+ T cell targeting and genetic recombination using engineered CD4+ cell-homing mRNA-LNPs.

Nucleoside-modified messenger RNA (mRNA)-lipid nanoparticles (LNPs) are the basis for the first two EUA (Emergency Use Authorization) COVID-19 vaccines. The use of nucleoside-modified mRNA as a pharmacological agent opens immense opportunities for therapeutic, prophylactic and diagnostic molecular interventions. In particular, mRNA-based drugs may specifically modulate immune cells, such as T lymphocytes, for immunotherapy of oncologic, infectious and other conditions. The key challenge, however, is that T cells are notoriously resistant to transfection by exogenous mRNA. Here, we report that conjugating CD4 antibody to LNPs enables specific targeting and mRNA interventions to CD4+ cells, including T cells. After systemic injection in mice, CD4-targeted radiolabeled mRNA-LNPs accumulated in spleen, providing ∼30-fold higher signal of reporter mRNA in T cells isolated from spleen as compared with non-targeted mRNA-LNPs. Intravenous injection of CD4-targeted LNPs loaded with Cre recombinase-encoding mRNA provided specific dose-dependent loxP-mediated genetic recombination, resulting in reporter gene expression in about 60% and 40% of CD4+ T cells in spleen and lymph nodes, respectively. T cell phenotyping showed uniform transfection of T cell subpopulations, with no variability in uptake of CD4-targeted mRNA-LNPs in naive, central memory, and effector cells. The specific and efficient targeting and transfection of mRNA to T cells established in this study provides a platform technology for immunotherapy of devastating conditions and HIV cure.

Lipid nanoparticle encapsulated nucleoside-modified mRNA vaccines elicit polyfunctional HIV-1 antibodies comparable to proteins in nonhuman primates.

The development of an effective AIDS vaccine remains a challenge. Nucleoside-modified mRNAs formulated in lipid nanoparticles (mRNA-LNP) have proved to be a potent mode of immunization against infectious diseases in preclinical studies, and are being tested for SARS-CoV-2 in humans. A critical question is how mRNA-LNP vaccine immunogenicity compares to that of traditional adjuvanted protein vaccines in primates. Here, we show that mRNA-LNP immunization compared to protein immunization elicits either the same or superior magnitude and breadth of HIV-1 Env-specific polyfunctional antibodies. Immunization with mRNA-LNP encoding Zika premembrane and envelope or HIV-1 Env gp160 induces durable neutralizing antibodies for at least 41 weeks. Doses of mRNA-LNP as low as 5 µg are immunogenic in macaques. Thus, mRNA-LNP can be used to rapidly generate single or multi-component vaccines, such as sequential vaccines needed to protect against HIV-1 infection. Such vaccines would be as or more immunogenic than adjuvanted recombinant protein vaccines in primates.

Vaccination with Messenger RNA: A Promising Alternative to DNA Vaccination.

The first proof-of-concept studies about the feasibility of genetic vaccines were published over three decades ago, opening the way for future development. The idea of nonviral antigen delivery had multiple advantages over the traditional live or inactivated pathogen-based vaccines, but a great deal of effort had to be invested to turn the idea of genetic vaccination into reality. Although early proof-of-concept studies were groundbreaking, they also showed that numerous aspects of genetic vaccines needed to be improved. Until the early 2000s, the vast majority of effort was invested into the development of DNA vaccines due to the potential issues of instability and low in vivo translatability of messenger RNA (mRNA). In recent years, numerous studies have demonstrated the outstanding abilities of mRNA to elicit potent immune responses against infectious pathogens and different types of cancer, making it a viable platform for vaccine development. Multiple mRNA vaccine platforms have been developed and evaluated in small and large animals and humans and the results seem to be promising. RNA-based vaccines have important advantages over other vaccine approaches including outstanding efficacy, safety, and the potential for rapid, inexpensive, and scalable production. There is a substantial investment by new mRNA companies into the development of mRNA therapeutics, particularly vaccines, increasing the number of basic and translational research publications and human clinical trials underway. This review gives a broad overview about genetic vaccines and mainly focuses on the past and present of mRNA vaccines along with the future directions to bring this potent vaccine platform closer to therapeutic use.

A Single Immunization with Nucleoside-Modified mRNA Vaccines Elicits Strong Cellular and Humoral Immune Responses against SARS-CoV-2 in Mice.

