RESUMEN
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.
Asunto(s)
Vacunas contra el SIDA , VIH-1 , Animales , Humanos , Anticuerpos ampliamente neutralizantes , Antígenos CD4 , Moléculas de Adhesión Celular , VIH-1/fisiología , Macaca , Vacunas contra el SIDA/inmunologíaRESUMEN
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.
Asunto(s)
Linfocitos B/inmunología , Vacunas contra la COVID-19/inmunología , COVID-19/inmunología , Centro Germinal/inmunología , SARS-CoV-2/fisiología , Linfocitos T Colaboradores-Inductores/inmunología , Vacunas de ARNm/inmunología , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Adyuvantes Inmunológicos , Animales , Células HEK293 , Humanos , Inmunidad Humoral , Interleucina-6/genética , Interleucina-6/metabolismo , Liposomas/administración & dosificación , Ratones , Ratones Endogámicos BALB C , Nanopartículas/administración & dosificación , Subunidades de Proteína/genética , Vacunas de ARNm/genéticaRESUMEN
The deployment of effective vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is critical to eradicate the coronavirus disease 2019 (COVID-19) pandemic. Many licensed vaccines confer protection by inducing long-lived plasma cells (LLPCs) and memory B cells (MBCs), cell types canonically generated during germinal center (GC) reactions. Here, we directly compared two vaccine platforms-mRNA vaccines and a recombinant protein formulated with an MF59-like adjuvant-looking for their abilities to quantitatively and qualitatively shape SARS-CoV-2-specific primary GC responses over time. We demonstrated that a single immunization with SARS-CoV-2 mRNA, but not with the recombinant protein vaccine, elicited potent SARS-CoV-2-specific GC B and T follicular helper (Tfh) cell responses as well as LLPCs and MBCs. Importantly, GC responses strongly correlated with neutralizing antibody production. mRNA vaccines more efficiently induced key regulators of the Tfh cell program and influenced the functional properties of Tfh cells. Overall, this study identifies SARS-CoV-2 mRNA vaccines as strong candidates for promoting robust GC-derived immune responses.
Asunto(s)
Anticuerpos Neutralizantes/metabolismo , Linfocitos B/inmunología , Vacunas contra la COVID-19/inmunología , COVID-19/inmunología , Centro Germinal/inmunología , SARS-CoV-2/fisiología , Linfocitos T Colaboradores-Inductores/inmunología , Vacunas Sintéticas/inmunología , Antígenos Virales/genética , Antígenos Virales/inmunología , Células Cultivadas , Epítopos , Humanos , Activación de Linfocitos , Polisorbatos , ARN Viral/inmunología , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Escualeno , Vacunación , Vacunas de ARNmRESUMEN
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.
Asunto(s)
Anticuerpos Neutralizantes/biosíntesis , Anticuerpos Antivirales/biosíntesis , Betacoronavirus/efectos de los fármacos , Infecciones por Coronavirus/prevención & control , Pandemias/prevención & control , Neumonía Viral/prevención & control , ARN Mensajero/inmunología , ARN Viral/inmunología , Vacunas Virales/administración & dosificación , Animales , Linfocitos B/efectos de los fármacos , Linfocitos B/inmunología , Linfocitos B/virología , Betacoronavirus/inmunología , Betacoronavirus/patogenicidad , Linfocitos T CD4-Positivos/efectos de los fármacos , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/virología , Linfocitos T CD8-positivos/efectos de los fármacos , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/virología , COVID-19 , Vacunas contra la COVID-19 , Infecciones por Coronavirus/genética , Infecciones por Coronavirus/inmunología , Infecciones por Coronavirus/patología , Modelos Animales de Enfermedad , Furina/genética , Furina/inmunología , Humanos , Inmunidad Humoral/efectos de los fármacos , Inmunización/métodos , Inmunogenicidad Vacunal , Memoria Inmunológica/efectos de los fármacos , Activación de Linfocitos/efectos de los fármacos , Ratones , Ratones Endogámicos BALB C , Nanopartículas/administración & dosificación , Nanopartículas/química , Neumonía Viral/inmunología , Neumonía Viral/patología , ARN Mensajero/genética , ARN Viral/genética , SARS-CoV-2 , Glicoproteína de la Espiga del Coronavirus/genética , Glicoproteína de la Espiga del Coronavirus/inmunología , Vacunas Sintéticas , Vacunas Virales/biosíntesis , Vacunas Virales/genéticaRESUMEN
