RESUMO
The development of a portfolio of COVID-19 vaccines to vaccinate the global population remains an urgent public health imperative1. Here we demonstrate the capacity of a subunit vaccine, comprising the SARS-CoV-2 spike protein receptor-binding domain displayed on an I53-50 protein nanoparticle scaffold (hereafter designated RBD-NP), to stimulate robust and durable neutralizing-antibody responses and protection against SARS-CoV-2 in rhesus macaques. We evaluated five adjuvants including Essai O/W 1849101, a squalene-in-water emulsion; AS03, an α-tocopherol-containing oil-in-water emulsion; AS37, a Toll-like receptor 7 (TLR7) agonist adsorbed to alum; CpG1018-alum, a TLR9 agonist formulated in alum; and alum. RBD-NP immunization with AS03, CpG1018-alum, AS37 or alum induced substantial neutralizing-antibody and CD4 T cell responses, and conferred protection against SARS-CoV-2 infection in the pharynges, nares and bronchoalveolar lavage. The neutralizing-antibody response to live virus was maintained up to 180 days after vaccination with RBD-NP in AS03 (RBD-NP-AS03), and correlated with protection from infection. RBD-NP immunization cross-neutralized the B.1.1.7 SARS-CoV-2 variant efficiently but showed a reduced response against the B.1.351 variant. RBD-NP-AS03 produced a 4.5-fold reduction in neutralization of B.1.351 whereas the group immunized with RBD-NP-AS37 produced a 16-fold reduction in neutralization of B.1.351, suggesting differences in the breadth of the neutralizing-antibody response induced by these adjuvants. Furthermore, RBD-NP-AS03 was as immunogenic as a prefusion-stabilized spike immunogen (HexaPro) with AS03 adjuvant. These data highlight the efficacy of the adjuvanted RBD-NP vaccine in promoting protective immunity against SARS-CoV-2 and have led to phase I/II clinical trials of this vaccine (NCT04742738 and NCT04750343).
Assuntos
Adjuvantes Imunológicos , Anticorpos Neutralizantes/imunologia , Vacinas contra COVID-19/imunologia , COVID-19/imunologia , COVID-19/prevenção & controle , SARS-CoV-2/imunologia , Vacinas de Subunidades Antigênicas/imunologia , Compostos de Alúmen , Animais , Anticorpos Antivirais/imunologia , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/imunologia , COVID-19/virologia , Ensaios Clínicos Fase I como Assunto , Ensaios Clínicos Fase II como Assunto , Modelos Animais de Doenças , Imunidade Celular , Imunidade Humoral , Macaca mulatta/imunologia , Masculino , Oligodesoxirribonucleotídeos , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/imunologia , EsqualenoRESUMO
Preclinical mouse models that recapitulate some characteristics of coronavirus disease (COVID-19) will facilitate focused study of pathogenesis and virus-host responses. Human agniotensin-converting enzyme 2 (hACE2) serves as an entry receptor for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) to infect people via binding to envelope spike proteins. Herein we report development and characterization of a rapidly deployable COVID-19 mouse model. C57BL/6J (B6) mice expressing hACE2 in the lung were transduced by oropharyngeal delivery of the recombinant human adenovirus type 5 that expresses hACE2 (Ad5-hACE2). Mice were infected with SARS-CoV-2 at Day 4 after transduction and developed interstitial pneumonia associated with perivascular inflammation, accompanied by significantly higher viral load in lungs at Days 3, 6, and 12 after infection compared with Ad5-empty control group. SARS-CoV-2 was detected in pneumocytes in alveolar septa. Transcriptomic analysis of lungs demonstrated that the infected Ad5-hACE mice had a significant increase in IFN-dependent chemokines Cxcl9 and Cxcl10, and genes associated with effector T-cell populations including Cd3 g, Cd8a, and Gzmb. Pathway analysis showed that several Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways were enriched in the data set, including cytokine-cytokine receptor interaction, the chemokine signaling pathway, the NOD-like receptor signaling pathway, the measles pathway, and the IL-17 signaling pathway. This response is correlative to clinical response in lungs of patients with COVID-19. These results demonstrate that expression of hACE2 via adenovirus delivery system sensitized the mouse to SARS-CoV-2 infection and resulted in the development of a mild COVID-19 phenotype, highlighting the immune and inflammatory host responses to SARS-CoV-2 infection. This rapidly deployable COVID-19 mouse model is useful for preclinical and pathogenesis studies of COVID-19.
