Nucleic acid-based vaccines such as viral vectors, plasmid DNA, and mRNA are being developed as a means to address a number of unmet medical needs that current vaccine technologies have been unable to address. Here, we describe a cationic nanoemulsion (CNE) delivery system developed to deliver a self-amplifying mRNA vaccine. This nonviral delivery system is based on Novartis's proprietary adjuvant MF59, which has an established clinical safety profile and is well tolerated in children, adults, and the elderly. We show that nonviral delivery of a 9 kb self-amplifying mRNA elicits potent immune responses in mice, rats, rabbits, and nonhuman primates comparable to a viral delivery technology, and demonstrate that, relatively low doses (75 µg) induce antibody and T-cell responses in primates. We also show the CNE-delivered self-amplifying mRNA enhances the local immune environment through recruitment of immune cells similar to an MF59 adjuvanted subunit vaccine. Lastly, we show that the site of protein expression within the muscle and magnitude of protein expression is similar to a viral vector. Given the demonstration that self-amplifying mRNA delivered using a CNE is well tolerated and immunogenic in a variety of animal models, we are optimistic about the prospects for this technology.
Exosomes are secreted cellular vesicles that can be internalized by dendritic cells (DCs), contributing to antigen-specific naive CD4 ؉ T-cell activation. Here, we demonstrate that human immunodeficiency virus type 1 (HIV-1) can exploit this exosome antigen-dissemination pathway intrinsic to mature DCs (mDCs) for mediating trans-infection of T lymphocytes. Capture of HIV-1, HIV-1 Gag-enhanced green fluorescent protein (eGFP) virallike particles (VLPs), and exosomes by DCs was up-regulated upon maturation, resulting in localization within a CD81 ؉ compartment. Uptake of VLPs or exosomes could be inhibited by a challenge with either particle, suggesting that the expression of common determinant(s) on VLP or exosome surface is necessary for internalization by mDCs. Capture by mDCs was insensitive to proteolysis but blocked when virus, VLPs, or exosomes were produced from cells treated with sphingolipid biosynthesis inhibitors that modulate the lipid composition of the budding particles. Finally, VLPs and exosomes captured by mDCs were transmitted to T lymphocytes in an envelope glycoproteinindependent manner, underscoring a new potential viral dissemination pathway. IntroductionDendritic cells (DCs) are specialized antigen-presenting cells that orchestrate innate and adaptive immune responses to invading pathogens. Immature DCs located in the peripheral mucosal tissues recognize and capture microbial pathogens, undergo maturation, and traffic to lymphoid tissues, where they induce adaptive immunity through antigen presentation to naive T cells. Although DCs are required to combat viral infections, viruses, including human immunodeficiency virus type 1 (HIV-1), have evolved strategies to evade their antiviral activity. HIV can gain access into DCs via a nonfusogenic endocytic mechanism, evade classical degradation pathways, and establish productive infection of DCinteracting T cells, a well-studied but poorly understood mechanism of HIV trans-infection of CD4 ϩ T cells. [1][2][3] The efficiency of DC-mediated HIV-1 transmission to T cells can be enhanced by maturing DCs in vitro, 2,4,5 although the mechanism underlying this process has not been well defined. 6 Previous studies have associated HIV trans-infection with the binding of the viral envelope glycoprotein (gp120) to C-type lectin receptors (CLR) such as DC-SIGN, trypsin-sensitive CLR, and CD4-independent receptors expressed on the DC surface. 3,7-11 However, we have recently identified an HIV gp120-independent mechanism of viral binding and endocytosis that is up-regulated upon DC maturation, 12 suggesting that HIV-1 might exploit a preexisting cellular pathway of antigen uptake and transmission. Interestingly, previous reports have shown that DCs can endocytose viral-like particles (VLPs) and induce immune responses. 13,14 Likewise, small secreted cellular organelles, termed exosomes, are also internalized by DCs and sorted into an endocytic compartment, stimulating antigenspecific naive CD4 ϩ T-cell activation in vivo. 15,16 On the basis of similarities i...
The COVID-19 pandemic, caused by infection with the SARS-CoV-2 coronavirus, is having a deleterious impact on health services and the global economy, highlighting the urgent need for an effective vaccine. Such a vaccine would need to rapidly confer protection after one or two doses and would need to be manufactured using components suitable for scale-up. Here, we developed an alphavirus-derived replicon RNA vaccine candidate, repRNA-CoV2S, encoding the SARS-CoV-2 spike (S) protein. The RNA replicons were formulated with Lipid InOrganic Nanoparticles (LION) that were designed to enhance vaccine stability, delivery, and immunogenicity. We show that a single intramuscular injection of the LION/repRNA-CoV2S vaccine in mice elicited robust production of anti-SARS-CoV-2 S protein IgG antibody isotypes indicative of a Type 1 T helper cell response. A prime/boost regimen induced potent T cell responses in mice including antigen-specific responses in lung and spleen. Prime-only immunization of aged (17-month old) mice induced smaller immune responses compared to young mice, but this difference was abrogated by booster immunization. Importantly, in nonhuman primates, prime-only immunization in one intramuscular injection site or prime/boost immunizations in 5 intramuscular injection sites elicited modest T cell responses and robust antibody responses. The antibody responses persisted for at least 70 days and neutralized SARS-CoV-2 at titers comparable to those in human serum samples collected from individuals convalescing from COVID-19. These data support further development of LION/repRNA-CoV2S as a vaccine candidate for prophylactic protection against SARS-CoV-2 infection.