The protective efficacy of serum antibodies results from the interplay of antigen-specific B cell clones of different affinities and specificities. These cellular dynamics underlie serum-level phenomena such as original antigenic sin (OAS)-a proposed propensity of the immune system to rely repeatedly on the first cohort of B cells engaged by an antigenic stimulus when encountering related antigens, in detriment to the induction of de novo responses1-5. OAS-type suppression of new, variant-specific antibodies may pose a barrier to vaccination against rapidly evolving viruses such as influenza and SARS-CoV-26,7. Precise measurement of OAS-type suppression is challenging because cellular and temporal origins cannot readily be ascribed to antibodies in circulation; its effect on subsequent antibody responses therefore remains unclear5,8. Here we introduce a molecular fate-mapping approach with which serum antibodies derived from specific cohorts of B cells can be differentially detected. We show that serum responses to sequential homologous boosting derive overwhelmingly from primary cohort B cells, while later induction of new antibody responses from naive B cells is strongly suppressed. Such 'primary addiction' decreases sharply as a function of antigenic distance, allowing reimmunization with divergent viral glycoproteins to produce de novo antibody responses targeting epitopes that are absent from the priming variant. Our findings have implications for the understanding of OAS and for the design and testing of vaccines against evolving pathogens.
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Formación de Anticuerpos , Linfocitos B , Inmunización Secundaria , Humanos , Anticuerpos Antivirales/biosíntesis , Anticuerpos Antivirales/sangre , Anticuerpos Antivirales/inmunología , Antígenos Virales/inmunología , Vacunas contra la Influenza/inmunología , SARS-CoV-2/inmunología , Vacunación , Linfocitos B/inmunología , Vacunas Virales/inmunologíaRESUMEN
Gene-editing technologies, which include the CRISPR-Cas nucleases1-3 and CRISPR base editors4,5, have the potential to permanently modify disease-causing genes in patients6. The demonstration of durable editing in target organs of nonhuman primates is a key step before in vivo administration of gene editors to patients in clinical trials. Here we demonstrate that CRISPR base editors that are delivered in vivo using lipid nanoparticles can efficiently and precisely modify disease-related genes in living cynomolgus monkeys (Macaca fascicularis). We observed a near-complete knockdown of PCSK9 in the liver after a single infusion of lipid nanoparticles, with concomitant reductions in blood levels of PCSK9 and low-density lipoprotein cholesterol of approximately 90% and about 60%, respectively; all of these changes remained stable for at least 8 months after a single-dose treatment. In addition to supporting a 'once-and-done' approach to the reduction of low-density lipoprotein cholesterol and the treatment of atherosclerotic cardiovascular disease (the leading cause of death worldwide7), our results provide a proof-of-concept for how CRISPR base editors can be productively applied to make precise single-nucleotide changes in therapeutic target genes in the liver, and potentially in other organs.