SARS-CoV-2 infection has emerged as a serious global pandemic. Because of the high transmissibility of the virus and the high rate of morbidity and mortality associated with COVID-19, developing effective and safe vaccines is a top research priority. Here, we provide a detailed evaluation of the immunogenicity of lipid nanoparticle-encapsulated, nucleoside-modified mRNA (mRNA-LNP) vaccines encoding the full-length SARS-CoV-2 spike protein or the spike receptor binding domain in mice. We demonstrate that a single dose of these vaccines induces strong type 1 CD4+ and CD8+ T cell responses, as well as long-lived plasma and memory B cell responses. Additionally, we detect robust and sustained neutralizing antibody responses and the antibodies elicited by nucleoside-modified mRNA vaccines do not show antibody-dependent enhancement of infection in vitro. Our findings suggest that the nucleoside-modified mRNA-LNP vaccine platform can induce robust immune responses and is a promising candidate to combat COVID-19.

Selective targeting of nanomedicine to inflamed cerebral vasculature to enhance the blood-brain barrier.

Drug targeting to inflammatory brain pathologies such as stroke and traumatic brain injury remains an elusive goal. Using a mouse model of acute brain inflammation induced by local tumor necrosis factor alpha (TNFα), we found that uptake of intravenously injected antibody to vascular cell adhesion molecule 1 (anti-VCAM) in the inflamed brain is >10-fold greater than antibodies to transferrin receptor-1 and intercellular adhesion molecule 1 (TfR-1 and ICAM-1). Furthermore, uptake of anti-VCAM/liposomes exceeded that of anti-TfR and anti-ICAM counterparts by ∼27- and ∼8-fold, respectively, achieving brain/blood ratio >300-fold higher than that of immunoglobulin G/liposomes. Single-photon emission computed tomography imaging affirmed specific anti-VCAM/liposome targeting to inflamed brain in mice. Intravital microscopy via cranial window and flow cytometry showed that in the inflamed brain anti-VCAM/liposomes bind to endothelium, not to leukocytes. Anti-VCAM/LNP selectively accumulated in the inflamed brain, providing de novo expression of proteins encoded by cargo messenger RNA (mRNA). Anti-VCAM/LNP-mRNA mediated expression of thrombomodulin (a natural endothelial inhibitor of thrombosis, inflammation, and vascular leakage) and alleviated TNFα-induced brain edema. Thus VCAM-directed nanocarriers provide a platform for cerebrovascular targeting to inflamed brain, with the goal of normalizing the integrity of the blood-brain barrier, thus benefiting numerous brain pathologies.

Lipid nanoparticle encapsulated nucleoside-modified mRNA vaccines elicit polyfunctional HIV-1 antibodies comparable to proteins in nonhuman primates.

Development of an effective AIDS vaccine remains a challenge. Nucleoside-modified mRNAs formulated in lipid nanoparticles (mRNA-LNP) have proved to be a potent mode of immunization against infectious diseases in preclinical studies, and are being tested for SARS-CoV-2 in humans. A critical question is how mRNA-LNP vaccine immunogenicity compares to that of traditional adjuvanted protein vaccines in primates. Here, we found that mRNA-LNP immunization compared to protein immunization elicited either the same or superior magnitude and breadth of HIV-1 Env-specific polyfunctional antibodies. Immunization with mRNA-LNP encoding Zika premembrane and envelope (prM-E) or HIV-1 Env gp160 induced durable neutralizing antibodies for at least 41 weeks. Doses of mRNA-LNP as low as 5 µg were immunogenic in macaques. Thus, mRNA-LNP can be used to rapidly generate single or multi-component vaccines, such as sequential vaccines needed to protect against HIV-1 infection. Such vaccines would be as or more immunogenic than adjuvanted recombinant protein vaccines in primates.

Characterization of HIV-1 Nucleoside-Modified mRNA Vaccines in Rabbits and Rhesus Macaques.

Despite the enormous effort in the development of effective vaccines against HIV-1, no vaccine candidate has elicited broadly neutralizing antibodies in humans. Thus, generation of more effective anti-HIV vaccines is critically needed. Here we characterize the immune responses induced by nucleoside-modified and purified mRNA-lipid nanoparticle (mRNA-LNP) vaccines encoding the clade C transmitted/founder HIV-1 envelope (Env) 1086C. Intradermal vaccination with nucleoside-modified 1086C Env mRNA-LNPs elicited high levels of gp120-specific antibodies in rabbits and rhesus macaques. Antibodies generated in rabbits neutralized a tier 1 virus, but no tier 2 neutralization activity could be measured. Importantly, three of six non-human primates developed antibodies that neutralized the autologous tier 2 strain. Despite stable anti-gp120 immunoglobulin G (IgG) levels, tier 2 neutralization titers started to drop 4 weeks after booster immunizations. Serum from both immunized rabbits and non-human primates demonstrated antibody-dependent cellular cytotoxicity activity. Collectively, these results are supportive of continued development of nucleoside-modified and purified mRNA-LNP vaccines for HIV. Optimization of Env immunogens and vaccination protocols are needed to increase antibody neutralization breadth and durability.

Nucleoside-modified mRNA vaccines induce potent T follicular helper and germinal center B cell responses.

