Computationally designed mRNA-launched protein nanoparticle vaccines.

Both protein nanoparticle and mRNA vaccines were clinically de-risked during the COVID-19 pandemic1-6. These vaccine modalities have complementary strengths: antigen display on protein nanoparticles can enhance the magnitude, quality, and durability of antibody responses7-10, while mRNA vaccines can be rapidly manufactured11 and elicit antigen-specific CD4 and CD8 T cells12,13. Here we leverage a computationally designed icosahedral protein nanoparticle that was redesigned for optimal secretion from eukaryotic cells14 to develop an mRNA-launched nanoparticle vaccine for SARS-CoV-2. The nanoparticle, which displays 60 copies of a stabilized variant of the Wuhan-Hu-1 Spike receptor binding domain (RBD)15, formed monodisperse, antigenically intact assemblies upon secretion from transfected cells. An mRNA vaccine encoding the secreted RBD nanoparticle elicited 5- to 28-fold higher levels of neutralizing antibodies than an mRNA vaccine encoding membrane-anchored Spike, induced higher levels of CD8 T cells than the same immunogen when delivered as an adjuvanted protein nanoparticle, and protected mice from vaccine-matched and -mismatched SARS-CoV-2 challenge. Our data establish that delivering protein nanoparticle immunogens via mRNA vaccines can combine the benefits of each modality and, more broadly, highlight the utility of computational protein design in genetic immunization strategies.

Hypertension Severity as Quantified by Hypertension Daily Dose and Blood Pressure With Risk of Stroke in REGARDS.

BACKGROUND: It is unknown how blood pressure (BP) relates to stroke risk across levels of hypertension daily dose (HDD)-quantified antihypertensive medication intensity. METHODS AND RESULTS: The REGARDS (Reasons for Geographic and Racial Differences in Stroke) study enrolled 30 239 participants from the 48 contiguous US states in 2003 to 2007 with in-person follow-up in 2013 to 2016 (Visit 2). We included those without prior stroke at Visit 2, treating this visit as T0. Biannual phone calls and medical record review ascertained incident stroke events. Cox proportional hazard models estimated the hazard ratio (HR) of incident stroke by treatment intensity defined by systolic BP stages and HDD groupings. There were 344 stroke events over a median 5.5 years. Relative to systolic BP <120 mm Hg and no antihypertensive medications, the stroke HR was 2.86 (95% CI, 1.68-4.85) for systolic BP 140 to 159 mm Hg and HDD tertile 2, 2.33 (1.37-3.97) for systolic BP 140 to 159 mm Hg and HDD tertile 3, 3.08 (1.20-7.88) for systolic BP ≥160 mm Hg and HDD tertile 2, and 3.66 (1.61-8.30) for systolic BP ≥160 mm Hg and HDD tertile 3. Stroke risk was similar across HDD levels for people with systolic BP <140 mm Hg. CONCLUSIONS: Among adults without prior stroke, systolic BP ≥140 mm Hg and HDD tertile ≥2 was associated with greater stroke risk. For adults with BP <140 mm Hg, stroke risk was similar despite cumulative dose of antihypertensive medications used. These findings support the practice of BP-lowering medications to mitigate stroke risk.

The roles of CD4+ T cell help, sex, and dose in the induction of protective CD8+ T cells against a lethal poxvirus by mRNA-LNP vaccines.

The role of CD4+ T cells in the induction of protective CD8+ T cells by mRNA lipid nanoparticle (LNP) vaccines is unknown. We used B6 or Tlr9 -/- mice depleted or not of CD4+ T cells and LNP vaccines loaded with mRNAs encoding the ectromelia virus (ECTV) MHC class I H-2 Kb-restricted immunodominant CD8+ T cell epitope TSYKFESV (TSYKFESV mRNA-LNPs) or the ECTV EVM158 protein, which contains TSYKFESV (EVM-158 mRNA-LNPs). Following prime and boost with 10 µg of either vaccine, Kb-TSYKFESV-specific CD8+ T cells fully protected male and female mice from ECTV at 29 (both mRNA-LNPs) or 90 days (EVM158 mRNA-LNPs) post boost (dpb) independently of CD4+ T cells. However, at 29 dpb with 1 µg mRNA-LNPs, males had lower frequencies of Kb-TSYKFESV-specific CD8+ T cells and were much less well protected than females from ECTV, also independently of CD4+ T cells. At 90 dpb with 1 µg EVM158 mRNA-LNPs, the frequencies of Kb-TSYKFESV-specific CD8+ T cells in males and females were similar, and both were similarly partially protected from ECTV, independently of CD4+ T cells. Therefore, at optimal or suboptimal doses of mRNA-LNP vaccines, CD4+ T cell help is unnecessary to induce protective anti-poxvirus CD8+ T cells specific to a dominant epitope. At suboptimal doses, protection of males requires more time to develop.