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Sistemas CRISPR-Cas , LDL-Colesterol/sangre , Edición Génica , Modelos Animales , Proproteína Convertasa 9/genética , Adenina/metabolismo , Animales , Células Cultivadas , Femenino , Hepatocitos/metabolismo , Humanos , Hígado/enzimología , Mutación con Pérdida de Función , Macaca fascicularis/sangre , Macaca fascicularis/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Mutagénesis Sitio-Dirigida , Proproteína Convertasa 9/sangre , Proproteína Convertasa 9/metabolismo , Factores de TiempoRESUMEN
The use of lipid nanoparticles (LNP) to encapsulate and deliver mRNA has become an important therapeutic advance. In addition to vaccines, LNP-mRNA can be used in many other applications. For example, targeting the LNP with anti-CD5 antibodies (CD5/tLNP) can allow for efficient delivery of mRNA payloads to T cells to express protein. As the percentage of protein expressing T cells induced by an intravenous injection of CD5/tLNP is relatively low (4-20%), our goal was to find ways to increase mRNA-induced translation efficiency. We showed that T cell activation using an anti-CD3 antibody improved protein expression after CD5/tLNP transfection in vitro but not in vivo. T cell health and activation can be increased with cytokines, therefore, using mCherry mRNA as a reporter, we found that culturing either mouse or human T cells with the cytokine IL7 significantly improved protein expression of delivered mRNA in both CD4+ and CD8+ T cells in vitro. By pre-treating mice with systemic IL7 followed by tLNP administration, we observed significantly increased mCherry protein expression by T cells in vivo. Transcriptomic analysis of mouse T cells treated with IL7 in vitro revealed enhanced genomic pathways associated with protein translation. Improved translational ability was demonstrated by showing increased levels of protein expression after electroporation with mCherry mRNA in T cells cultured in the presence of IL7, but not with IL2 or IL15. These data show that IL7 selectively increases protein translation in T cells, and this property can be used to improve expression of tLNP-delivered mRNA in vivo.
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Linfocitos T CD4-Positivos , Linfocitos T CD8-positivos , Interleucina-7 , Liposomas , Nanopartículas , Biosíntesis de Proteínas , ARN Mensajero , Animales , Humanos , Ratones , Linfocitos T CD8-positivos/efectos de los fármacos , Linfocitos T CD8-positivos/metabolismo , Interleucina-7/farmacología , Biosíntesis de Proteínas/efectos de los fármacos , ARN Mensajero/metabolismo , Ratones Endogámicos C57BL , Células Cultivadas , Linfocitos T CD4-Positivos/efectos de los fármacos , Linfocitos T CD4-Positivos/inmunologíaRESUMEN
ï¡-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 ï¡-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 (ALS20ï¡I). Myeloablated mice transplanted with mRNACreLNPCD117-treated HSC showed a complete knockout of ï¡-globin genes. They demonstrated a phenotype characterized by the synthesis of hemoglobin H (ï¢-tetramers, ï¢ï´ 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 ALS20ï¡I survived long-term with normalization of erythropoiesis, decreased the production of HbH, and ameliorated the abnormal organ morphology. Furthermore, we tested ALS20ï¡I in erythroid progenitors derived from ï¡-globin-KO CD34+ and cells isolated from patients with both deletional and non-deletional HbH disease, demonstrating improvement in ï¡-globin/ï¢-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 ALS20ï¡I for treating AT.
RESUMEN
Malaria caused by Plasmodium falciparum remains the leading single-agent cause of mortality in children1, yet the promise of an effective vaccine has not been fulfilled. Here, using our previously described differential screening method to analyse the proteome of blood-stage P. falciparum parasites2, we identify P. falciparum glutamic-acid-rich protein (PfGARP) as a parasite antigen that is recognized by antibodies in the plasma of children who are relatively resistant-but not those who are susceptible-to malaria caused by P. falciparum. PfGARP is a parasite antigen of 80 kDa that is expressed on the exofacial surface of erythrocytes infected by early-to-late-trophozoite-stage parasites. We demonstrate that antibodies against PfGARP kill trophozoite-infected erythrocytes in culture by inducing programmed cell death in the parasites, and that vaccinating non-human primates with PfGARP partially protects against a challenge with P. falciparum. Furthermore, our longitudinal cohort studies showed that, compared to individuals who had naturally occurring anti-PfGARP antibodies, Tanzanian children without anti-PfGARP antibodies had a 2.5-fold-higher risk of severe malaria and Kenyan adolescents and adults without these antibodies had a twofold-higher parasite density. By killing trophozoite-infected erythrocytes, PfGARP could synergize with other vaccines that target parasite invasion of hepatocytes or the invasion of and egress from erythrocytes.