Assuntos
Células Epiteliais Alveolares/imunologia , COVID-19/imunologia , Expressão Gênica , SARS-CoV-2/imunologia , Transdução de Sinais/imunologia , Adenoviridae/genética , Adenoviridae/metabolismo , Células Epiteliais Alveolares/metabolismo , Células Epiteliais Alveolares/virologia , Enzima de Conversão de Angiotensina 2/biossíntese , Enzima de Conversão de Angiotensina 2/genética , Enzima de Conversão de Angiotensina 2/imunologia , Animais , COVID-19/genética , COVID-19/metabolismo , COVID-19/patologia , Citocinas/genética , Citocinas/imunologia , Modelos Animais de Doenças , Humanos , Camundongos , Camundongos Transgênicos , SARS-CoV-2/genética , SARS-CoV-2/metabolismo , Transdução de Sinais/genética , Transdução GenéticaRESUMO
Although antivirals are important tools to control severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, effective vaccines are essential to control the current coronavirus disease 2019 (COVID-19) pandemic. Plant-derived virus-like particle (VLP) vaccine candidates have previously demonstrated immunogenicity and efficacy against influenza. Here, we report the immunogenicity and protection induced in rhesus macaques by intramuscular injections of a VLP bearing a SARS-CoV-2 spike protein (CoVLP) vaccine candidate formulated with or without Adjuvant System 03 (AS03) or cytidine-phospho-guanosine (CpG) 1018. Although a single dose of the unadjuvanted CoVLP vaccine candidate stimulated humoral and cell-mediated immune responses, booster immunization (at 28 days after priming) and adjuvant administration significantly improved both responses, with higher immunogenicity and protection provided by the AS03-adjuvanted CoVLP. Fifteen micrograms of CoVLP adjuvanted with AS03 induced a polyfunctional interleukin-2 (IL-2)-driven response and IL-4 expression in CD4 T cells. Animals were challenged by multiple routes (i.e., intratracheal, intranasal, and ocular) with a total viral dose of 106 plaque-forming units of SARS-CoV-2. Lower viral replication in nasal swabs and bronchoalveolar lavage fluid (BALF) as well as fewer SARS-CoV-2-infected cells and immune cell infiltrates in the lungs concomitant with reduced levels of proinflammatory cytokines and chemotactic factors in the BALF were observed in animals immunized with the CoVLP adjuvanted with AS03. No clinical, pathologic, or virologic evidence of vaccine-associated enhanced disease was observed in vaccinated animals. The CoVLP adjuvanted with AS03 was therefore selected for vaccine development and clinical trials.
