Adjuvant effects of a sequence-engineered mRNA vaccine: translational profiling demonstrates similar human and murine innate response.

BACKGROUND: Prophylactic and therapeutic vaccines often depend upon a strong activation of the innate immune system to drive a potent adaptive immune response, often mediated by a strong adjuvant. For a number of adjuvants immunological readouts may not be consistent across species. METHODS: In this study, we evaluated the innate immunostimulatory potential of mRNA vaccines in both humans and mice, using a novel mRNA-based vaccine encoding influenza A hemagglutinin of the pandemic strain H1N1pdm09 as a model. This evaluation was performed using an in vitro model of human innate immunity and in vivo in mice after intradermal injection. RESULTS: Results suggest that immunostimulation from the mRNA vaccine in humans is similar to that in mice and acts through cellular RNA sensors, with genes for RLRs [ddx58 (RIG-1) and ifih1 (MDA-5)], TLRs (tlr3, tlr7, and tlr8-human only), and CLRs (clec4gp1, clec2d, cledl1) all significantly up-regulated by the mRNA vaccine. The up-regulation of TLR8 and TLR7 points to the involvement of both mDCs and pDCs in the response to the mRNA vaccine in humans. In both humans and mice activation of these pathways drove maturation and activation of immune cells as well as production of cytokines and chemokines known to attract and activate key players of the innate and adaptive immune system. CONCLUSION: This translational approach not only allowed for identification of the basic mechanisms of self-adjuvantation from the mRNA vaccine but also for comparison of the response across species, a response that appears relatively conserved or at least convergent between the in vitro human and in vivo mouse models.

Immunogenicity and protective efficacy of RSV G central conserved domain vaccine with a prefusion nanoparticle.

Respiratory syncytial virus (RSV) G glycoprotein has recently reemerged as a vaccine antigen due to its ability to elicit potent neutralizing antibodies and ameliorate disease in animal models. Here we designed three constructs to display the G central conserved domain (Gcc) focused on inducing broad and potent neutralizing antibodies. One construct displaying Gcc from both RSV subgroups trimerized via a C-terminal foldon (Gcc-Foldon) was highly immunogenic in mice and in MIMIC, a pre-immune human in vitro model. To explore an optimal RSV vaccine, we combined the Gcc-Foldon antigen with a stabilized pre-fusion-F nanoparticle (pre-F-NP) as a bivalent vaccine and detected no antigenic interference between the two antigens in the MIMIC model. In RSV-primed macaques, the bivalent vaccine elicited potent humoral responses. Furthermore, both Gcc-Foldon and the bivalent vaccine conferred effective protection against RSV challenge in mice. This two-component vaccine could potentially provide effective protection against RSV infection in humans and warrants further clinical evaluation.

Development of multivalent mRNA vaccine candidates for seasonal or pandemic influenza.

Recent approval of mRNA vaccines for emergency use against COVID-19 is likely to promote rapid development of mRNA-based vaccines targeting a wide range of infectious diseases. Compared to conventional approaches, this vaccine modality promises comparable potency while substantially accelerating the pace of development and deployment of vaccine doses. Already demonstrated successfully for single antigen vaccines such as for COVID-19, this technology could be optimized for complex multi-antigen vaccines. Herein, utilizing multiple influenza antigens, we demonstrated the suitability of the mRNA therapeutic (MRT) platform for such applications. Seasonal influenza vaccines have three or four hemagglutinin (HA) antigens of different viral subtypes. In addition, influenza neuraminidase (NA), a tetrameric membrane protein, is identified as an antigen that has been linked to protective immunity against severe viral disease. We detail the efforts in optimizing formulations of influenza candidates that use unmodified mRNA encoding full-length HA or full-length NA encapsulated in lipid nanoparticles (LNPs). HA and NA mRNA-LNP formulations, either as monovalent or as multivalent vaccines, induced strong functional antibody and cellular responses in non-human primates and such antigen-specific antibody responses were associated with protective efficacy against viral challenge in mice.

A respiratory syncytial virus (RSV) F protein nanoparticle vaccine focuses antibody responses to a conserved neutralization domain.

A stabilized form of the respiratory syncytial virus (RSV) fusion (F) protein has been explored as a vaccine to prevent viral infection because it presents several potent neutralizing epitopes. Here, we used a structure-based rational design to optimize antigen presentation and focus antibody (Ab) responses to key epitopes on the pre-fusion (pre-F) protein. This protein was fused to ferritin nanoparticles (pre-F-NP) and modified with glycans to mask nonneutralizing or poorly neutralizing epitopes to further focus the Ab response. The multimeric pre-F-NP elicited durable pre-F-specific Abs in nonhuman primates (NHPs) after >150 days and elicited potent neutralizing Ab (NAb) responses in mice and NHPs in vivo, as well as in human cells evaluated in the in vitro MIMIC system. This optimized pre-F-NP stimulated a more potent Ab response than a representative pre-F trimer, DS-Cav1. Collectively, this pre-F vaccine increased the generation of NAbs targeting the desired pre-F conformation, an attribute that facilitates the development of an effective RSV vaccine.