Asunto(s)
Apoptosis/inmunología , Péptidos y Proteínas de Señalización Intercelular/inmunología , Malaria Falciparum/inmunología , Malaria Falciparum/prevención & control , Parásitos/inmunología , Plasmodium falciparum/citología , Plasmodium falciparum/inmunología , Proteínas Protozoarias/inmunología , Adolescente , Adulto , Animales , Anticuerpos Antiprotozoarios/inmunología , Antígenos de Protozoos/química , Antígenos de Protozoos/inmunología , Aotidae/inmunología , Aotidae/parasitología , Caspasas/metabolismo , Niño , Estudios de Cohortes , ADN Protozoario/química , ADN Protozoario/metabolismo , Activación Enzimática , Eritrocitos/parasitología , Femenino , Humanos , Péptidos y Proteínas de Señalización Intercelular/química , Kenia , Vacunas contra la Malaria/inmunología , Malaria Falciparum/parasitología , Masculino , Ratones , Parásitos/citología , Parásitos/crecimiento & desarrollo , Plasmodium falciparum/crecimiento & desarrollo , Proteínas Protozoarias/química , Tanzanía , Trofozoítos/citología , Trofozoítos/crecimiento & desarrollo , Trofozoítos/inmunología , Vacuolas/inmunologíaRESUMEN
Nucleoside-modified mRNA technology has revolutionized vaccine development with the success of mRNA COVID-19 vaccines. We used modified mRNA technology for the design of envelopes (Env) to induce HIV-1 broadly neutralizing antibodies (bnAbs). However, unlike SARS-CoV-2 neutralizing antibodies that are readily made, HIV-1 bnAb induction is disfavored by the immune system because of the rarity of bnAb B cell precursors and the cross-reactivity of bnAbs targeting certain Env epitopes with host molecules, thus requiring optimized immunogen design. The use of protein nanoparticles (NPs) has been reported to enhance B cell germinal center responses to HIV-1 Env. Here, we report our experience with the expression of Env-ferritin NPs compared with membrane-bound Env gp160 when encoded by modified mRNA. We found that well-folded Env-ferritin NPs were a minority of the protein expressed by an mRNA design and were immunogenic at 20 µg but minimally immunogenic in mice at 1 µg dose in vivo and were not expressed well in draining lymph nodes (LNs) following intramuscular immunization. In contrast, mRNA encoding gp160 was more immunogenic than mRNA encoding Env-NP at 1 µg dose and was expressed well in draining LN following intramuscular immunization. Thus, analysis of mRNA expression in vitro and immunogenicity at low doses in vivo are critical for the evaluation of mRNA designs for optimal immunogenicity of HIV-1 immunogens.IMPORTANCEAn effective HIV-1 vaccine that induces protective antibody responses remains elusive. We have used mRNA technology for designs of HIV-1 immunogens in the forms of membrane-bound full-length envelope gp160 and envelope ferritin nanoparticle. Here, we demonstrated in a mouse model that the membrane-bound form induced a better response than envelope ferritin nanoparticle because of higher in vivo protein expression. The significance of our research is in highlighting the importance of analysis of mRNA design expression and low-dose immunogenicity studies for HIV-1 immunogens before moving to vaccine clinical trials.
RESUMEN
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.
Asunto(s)
Microbiota , Vacunas de ADN , Ratones , Animales , Vacunación Basada en Ácidos Nucleicos , Linfocitos T CD8-positivos , ADN , ARN Mensajero , Inmunización SecundariaRESUMEN
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.