Assuntos
Adjuvantes Imunológicos/efeitos adversos , Vacinas contra COVID-19/efeitos adversos , COVID-19/imunologia , COVID-19/prevenção & controle , Imunogenicidade da Vacina/imunologia , Nicotiana/metabolismo , Pandemias/prevenção & controle , Polissorbatos/efeitos adversos , SARS-CoV-2/imunologia , Esqualeno/efeitos adversos , Vacinação/métodos , Vacinas de Partículas Semelhantes a Vírus/efeitos adversos , alfa-Tocoferol/efeitos adversos , Adjuvantes Imunológicos/administração & dosagem , Animais , Anticorpos Neutralizantes/sangue , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/sangue , Anticorpos Antivirais/imunologia , COVID-19/epidemiologia , COVID-19/virologia , Vacinas contra COVID-19/administração & dosagem , Modelos Animais de Doenças , Combinação de Medicamentos , Composição de Medicamentos/métodos , Imunidade Humoral , Macaca mulatta , Masculino , Polissorbatos/administração & dosagem , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismo , Glicoproteína da Espícula de Coronavírus/imunologia , Glicoproteína da Espícula de Coronavírus/metabolismo , Esqualeno/administração & dosagem , Resultado do Tratamento , Vacinas de Partículas Semelhantes a Vírus/administração & dosagem , alfa-Tocoferol/administração & dosagemRESUMO
Neurological manifestations are a significant complication of coronavirus disease (COVID-19), but underlying mechanisms aren't well understood. The development of animal models that recapitulate the neuropathological findings of autopsied brain tissue from patients who died from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection are critical for elucidating the neuropathogenesis of infection and disease. Here, we show neuroinflammation, microhemorrhages, brain hypoxia, and neuropathology that is consistent with hypoxic-ischemic injury in SARS-CoV-2 infected non-human primates (NHPs), including evidence of neuron degeneration and apoptosis. Importantly, this is seen among infected animals that do not develop severe respiratory disease, which may provide insight into neurological symptoms associated with "long COVID". Sparse virus is detected in brain endothelial cells but does not associate with the severity of central nervous system (CNS) injury. We anticipate our findings will advance our current understanding of the neuropathogenesis of SARS-CoV-2 infection and demonstrate SARS-CoV-2 infected NHPs are a highly relevant animal model for investigating COVID-19 neuropathogenesis among human subjects.
Assuntos
COVID-19 , SARS-CoV-2 , Animais , Encéfalo , Células Endoteliais , Humanos , PrimatasRESUMO
The development of a portfolio of SARS-CoV-2 vaccines to vaccinate the global population remains an urgent public health imperative. Here, we demonstrate the capacity of a subunit vaccine under clinical development, comprising the SARS-CoV-2 Spike protein receptor-binding domain displayed on a two-component protein nanoparticle (RBD-NP), to stimulate robust and durable neutralizing antibody (nAb) responses and protection against SARS-CoV-2 in non-human primates. We evaluated five different adjuvants combined with RBD-NP including Essai O/W 1849101, a squalene-in-water emulsion; AS03, an alpha-tocopherol-containing squalene-based oil-in-water emulsion used in pandemic influenza vaccines; AS37, a TLR-7 agonist adsorbed to Alum; CpG 1018-Alum (CpG-Alum), a TLR-9 agonist formulated in Alum; or Alum, the most widely used adjuvant. All five adjuvants induced substantial nAb and CD4 T cell responses after two consecutive immunizations. Durable nAb responses were evaluated for RBD-NP/AS03 immunization and the live-virus nAb response was durably maintained up to 154 days post-vaccination. AS03, CpG-Alum, AS37 and Alum groups conferred significant protection against SARS-CoV-2 infection in the pharynges, nares and in the bronchoalveolar lavage. The nAb titers were highly correlated with protection against infection. Furthermore, RBD-NP when used in conjunction with AS03 was as potent as the prefusion stabilized Spike immunogen, HexaPro. Taken together, these data highlight the efficacy of the RBD-NP formulated with clinically relevant adjuvants in promoting robust immunity against SARS-CoV-2 in non-human primates.