Use of HSC Targeted LNP to Generate a Mouse Model of Lethal α-Thalassemia and Treatment via Lentiviral Gene Therapy.

-Thalassemia (AT) is one of the most commonly occurring inherited hematological diseases. However, few treatments are available, and allogeneic bone marrow transplantation (BMT) is the only available therapeutic option for patients with severe AT. Research into AT has remained limited due to a lack of adult mouse models, with severe AT typically resulting in in utero lethality. By using a lipid nanoparticle (LNP) targeting the receptor CD117 and delivering a Cre mRNA (mRNACreLNPCD117), we were able to delete floxed -globin genes at high efficiency in hematopoietic stem cells (HSC) ex vivo. These cells were then engrafted in the absence or presence of a novel α-globin expressing lentiviral vector (ALS20I). Myeloablated mice transplanted with mRNACreLNPCD117-treated HSC showed a complete knockout of -globin genes. They demonstrated a phenotype characterized by the synthesis of hemoglobin H (-tetramers,  or HbH), aberrant erythropoiesis, and abnormal organ morphology, culminating in lethality approximately eight weeks following engraftment. Mice receiving mRNACreLNPCD117-treated HSC with at least one copy of ALS20I survived long-term with normalization of erythropoiesis, decreased the production of HbH, and ameliorated the abnormal organ morphology. Furthermore, we tested ALS20I in erythroid progenitors derived from -globin-KO CD34+ and cells isolated from patients with both deletional and non-deletional HbH disease, demonstrating improvement in -globin/-globin mRNA ratio and reduction in the formation of HbH by HPLC. Our results demonstrate the broad applicability of LNP for disease modeling, characterization of a novel severe mouse model of AT, and the efficacy of ALS20I for treating AT.

Distinct components of nucleoside-modified messenger RNA vaccines cooperate to instruct efficient germinal center responses.

Nucleoside-modified mRNA vaccines elicit protective antibodies through their ability to promote T follicular helper (Tfh) cells. The lipid nanoparticle (LNP) component of mRNA vaccines possesses inherent adjuvant activity. However, to what extent the nucleoside-modified mRNA can be sensed and contribute to Tfh cell responses remains largely undefined. Herein, we deconvoluted the signals induced by LNP and mRNA that instruct dendritic cells (DCs) to promote Tfh cell differentiation. We demonstrated that the nucleoside-modified mRNA drives the production of type I interferons that act on DCs to induce their maturation and the induction of Th1-biased Tfh responses. Conversely, LNP favors the acquisition of a Tfh cell-inducing program in DCs, a stronger Th2 polarization in Tfh cells, and allows for rapid mRNA translation by DCs within the draining lymph node. Our work unravels distinct adjuvant features of mRNA and LNP necessary for the induction of Tfh cells, with implications for vaccine design.

mRNA-LNP vaccine-induced CD8+ T cells protect mice from lethal SARS-CoV-2 infection in the absence of specific antibodies.

The role of CD8+ T cells in SARS-CoV-2 pathogenesis or mRNA-LNP vaccine-induced protection from lethal COVID-19 is unclear. Using mouse-adapted SARS-CoV-2 virus (MA30) in C57BL/6 mice, we show that CD8+ T cells are unnecessary for the intrinsic resistance of female or the susceptibility of male mice to lethal SARS-CoV-2 infection. Also, mice immunized with a di-proline prefusion-stabilized full-length SARS-CoV-2 Spike (S-2P) mRNA-LNP vaccine, which induces Spike-specific antibodies and CD8+ T cells specific for the Spike-derived VNFNFNGL peptide, are protected from SARS-CoV-2 infection-induced lethality and weight loss, while mice vaccinated with mRNA-LNPs encoding only VNFNFNGL are protected from lethality but not weight loss. CD8+ T cell depletion ablates protection in VNFNFNGL but not in S-2P mRNA-LNP-vaccinated mice. Therefore, mRNA-LNP vaccine-induced CD8+ T cells are dispensable when protective antibodies are present but essential for survival in their absence. Hence, vaccine-induced CD8+ T cells may be critical to protect against SARS-CoV-2 variants that mutate epitopes targeted by protective antibodies.