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Anticuerpos Antivirales , Linfocitos T CD8-positivos , Vacunas contra la COVID-19 , COVID-19 , SARS-CoV-2 , Glicoproteína de la Espiga del Coronavirus , Animales , Linfocitos T CD8-positivos/inmunología , Ratones , SARS-CoV-2/inmunología , COVID-19/inmunología , COVID-19/prevención & control , Femenino , Glicoproteína de la Espiga del Coronavirus/inmunología , Glicoproteína de la Espiga del Coronavirus/genética , Vacunas contra la COVID-19/inmunología , Masculino , Anticuerpos Antivirales/inmunología , Ratones Endogámicos C57BL , Humanos , Modelos Animales de EnfermedadRESUMEN
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.
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Barrera Hematoencefálica , Modelos Animales de Enfermedad , Accidente Cerebrovascular Isquémico , Liposomas , Nanopartículas , Molécula 1 de Adhesión Celular Vascular , Barrera Hematoencefálica/metabolismo , Barrera Hematoencefálica/efectos de los fármacos , Animales , Ratones , Molécula 1 de Adhesión Celular Vascular/metabolismo , Molécula 1 de Adhesión Celular Vascular/genética , Nanopartículas/química , Accidente Cerebrovascular Isquémico/metabolismo , Accidente Cerebrovascular Isquémico/tratamiento farmacológico , Lípidos/química , Sistemas de Liberación de Medicamentos/métodos , Infarto de la Arteria Cerebral Media/metabolismo , Infarto de la Arteria Cerebral Media/tratamiento farmacológico , HumanosRESUMEN
Combined vaccine formulations targeting not only hemagglutinin but also other influenza virus antigens could form the basis for a universal influenza virus vaccine that has the potential to elicit long-lasting, broadly cross-reactive immune responses. Lipid nanoparticle (LNP)-encapsulated messenger RNA (mRNA) vaccines can be utilized to efficiently target multiple antigens with a single vaccine. Here, we assessed the immunogenicity and protective efficacy of nucleoside-modified mRNA-LNP vaccines that contain four influenza A group 2 virus antigens (hemagglutinin stalk, neuraminidase, matrix protein 2, and nucleoprotein) in mice. We found that all vaccine components induced antigen-specific cellular and humoral immune responses after administration of a single dose. While the monovalent formulations were not exclusively protective, the combined quadrivalent formulation protected mice from all challenge viruses, including a relevant H1N1 influenza virus group 1 strain, with minimal weight loss. Importantly, the combined vaccine protected from morbidity at a dose of 125 ng per antigen after a single vaccination in mice. With these findings, we confidently conclude that the nucleoside-modified mRNA-LNP platform can be used to elicit protection against a large panel of influenza viruses.
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Subtipo H1N1 del Virus de la Influenza A , Vacunas contra la Influenza , Gripe Humana , Infecciones por Orthomyxoviridae , Ratones , Animales , Humanos , Subtipo H1N1 del Virus de la Influenza A/genética , Nucleósidos , Hemaglutininas , Vacunas Combinadas , ARN Mensajero/genética , Anticuerpos Antivirales , Vacunación , Glicoproteínas Hemaglutininas del Virus de la Influenza , Vacunas de ARNmRESUMEN
Infection by Kaposi sarcoma-associated herpesvirus (KSHV) can cause severe consequences, such as cancers and lymphoproliferative diseases. Whole inactivated viruses (WIV) with chemically destroyed genetic materials have been used as antigens in several licensed vaccines. During KSHV productive replication, virus-like vesicles (VLVs) that lack capsids and viral genomes are generated along with virions. Here, we investigated the immunogenicity of KSHV VLVs produced from a viral mutant that was defective in capsid formation and DNA packaging. Mice immunized with adjuvanted VLVs generated KSHV-specific T cell and antibody responses. Neutralization of KSHV infection by the VLV immune serum was low but was markedly enhanced in the presence of the complement system. Complement-enhanced neutralization and complement deposition on KSHV-infected cells was dependent on antibodies targeting viral open reading frame 4 (ORF4). However, limited complement-mediated enhancement was detected in the sera of a small cohort of KSHV-infected humans which contained few neutralizing antibodies. Therefore, vaccination that induces antibody effector functions can potentially improve infection-induced humoral immunity. Overall, our study highlights a potential benefit of engaging complement-mediated antibody functions in future KSHV vaccine development. IMPORTANCE KSHV is a virus that can lead to cancer after infection. A vaccine that prevents KSHV infection or transmission would be helpful in preventing the development of these cancers. We investigated KSHV VLV as an immunogen for vaccination. We determined that antibodies targeting the viral protein ORF4 induced by VLV immunization could engage the complement system and neutralize viral infection. However, ORF4-specific antibodies were seldom detected in the sera of KSHV-infected humans. Moreover, these human sera did not potently trigger complement-mediated neutralization, indicating an improvement that immunization can confer. Our study suggests a new antibody-mediated mechanism to control KSHV infection and underscores the benefit of activating the complement system in a future KSHV vaccine.