T follicular helper (Tfh) cells are required to develop germinal center (GC) responses and drive immunoglobulin class switch, affinity maturation, and long-term B cell memory. In this study, we characterize a recently developed vaccine platform, nucleoside-modified, purified mRNA encapsulated in lipid nanoparticles (mRNA-LNPs), that induces high levels of Tfh and GC B cells. Intradermal vaccination with nucleoside-modified mRNA-LNPs encoding various viral surface antigens elicited polyfunctional, antigen-specific, CD4+ T cell responses and potent neutralizing antibody responses in mice and nonhuman primates. Importantly, the strong antigen-specific Tfh cell response and high numbers of GC B cells and plasma cells were associated with long-lived and high-affinity neutralizing antibodies and durable protection. Comparative studies demonstrated that nucleoside-modified mRNA-LNP vaccines outperformed adjuvanted protein and inactivated virus vaccines and pathogen infection. The incorporation of noninflammatory, modified nucleosides in the mRNA is required for the production of large amounts of antigen and for robust immune responses.

High Fcp1 phosphatase activity contributes to setting an intense transcription rate required in Drosophila nurse and follicular cells for egg production.

During transcription cycles serine side chains in the carboxyl terminal domain (CTD) of the largest subunit of RNA polymerase II undergo dynamic phosphorylation-de-phosphorylation changes, and the modification status of the CTD serves as a signal for proteins involved in transcription and RNA maturation. We show here that the major CTD de-phosphorylating enzyme Fcp1 is expressed at high levels in germline cells of Drosophila. We used transgene constructs to modify the Fcp1 phosphatase level in Drosophila ovaries and found that high levels of Fcp1 are required for intensive gene expression in nurse cells. On the contrary, low Fcp1 levels might limit the rate of transcription. Fcp1 over-expression results in increased expression of microtubules in nurse cells. Our results show that tightly controlled high level Fcp1 expression in the nurse cells of Drosophila ovaries is required for proper egg maturation.

The RNA Pol II CTD phosphatase Fcp1 is essential for normal development in Drosophila melanogaster.

The reversible phosphorylation-dephosphorylation of RNA polymerase II (Pol II) large subunit carboxyl terminal domain (CTD) during transcription cycles in eukaryotic cells generates signals for the steps of RNA synthesis and maturation. The major phosphatase specific for CTD dephosphorylation from yeast to mammals is the TFIIF-interacting CTD-phosphatase, Fcp1. We report here on the in vivo analysis of Fcp1 function in Drosophila using transgenic lines in which the phosphatase production is misregulated. Fcp1 function is essential throughout Drosophila development and ectopic up- or downregulation of fcp1 results in lethality. The fly Fcp1 binds to specific regions of the polytene chromosomes at many sites colocalized with Pol II. In accord with the strong evolutional conservation of Fcp1: (1) the Xenopus fcp1 can substitute the fly fcp1 function, (2) similarly to its S. pombe homologue, Drosophila melanogaster (Dm)Fcp1 interacts with the RPB4 subunit of Pol II, and (3) transient expression of DmFcp1 has a negative effect on transcription in mammalian cells. The in vivo experimental system described here suggests that fly Fcp1 is associated with the transcription engaged Pol II and offers versatile possibilities for studying this evolutionary conserved essential enzyme.

GAL4 induces transcriptionally active puff in the absence of dSAGA- and ATAC-specific chromatin acetylation in the Drosophila melanogaster polytene chromosome.

Puffs in the polytene chromosome of Drosophila melanogaster are characteristic of sites of high-level active transcription which can be observed directly under the microscope. We studied the dependence of puff formation on chromatin modifications at a site where a GAL4-inducible transgene is located in the 61C7 cytological region. Immunostaining of salivary gland polytene chromosomes indicated no increase of either dSAGA-specific histone H3 lysine 14, or ATAC-specific histone H4 lysine 5 and 12 acetylation in the puffed region. Nor did we observe increased levels of H4K8ac, H3K18ac, or H4K16ac in the puff. In accordance with the above, puff formation as well as localization of Pol II and GAL4 was detectable at the 61C region in dAda2b and dAda2a null homozygotes, which are dSAGA- and ATAC-specific mutants, respectively. Moreover, the reduced level of JIL-1-specific H3 serine 10 phosphorylation did not abolish puff formation in ATAC mutants. Surprisingly, in wild-type animals dADA3 and GCN5 shared constituents of dSAGA and ATAC, as well as JIL-1 localized specifically to the puff, where the JIL-1-phosphorylated H3S10ph level was also high. Altogether these data strongly suggest that the GAL4 activator can induce transcription and chromatin reorganization seen as a puff without dSAGA- and ATAC-specific histone acetylation and JIL-1-specific histone H3 phosphorylation.