Mutation-guided vaccine design: A process for developing boosting immunogens for HIV broadly neutralizing antibody induction.

A major goal of HIV-1 vaccine development is the induction of broadly neutralizing antibodies (bnAbs). Although success has been achieved in initiating bnAb B cell lineages, design of boosting immunogens that select for bnAb B cell receptors with improbable mutations required for bnAb affinity maturation remains difficult. Here, we demonstrate a process for designing boosting immunogens for a V3-glycan bnAb B cell lineage. The immunogens induced affinity-matured antibodies by selecting for functional improbable mutations in bnAb precursor knockin mice. Moreover, we show similar success in prime and boosting with nucleoside-modified mRNA-encoded HIV-1 envelope trimer immunogens, with improved selection by mRNA immunogens of improbable mutations required for bnAb binding to key envelope glycans. These results demonstrate the ability of both protein and mRNA prime-boost immunogens for selection of rare B cell lineage intermediates with neutralizing breadth after bnAb precursor expansion, a key proof of concept and milestone toward development of an HIV-1 vaccine.

Targeting lipid nanoparticles to the blood-brain barrier to ameliorate acute ischemic stroke.

Effective delivery of mRNA or small molecule drugs to the brain is a significant challenge in developing treatment for acute ischemic stroke (AIS). To address the problem, we have developed targeted nanomedicine to increase drug concentrations in endothelial cells of the blood-brain barrier (BBB) of the injured brain. Inflammation during ischemic stroke causes continuous neuronal death and an increase in the infarct volume. To enable targeted delivery to the inflamed BBB, we conjugated lipid nanocarriers (NCs) with antibodies that bind cell adhesion molecules expressed at the BBB. In the transient middle cerebral artery occlusion mouse model, NCs targeted to vascular cellular adhesion molecule-1 (VCAM) achieved the highest level of brain delivery, nearly two orders of magnitude higher than untargeted ones. VCAM-targeted lipid nanoparticles with luciferase-encoding mRNA and Cre-recombinase showed selective expression in the ischemic brain. Anti-inflammatory drugs administered intravenously after ischemic stroke reduced cerebral infarct volume by 62% (interleukin-10 mRNA) or 35% (dexamethasone) only when they were encapsulated in VCAM-targeted NCs. Thus, VCAM-targeted lipid NCs represent a new platform for strongly concentrating drugs within the compromised BBB of penumbra, thereby ameliorating AIS.

Evolutionarily related host and microbial pathways regulate fat desaturation in C. elegans.

Fatty acid desaturation is central to metazoan lipid metabolism and provides building blocks of membrane lipids and precursors of diverse signaling molecules. Nutritional conditions and associated microbiota regulate desaturase expression, but the underlying mechanisms have remained unclear. Here, we show that endogenous and microbiota-dependent small molecule signals promote lipid desaturation via the nuclear receptor NHR-49/PPARα in C. elegans. Untargeted metabolomics of a ß-oxidation mutant, acdh-11, in which expression of the stearoyl-CoA desaturase FAT-7/SCD1 is constitutively increased, revealed accumulation of a ß-cyclopropyl fatty acid, becyp#1, that potently activates fat-7 expression via NHR-49. Biosynthesis of becyp#1 is strictly dependent on expression of cyclopropane synthase by associated bacteria, e.g., E. coli. Screening for structurally related endogenous metabolites revealed a ß-methyl fatty acid, bemeth#1, which mimics the activity of microbiota-dependent becyp#1 but is derived from a methyltransferase, fcmt-1, that is conserved across Nematoda and likely originates from bacterial cyclopropane synthase via ancient horizontal gene transfer. Activation of fat-7 expression by these structurally similar metabolites is controlled by distinct mechanisms, as microbiota-dependent becyp#1 is metabolized by a dedicated ß-oxidation pathway, while the endogenous bemeth#1 is metabolized via α-oxidation. Collectively, we demonstrate that evolutionarily related biosynthetic pathways in metazoan host and associated microbiota converge on NHR-49/PPARα to regulate fat desaturation.

Generation of superposed orbital angular momentum beams using a free-electron laser oscillator.