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Anticuerpos Neutralizantes , Herpesvirus Humano 8 , Animales , Humanos , Ratones , Anticuerpos Neutralizantes/inmunología , Infecciones por Herpesviridae , Herpesvirus Humano 8/inmunología , Sistemas de Lectura Abierta/inmunología , Vacunación , Proteínas Virales/inmunologíaRESUMEN
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.
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COVID-19 , Enfermedad de Lyme , Humanos , Animales , Ratones , Vacunas contra la COVID-19 , COVID-19/prevención & control , SARS-CoV-2 , Enfermedad de Lyme/prevención & control , Antígenos de Superficie/genética , Proteínas de la Membrana Bacteriana Externa/genéticaRESUMEN
Crimean-Congo hemorrhagic fever (CCHF), caused by Crimean-Congo hemorrhagic fever virus (CCHFV), is on the World Health Organizations' list of prioritized diseases and pathogens. With global distribution, high fatality rate, and no approved vaccine or effective treatment, CCHF constitutes a threat against global health. In the current study, we demonstrate that vaccination with nucleoside-modified mRNA-lipid nanoparticles (mRNA-LNP), encoding for the CCHFV nucleoprotein (N) or glycoproteins (GcGn) protect IFNAR-/- mice against lethal CCHFV infection. In addition, we found that both mRNA-LNP induced strong humoral and cellular immune responses in IFNAR-/- and immunocompetent mice and that neutralizing antibodies are not necessary for protection. When evaluating immune responses induced by immunization including CCHFV Gc and Gn antigens, we found the Gc protein to be more immunogenic compared with the Gn protein. Hepatic injury is prevalent in CCHF and contributes to the severity and mortality of the disease in humans. Thus, to understand the immune response in the liver after infection and the potential effect of the vaccine, we performed a proteomic analysis on liver samples from vaccinated and control mice after CCHFV infection. Similar to observations in humans, vaccination affected the metabolic pathways. In conclusion, this study shows that a CCHFV mRNA-LNP vaccine, based on viral nucleo- or glycoproteins, mediate protection against CCHFV induced disease. Consequently, genetic immunization is an attractive approach to prevent disease caused by CCHFV and we believe we have necessary evidence to bring this vaccine platform to the next step in the development of a vaccine against CCHFV infection. IMPORTANCE Crimean-Congo hemorrhagic fever virus (CCHFV) is a zoonotic pathogen causing Crimean-Congo hemorrhagic fever (CCHF), a severe fever disease. CCHFV has a wide distribution and is endemic in several areas around the world. Cases of CCHF are also being reported in new areas, indicating an expansion of the disease, which is of high concern. Dispersion of the disease, high fatality rate, and no approved vaccine makes CCHF a threat to global health. The development of a vaccine is thus of great importance. Here we show 100% protection against lethal CCHFV infection in mice immunized with mRNA-LNP encoding for different CCHFV proteins. The vaccination showed both robust humoral and cellular immunity. mRNA-LNP vaccines combine the ability to induce an effective immune response, the safety of a transient carrier, and the flexibility of genetic vaccines. This and our results from the current study support the development of a mRNA-LNP based vaccine against CCHFV.