RESUMO
Simian immunodeficiency virus SIVrcm, which naturally infects red-capped mangabeys (RCMs), is the only SIV that uses CCR2 as its main coreceptor due to the high frequency of a CCR5 deletion in RCMs. We investigated the dynamics of SIVrcm infection to identify specific pathogenic mechanisms associated with this major difference in SIV biology. Four pigtailed macaques (PTMs) were infected with SIVrcm, and infection was monitored for over 2 years. The dynamics of in vivo SIVrcm replication in PTMs was similar to that of other pathogenic and nonpathogenic lymphotropic SIVs. Plasma viral loads (VLs) peaked at 10(7) to 10(9) SIVrcm RNA copies/ml by day 10 postinoculation (p.i.). A viral set point was established by day 42 p.i. at 10(3) to 10(5) SIVrcm RNA copies/ml and lasted up to day 180 p.i., when plasma VLs decreased below the threshold of detection, with blips of viral replication during the follow-up. Intestinal SIVrcm replication paralleled that of plasma VLs. Up to 80% of the CD4(+) T cells were depleted by day 28 p.i. in the gut. The most significant depletion (>90%) involved memory CD4(+) T cells. Partial CD4(+) T-cell restoration was observed in the intestine at later time points. Effector memory CD4(+) T cells were the least restored. SIVrcm strains isolated from acutely infected PTMs used CCR2 coreceptor, as reported, but expansion of coreceptor usage to CCR4 was also observed. Selective depletion of effector memory CD4(+) T cells is in contrast with predicted in vitro tropism of SIVrcm for macrophages and is probably due to expansion of coreceptor usage. Taken together, these findings emphasize the importance of understanding the selective forces driving viral adaptation to a new host.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Infecções por HIV/imunologia , Receptores CCR2/imunologia , Receptores de HIV/imunologia , Síndrome de Imunodeficiência Adquirida dos Símios/imunologia , Vírus da Imunodeficiência Símia/fisiologia , Animais , Linfócitos T CD4-Positivos/virologia , Células Cultivadas , Modelos Animais de Doenças , Expressão Gênica , HIV/fisiologia , Infecções por HIV/virologia , Humanos , Leucócitos Mononucleares/metabolismo , Leucócitos Mononucleares/virologia , Macaca nemestrina , Dados de Sequência Molecular , Receptores CCR2/genética , Receptores CCR4/genética , Receptores CCR4/imunologia , Receptores de HIV/genética , Síndrome de Imunodeficiência Adquirida dos Símios/genética , Síndrome de Imunodeficiência Adquirida dos Símios/virologia , Vírus da Imunodeficiência Símia/genética , Vírus da Imunodeficiência Símia/imunologia , Carga Viral , Internalização do Vírus , Replicação ViralRESUMO
Chronically SIVagm-infected African green monkeys (AGMs) have a remarkably stable nonpathogenic disease course, with levels of immune activation in chronic SIVagm infection similar to those observed in uninfected monkeys and with stable viral loads for long periods of time. In vivo administration of LPS or an IL-2/diphtheria toxin fusion protein (Ontak) to chronically SIVagm-infected AGMs triggered increases in immune activation and subsequently of viral replication and depletion of intestinal CD4(+) T cells. Our study indicates that circulating microbial products can increase viral replication by inducing immune activation and increasing the number of viral target cells, thus demonstrating that immune activation and T cell proliferation are key factors in AIDS pathogenesis.