With wavelength tunability, free-electron lasers (FELs) are well-suited for generating orbital angular momentum (OAM) beams in a wide photon energy range. We report here the first experimental demonstration of OAM beam generation using an oscillator FEL with the tens of picosecond pulse duration. Lasing around 458 nm, we have produced the four lowest orders of superposed Laguerre-Gaussian beams using a very long FEL resonator of 53.73 m. The produced beams have good beam quality, excellent stability, and substantial average power. We have also developed a pulsed operation mode for these beams with a highly reproducible temporal structure for a range of repetition rate of 1-30 Hz. This development can be extended to short wavelengths, for example to x-rays using a future x-ray FEL oscillator. The OAM operation of such a storage-ring FEL also paves the way for the generation of OAM gamma-ray beams via inverse Compton scattering.

mRNA vaccination of rabbits alters the fecundity, but not the attachment, of adult Ixodes scapularis.

19ISP is a nucleoside-modified mRNA-lipid nanoparticle vaccine that targets 19 Ixodes scapularis proteins. We demonstrate that adult I. scapularis have impaired fecundity when allowed to engorge on 19ISP-immunized rabbits. 19ISP, therefore, has the potential to interrupt the tick reproductive cycle, without triggering some of the other effects associated with acquired tick resistance. This may lead to the development of new strategies to reduce I. scapularis populations in endemic areas.

Vaccine induction of CD4-mimicking HIV-1 broadly neutralizing antibody precursors in macaques.

The CD4-binding site (CD4bs) is a conserved epitope on HIV-1 envelope (Env) that can be targeted by protective broadly neutralizing antibodies (bnAbs). HIV-1 vaccines have not elicited CD4bs bnAbs for many reasons, including the occlusion of CD4bs by glycans, expansion of appropriate naive B cells with immunogens, and selection of functional antibody mutations. Here, we demonstrate that immunization of macaques with a CD4bs-targeting immunogen elicits neutralizing bnAb precursors with structural and genetic features of CD4-mimicking bnAbs. Structures of the CD4bs nAb bound to HIV-1 Env demonstrated binding angles and heavy-chain interactions characteristic of all known human CD4-mimicking bnAbs. Macaque nAb were derived from variable and joining gene segments orthologous to the genes of human VH1-46-class bnAb. This vaccine study initiated in primates the B cells from which CD4bs bnAbs can derive, accomplishing the key first step in the development of an effective HIV-1 vaccine.

A Pvs25 mRNA vaccine induces complete and durable transmission-blocking immunity to Plasmodium vivax.

Plasmodium vivax (P. vivax) is the major malaria parasite outside of Africa and no vaccine is available against it. A vaccine that interrupts parasite transmission (transmission-blocking vaccine, TBV) is considered highly desirable to reduce the spread of P. vivax and to accelerate its elimination. However, the development of a TBV against this pathogen has been hampered by the inability to culture the parasite as well as the low immunogenicity of the vaccines developed to date. Pvs25 is the most advanced TBV antigen candidate for P. vivax. However, in previous phase I clinical trials, TBV vaccines based on Pvs25 yielded low antibody responses or had unacceptable safety profiles. As the nucleoside-modified mRNA-lipid nanoparticle (mRNA-LNP) vaccine platform proved to be safe and effective in humans, we generated and tested mRNA-LNP vaccines encoding several versions of Pvs25 in mice. We found that in a prime-boost vaccination schedule, all Pvs25 mRNA-LNP vaccines elicited robust antigen-specific antibody responses. Furthermore, when compared with a Pvs25 recombinant protein vaccine formulated with Montanide ISA-51 adjuvant, the full-length Pvs25 mRNA-LNP vaccine induced a stronger and longer-lasting functional immunity. Seven months after the second vaccination, vaccine-induced antibodies retained the ability to fully block P. vivax transmission in direct membrane feeding assays, whereas the blocking activity induced by the protein/ISA-51 vaccine dropped significantly. Taken together, we report on mRNA vaccines targeting P. vivax and demonstrate that Pvs25 mRNA-LNP outperformed an adjuvanted Pvs25 protein vaccine suggesting that it is a promising candidate for further testing in non-human primates.

Neoadjuvant nivolumab plus chemotherapy versus chemotherapy for resectable NSCLC: subpopulation analysis of Chinese patients in CheckMate 816.