The timing of vaccine availability is essential for an effective response to pandemic influenza. In 2009, vaccine became available after the disease peak, and this has motivated the development of next generation vaccine technologies for more rapid responses. The SAM® vaccine platform, now in pre-clinical development, is based on a synthetic, self-amplifying mRNA, delivered by a synthetic lipid nanoparticle (LNP). When used to express seasonal influenza hemagglutinin (HA), a SAM vaccine elicited potent immune responses, comparable to those elicited by a licensed influenza subunit vaccine preparation. When the sequences coding for the HA and neuraminidase (NA) genes from the H7N9 influenza outbreak in China were posted on a web-based data sharing system, the combination of rapid and accurate cell-free gene synthesis and SAM vaccine technology allowed the generation of a vaccine candidate in 8 days. Two weeks after the first immunization, mice had measurable hemagglutinin inhibition (HI) and neutralizing antibody titers against the new virus. Two weeks after the second immunization, all mice had HI titers considered protective. If the SAM vaccine platform proves safe, potent, well tolerated and effective in humans, fully synthetic vaccine technologies could provide unparalleled speed of response to stem the initial wave of influenza outbreaks, allowing first availability of a vaccine candidate days after the discovery of a new virus.
Since the first demonstration of in vivo gene expression from an injected RNA molecule almost two decades ago, the field of RNA-based therapeutics is now taking significant strides, with many cancer and infectious disease targets entering clinical trials. Critical to this success has been advances in the knowledge and application of delivery formulations. Currently, various lipid nanoparticle (LNP) platforms are at the forefront, but the encapsulation approach underpinning LNP formulations offsets the synthetic and rapid-response nature of RNA vaccines. Second, limited stability of LNP formulated RNA precludes stockpiling for pandemic readiness. Here, we show the development of a two-vialed approach wherein the delivery formulation, a highly stable nanostructured lipid carrier (NLC), can be manufactured and stockpiled separate from the target RNA, which is admixed prior to administration. Furthermore, specific physicochemical modifications to the NLC modulate immune responses, either enhancing or diminishing neutralizing antibody responses. We have combined this approach with a replicating viral RNA (rvRNA) encoding Zika virus (ZIKV) antigens and demonstrated a single dose as low as 10 ng can completely protect mice against a lethal ZIKV challenge, representing what might be the most potent approach to date of any Zika vaccine.
Interactions of human immunodeficiency virus type 1 (HIV-1) with dendritic cells (DCs) are multifactorial and presumably require nonredundant interactions between the HIV-1 envelope glycoprotein gp120 and molecules expressed on the DC surface that define the cellular fate of the virus particle. Surprisingly, neutralization of HIV-1 gp120-dependent binding interactions with DCs was insufficient to prevent HIV-1 attachment. Besides gp120, HIV-1 particles also incorporate host cell-derived proteins and lipids in their particle membrane. In this study, we demonstrate a crucial role for host cell-derived glycosphingolipids (GSLs) for the initial interactions of HIV-1 particles with both immature and mature DCs. Production of HIV-1 particles from virus producer cells treated with ceramide synthase inhibitor fumonisin B1 or glucosylceramide synthase inhibitor 1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP) resulted in the production of virus particles that, although capable of binding previously defined HIV-1 gp120-specific attachment factors CD4, DC-SIGN, and syndecans, were attenuated in their ability to be captured by both immature and mature DCs. Furthermore, GSL-deficient HIV-1 particles were inhibited in their ability to establish productive infections in DC-T-cell cocultures. These studies provide initial evidence for the role of HIV-1 particle membrane-associated GSLs in virus invasion of DCs and also provide additional novel cellular targets, GSL biosynthetic pathways and GSL-dependent HIV-1 interactions with DCs, for development of antiviral therapy.
Monoclonal antibody (mAb) therapeutics are an effective modality for the treatment of infectious, autoimmune, and cancer-related diseases. However, the discovery, development, and manufacturing processes are complex, resource-consuming activities that preclude the rapid deployment of mAbs in outbreaks of emerging infectious diseases. Given recent advances in nucleic acid delivery technology, it is now possible to deliver exogenous mRNA encoding mAbs for in situ expression following intravenous (i.v.) infusion of lipid nanoparticle-encapsulated mRNA. However, the requirement for i.v. administration limits the application to settings where infusion is an option, increasing the cost of treatment. As an alternative strategy, and to enable intramuscular (IM) administration of mRNA-encoded mAbs, we describe a nanostructured lipid carrier for delivery of an alphavirus replicon encoding a previously described highly neutralizing human mAb, ZIKV-117. Using a lethal Zika virus challenge model in mice, our studies show robust protection following alphavirus-driven expression of ZIKV-117 mRNA when given by IM administration as pre-exposure prophylaxis or post-exposure therapy.
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