Assuntos
Adjuvantes Imunológicos , Linfócitos T CD4-Positivos/imunologia , Ativação Linfocitária/imunologia , Síndrome de Imunodeficiência Adquirida dos Símios/imunologia , Vírus da Imunodeficiência Símia/imunologia , Animais , Linfócitos T CD4-Positivos/virologia , Chlorocebus aethiops , Doença Crônica , Toxina Diftérica/imunologia , Citometria de Fluxo , Interleucina-2/imunologia , Lipopolissacarídeos/imunologia , Proteínas Recombinantes de Fusão/imunologia , Síndrome de Imunodeficiência Adquirida dos Símios/virologia , Carga Viral , Replicação Viral/imunologiaRESUMO
The mechanisms underlying the lack of disease progression in natural simian immunodeficiency virus (SIV) hosts are still poorly understood. To test the hypothesis that SIV-infected African green monkeys (AGMs) avoid AIDS due to virus replication occurring in long-lived infected cells, we infected six animals with SIVagm and treated them with potent antiretroviral therapy [ART; 9-R-(2-phosphonomethoxypropyl) adenine (tenofovir) and beta-2,3-dideoxy-3-thia-5-fluorocytidine (emtricitabine)]. All AGMs showed a rapid decay of plasma viremia that became undetectable 36 h after ART initiation. A significant decrease of viral load was observed in peripheral blood mononuclear cells and intestine. Mathematical modeling of viremia decay post-ART indicates a half-life of productively infected cells ranging from 4 to 9.5 h, i.e., faster than previously reported for human immunodeficiency virus and SIV. ART induced a slight but significant increase in peripheral CD4(+) T-cell counts but no significant changes in CD4(+) T-cell levels in lymph nodes and intestine. Similarly, ART did not significantly change the levels of cell proliferation, activation, and apoptosis, already low in AGMs chronically infected with SIVagm. Collectively, these results indicate that, in SIVagm-infected AGMs, the bulk of virus replication is sustained by short-lived cells; therefore, differences in disease outcome between SIVmac infection of macaques and SIVagm infection of AGMs are unlikely due to intrinsic differences in the in vivo cytopathicities between the two viruses.
Assuntos
Chlorocebus aethiops/virologia , Vírus da Imunodeficiência Símia/crescimento & desenvolvimento , Animais , Fármacos Anti-HIV/uso terapêutico , Contagem de Linfócito CD4 , Intestinos/imunologia , Intestinos/virologia , Leucócitos Mononucleares/virologia , Linfonodos/imunologia , Modelos Teóricos , Carga Viral , ViremiaRESUMO
Angiotensin (ANG) type 1A (AT(1A)) receptor-null (AT(1A)(-/-)) mice exhibit reduced afferent arteriolar (AA) constrictor responses to ANG II compared with wild-type (WT) mice, whereas efferent arteriolar (EA) responses are absent (Harrison-Bernard LM, Cook AK, Oliverio MI, and Coffman TM. Am J Physiol Renal Physiol 284: F538-F545, 2003). In the present study, the renal arteriolar constrictor responses to norepinephrine (NE) and/or ANG II were determined in blood-perfused juxtamedullary nephrons from kidneys of AT(1A)(-/-), AT(1B) receptor-null (AT(1B)(-/-)), and WT mice. Baseline AA diameter in AT(1A)(-/-) mice was not different from that in WT mice (13.1 +/- 0.9 and 12.6 +/- 0.9 microm, n = 7 and 8, respectively); however, EA diameters were significantly larger (17.3 +/- 1.4 vs. 11.7 +/- 0.4 microm, n = 10 and 8) in AT(1A)(-/-) than in WT mice. Constriction of AA (-40 +/- 8 and -51 +/- 6% at 1 microM NE) and EA (-29 +/- 6 and -38 +/- 3% at 1 microM NE) in response to 0.1-1 microM NE was similar in AT(1A)(-/-) and WT mice. Baseline diameters of AA (13.5 +/- 0.7 and 14.2 +/- 0.9 microm, n = 9 and 10) and EA (15.4 +/- 1.0 and 15.0 +/- 0.7 microm, n = 11 and 9) and ANG II (0.1-10 nM) constrictor responses of AA (-25 +/- 4 and -31 +/- 5% at 10 nM) and EA (-32 +/- 6 and -35 +/- 7% at 10 nM) were similar in AT(1B)(-/-) and WT mice, respectively. ANG II-induced constrictions were eliminated by AT(1) receptor blockade with 4 microM candesartan. Taken together, our data demonstrate that AA and EA responses to NE are unaltered in the absence of AT(1A) receptors, and ANG II responses remain intact in the absence of AT(1B) receptors. Therefore, we conclude that AT(1A) and AT(1B) receptors are functionally expressed on the AA, whereas the EA exclusively expresses the AT(1A) receptor.