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Virus de la Fiebre Hemorrágica de Crimea-Congo/inmunología , Fiebre Hemorrágica de Crimea/prevención & control , Receptor de Interferón alfa y beta/deficiencia , Vacunas Sintéticas/inmunología , Vacunas de ARNm/inmunología , Animales , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , Biología Computacional/métodos , Modelos Animales de Enfermedad , Relación Dosis-Respuesta Inmunológica , Femenino , Ensayos Analíticos de Alto Rendimiento , Inmunización , Inmunogenicidad Vacunal , Liposomas , Ratones , Ratones Noqueados , Nanopartículas , Proteómica/métodos , VacunaciónRESUMEN
Zika virus (ZIKV) has recently emerged as a pandemic associated with severe neuropathology in newborns and adults. There are no ZIKV-specific treatments or preventatives. Therefore, the development of a safe and effective vaccine is a high priority. Messenger RNA (mRNA) has emerged as a versatile and highly effective platform to deliver vaccine antigens and therapeutic proteins. Here we demonstrate that a single low-dose intradermal immunization with lipid-nanoparticle-encapsulated nucleoside-modified mRNA (mRNA-LNP) encoding the pre-membrane and envelope glycoproteins of a strain from the ZIKV outbreak in 2013 elicited potent and durable neutralizing antibody responses in mice and non-human primates. Immunization with 30 µg of nucleoside-modified ZIKV mRNA-LNP protected mice against ZIKV challenges at 2 weeks or 5 months after vaccination, and a single dose of 50 µg was sufficient to protect non-human primates against a challenge at 5 weeks after vaccination. These data demonstrate that nucleoside-modified mRNA-LNP elicits rapid and durable protective immunity and therefore represents a new and promising vaccine candidate for the global fight against ZIKV.
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ARN Mensajero/administración & dosificación , ARN Mensajero/química , Vacunas Virales/inmunología , Infección por el Virus Zika/prevención & control , Virus Zika/inmunología , Animales , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , Antígenos Virales/genética , Antígenos Virales/inmunología , Femenino , Glicoproteínas/genética , Glicoproteínas/inmunología , Inyecciones Intradérmicas , Macaca mulatta/inmunología , Macaca mulatta/virología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Nanopartículas/administración & dosificación , Nanopartículas/química , Estabilidad del ARN , ARN Mensajero/genética , ARN Viral/administración & dosificación , ARN Viral/química , ARN Viral/genética , Factores de Tiempo , Vacunación , Proteínas del Envoltorio Viral/genética , Proteínas del Envoltorio Viral/inmunología , Vacunas Virales/administración & dosificación , Virus Zika/química , Virus Zika/genética , Infección por el Virus Zika/inmunologíaRESUMEN
Persistence of chronic hepatitis B (CHB) is attributed to maintenance of the intrahepatic pool of the viral covalently closed circular DNA (cccDNA), which serves as the transcriptional template for all viral gene products required for replication. Current nucleos(t)ide therapies for CHB prevent virus production and spread but have no direct impact on cccDNA or expression of viral genes. We describe a potential curative approach using a highly specific engineered ARCUS nuclease (ARCUS-POL) targeting the hepatitis B virus (HBV) genome. Transient ARCUS-POL expression in HBV-infected primary human hepatocytes produced substantial reductions in both cccDNA and hepatitis B surface antigen (HBsAg). To evaluate ARCUS-POL in vivo, we developed episomal adeno-associated virus (AAV) mouse and non-human primate (NHP) models containing a portion of the HBV genome serving as a surrogate for cccDNA. Clinically relevant delivery was achieved through systemic administration of lipid nanoparticles containing ARCUS-POL mRNA. In both mouse and NHP, we observed a significant decrease in total AAV copy number and high on-target indel frequency. In the case of the mouse model, which supports HBsAg expression, circulating surface antigen was durably reduced by 96%. Together, these data support a gene-editing approach for elimination of cccDNA toward an HBV cure.