BACKGROUND: Neoadjuvant nivolumab plus chemotherapy significantly improved event-free survival (EFS) and pathologic complete response (pCR) versus chemotherapy alone in patients with resectable non-small-cell lung cancer (NSCLC) in the global phase III CheckMate 816 study. Here, we report post hoc exploratory efficacy, safety, and surgical outcomes in the Chinese subpopulation of this study. METHODS: Adults with stage IB-IIIA resectable NSCLC were randomized to receive nivolumab 360 mg plus chemotherapy or chemotherapy alone every 3 weeks for three cycles followed by surgery. Primary endpoints included EFS and pCR (both per blinded independent review). EFS and pCR results were from 14 October 2022, and 16 September 2020, database locks, respectively. RESULTS: The Chinese subpopulation comprised 97 patients (nivolumab plus chemotherapy, 44; chemotherapy, 53). At 38.2 months of minimum follow-up, median EFS was not reached [95% confidence interval (CI) 23.4 months-not reached] in the nivolumab plus chemotherapy arm and 13.9 months (95% CI 8.3-34.3 months) in the chemotherapy arm (hazard ratio 0.47, 95% CI 0.25-0.88). pCR rates were 25.0% (95% CI 13.2% to 40.3%) and 1.9% (95% CI 0.0% to 10.1%), respectively (odds ratio 11.05; 95% CI 1.41-86.49). Of 97 Chinese patients, 36 (82%) in the nivolumab plus chemotherapy arm and 41 (77%) in the chemotherapy arm underwent definitive surgery. Grade 3-4 treatment-related adverse events occurred in 18/43 patients (42%) treated with nivolumab plus chemotherapy and 22/53 patients (42%) treated with chemotherapy. CONCLUSIONS: Consistent with findings in the global study population of CheckMate 816, neoadjuvant nivolumab plus chemotherapy improved EFS and pCR versus chemotherapy in the Chinese subpopulation without impacting treatment tolerability or the feasibility of surgery. These findings support the use of nivolumab plus chemotherapy as a standard neoadjuvant treatment option for Chinese patients with resectable NSCLC.

The Regulation of Nucleic Acid Vaccine Responses by the Microbiome.

Nucleic acid vaccines, including both RNA and DNA platforms, are key technologies that have considerable promise in combating both infectious disease and cancer. However, little is known about the extrinsic factors that regulate nucleic acid vaccine responses and which may determine their effectiveness. The microbiome is recognized as a significant regulator of immune development and response, whose role in regulating some traditional vaccine platforms has recently been discovered. Using germ-free and specific pathogen-free mouse models in combination with different protein, DNA, and mRNA vaccine regimens, we demonstrate that the microbiome is a significant regulator of nucleic acid vaccine immunogenicity. Although the presence of the microbiome enhances CD8+ T cell responses to mRNA lipid nanoparticle immunization, the microbiome suppresses Ig and CD4+ T cell responses to DNA-prime, DNA-protein-boost immunization, indicating contrasting roles for the microbiome in the regulation of these different nucleic acid vaccine platforms. In the case of mRNA lipid nanoparticle vaccination, germ-free mice display reduced dendritic cell/macrophage activation that may underlie the deficient vaccine response. Our study identifies the microbiome as a relevant determinant of nucleic acid vaccine response with implications for continued therapeutic development and deployment of these vaccines.

Evolutionarily related host and microbial pathways regulate fat desaturation.

Fatty acid desaturation is central to metazoan lipid metabolism and provides building blocks of membrane lipids and precursors of diverse signaling molecules. Nutritional conditions and associated microbiota regulate desaturase expression1-4, but the underlying mechanisms have remained unclear. Here, we show that endogenous and microbiota-dependent small molecule signals promote lipid desaturation via the nuclear receptor NHR-49/PPARα in C. elegans. Untargeted metabolomics of a ß-oxidation mutant, acdh-11, in which expression of the stearoyl-CoA desaturase FAT-7/SCD1 is constitutively increased, revealed accumulation of a ß-cyclopropyl fatty acid, becyp#1, that potently activates fat-7 expression via NHR-49. Biosynthesis of becyp#1 is strictly dependent on expression of cyclopropane synthase by associated bacteria, e.g., E. coli. Screening for structurally related endogenous metabolites revealed a ß-methyl fatty acid, bemeth#1, whose activity mimics that of microbiota-dependent becyp#1, but is derived from a methyltransferase, fcmt-1, that is conserved across Nematoda and likely originates from bacterial cyclopropane synthase via ancient horizontal gene transfer. Activation of fat-7 expression by these structurally similar metabolites is controlled by distinct mechanisms, as microbiota-dependent becyp#1 is metabolized by a dedicated ß-oxidation pathway, while the endogenous bemeth#1 is metabolized via α-oxidation. Collectively, we demonstrate that evolutionarily related biosynthetic pathways in metazoan host and associated microbiota converge on NHR-49/PPARα to regulate fat desaturation.

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.