High level expression of the bioactive human interleukin-10 in milk of transgenic mice.

Human interleukin-10 (hIL-10) has wide spectrum of anti-inflammatory activities and has shown a potential to be used for treatment of inflammatory or immune illness. In this study, transgenic mice that over-express human interleukin-10 (IL-10) in their milk were generated using a bovine beta-casein/human IL-10 hybrid gene. After cloning of the IL-10 gene, a 22 kb hybrid gene was constructed by linking a 10 kb promoter sequence of the bovine beta-casein gene to the cloned 12 kb IL-10 gene. In six of the eight transgenic mice, the transgene RNA was expressed only in the mammary gland and in the other two mice, it was also slightly expressed in the lung. The highest human IL-10 level in milk was 1620 microg x ml(-1). Notably, transgenes in all the eight transgenic mice were expressed regardless of the integration site even though no correlation was shown between the copy numbers of the transgene and expression level. These results suggest that the genomic sequence of the human IL-10 gene can induce the IL-10 expression at high levels under the control of the bovine beta-casein promoter.

The cell cycle time of CD8+ T cells responding in vivo is controlled by the type of antigenic stimulus.

A hallmark of cells comprising the mammalian adaptive immune system is the requirement for these rare naïve T (and B) lymphocytes directed to a specific microorganism to undergo proliferative expansion upon first encounter with this antigen. In the case of naïve CD8(+) T cells the ability of these rare quiescent lymphocytes to rapidly activate and expand into effector T cells in numbers sufficient to control viral and certain bacterial infections can be essential for survival. In this report we examined the activation, cell cycle time and initial proliferative response of naïve murine CD8(+) T cells responding in vivo to Influenza and Vaccinia virus infection or vaccination with viral antigens. Remarkably, we observed that CD8(+) T cells could divide and proliferate with an initial cell division time of as short as 2 hours. The initial cell cycle time of responding CD8(+) T cells is not fixed but is controlled by the antigenic stimulus provided by the APC in vivo. Initial cell cycle time influences the rate of T cell expansion and the numbers of effector T cells subsequently accumulating at the site of infection. The T cell cycle time varies with duration of the G(1) phase of the cell cycle. The duration of G(1) is inversely correlated with the phosphorylation state of the retinoblastoma (Rb) protein in the responding T cells. The implication of these findings for the development of adaptive immune responses and the regulation of cell cycle in higher eukaryotic cells is discussed.

Sequential activation of CD8+ T cells in the draining lymph nodes in response to pulmonary virus infection.

We have used a TCR-transgenic CD8+ T cell adoptive transfer model to examine the tempo of T cell activation and proliferation in the draining lymph nodes (DLN) in response to respiratory virus infection. The T cell response in the DLN differed for mice infected with different type A influenza strains with the onset of T cell activation/proliferation to the A/JAPAN virus infection preceding the A/PR8 response by 12-24 h. This difference in T cell activation/proliferation correlated with the tempo of accelerated respiratory DC (RDC) migration from the infected lungs to the DLN in response to influenza virus infection, with the migrant RDC responding to the A/JAPAN infection exhibiting a more rapid accumulation in the lymph nodes (i.e., peak migration for A/JAPAN at 18 h, A/PR8 at 24-36 h). Furthermore, in vivo administration of blocking anti-CD62L Ab at various time points before/after infection revealed that the virus-specific CD8+ T cells entered the DLN and activated in a sequential "conveyor belt"-like fashion. These results indicate that the tempo of CD8+ T cell activation/proliferation after viral infection is dependent on the tempo of RDC migration to the DLN and that T cell activation occurs in an ordered sequential fashion.

Cutting edge: pulmonary immunopathology mediated by antigen-specific expression of TNF-alpha by antiviral CD8+ T cells.

Respiratory virus infection results in considerable pulmonary immunopathology, a component of which results from the host immune responses. We have developed a murine model to specifically examine the lung injury due to CD8(+) T cell recognition of an influenza hemagglutinin (HA) transgene on lung epithelium in the absence of replicating virus, after adoptive transfer. Lung injury is largely mediated by chemokines expressed by the epithelial cells upon T cell recognition mediated by TNF-alpha. To determine the critical source of TNF-alpha, HA-specific TNF(-/-) CD8(+) T cells were transferred into HA transgenic animals, and lung injury was not observed, though these T cells exhibited no defect in antiviral activity in vivo. This indicates that the initiating event in the injury process is Ag-specific expression of TNF-alpha by antiviral CD8(+) T cells upon recognition of alveolar epithelial Ag, and that the effector activities responsible for viral clearance may be dissociable from those resulting in immunopathology.