RESUMEN
Mpox virus (MPXV) caused a global outbreak in 2022. Although smallpox vaccines were rapidly deployed to curb spread and disease among those at highest risk, breakthrough disease was noted after complete immunization. Given the threat of additional zoonotic events and the virus's evolving ability to drive human-to-human transmission, there is an urgent need for an MPXV-specific vaccine that confers protection against evolving MPXV strains and related orthopoxviruses. Here, we demonstrate that an mRNA-lipid nanoparticle vaccine encoding a set of four highly conserved MPXV surface proteins involved in virus attachment, entry, and transmission can induce MPXV-specific immunity and heterologous protection against a lethal vaccinia virus (VACV) challenge. Compared with modified vaccinia virus Ankara (MVA), which forms the basis for the current MPXV vaccine, immunization with an mRNA-based MPXV vaccine generated superior neutralizing activity against MPXV and VACV and more efficiently inhibited spread between cells. We also observed greater Fc effector TH1-biased humoral immunity to the four MPXV antigens encoded by the vaccine, as well as to the four VACV homologs. Single MPXV antigen-encoding mRNA vaccines provided partial protection against VACV challenge, whereas multivalent vaccines combining mRNAs encoding two, three, or four MPXV antigens protected against disease-related weight loss and death equal or superior to MVA vaccination. These data demonstrate that an mRNA-based MPXV vaccine confers robust protection against VACV.
Asunto(s)
Vacuna contra Viruela , Vacunas Virales , Humanos , Monkeypox virus/genética , Virus Vaccinia/genética , Vacuna contra Viruela/genética , Antígenos Virales , ARN Mensajero/genéticaRESUMEN
SARS-CoV-2 vaccines prevent severe disease but are less efficient in averting infection and transmission of variant strains, making it imperative to explore ways of enhancing protection. Use of inbred mice expressing the human SARS-CoV-2 receptor facilitates such investigations. We employed recombinant MVAs (rMVAs) expressing modified S of several SARS-CoV-2 strains and compared their ability to neutralize variants, bind S proteins and protect K18-hACE2 mice against SARS-CoV-2 challenge when administered intramuscularly or intranasally. The rMVAs expressing Wuhan, Beta and Delta S induced substantial cross neutralizing activities to each other but very low neutralization of Omicron; while rMVA expressing Omicon S induced neutralizing antibody predominanly to Omicron. In mice primed and boosted with rMVA expressing the Wuhan S, neutralizing antibodies to Wuhan increased after one immunization with rMVA expressing Omicron S due to original antigenic sin, but substantial neutralizing antibody to Omicron required a second immunization. Nevertheless, monovalent vaccines with S mismatched to the challenge virus still protected against severe disease and reduced the amounts of virus and subgenomic RNAs in the lungs and nasal turbinates, though not as well as vaccines with matched S. Passive transfer of Wuhan immune serum with Omicron S binding but undetectable neutralizing activity reduced infection of the l-ungs by Omicron suggesting additional effector functions. Notably, there was less infectious virus and viral subgenomic RNAs in the nasal turbinates and lungs when the rMVAs were administered intranasally rather than intramuscularly and this held true for vaccines that were matched or mismatched to the challenge strain of SARS-CoV-2.
RESUMEN
Human mpox (monkeypox), a disease with similarities to smallpox, is endemic in Africa where it has persisted as a zoonosis with limited human-to-human spread. Unexpectedly, the disease expanded globally in 2022 driven by human-to-human transmission outside of Africa. It is not yet known whether the latter is due solely to behavioral and environmental factors or whether the mpox virus is adapting to a new host. Genome sequencing has revealed differences between the current outbreak strains, classified as clade IIb, and the prior clade IIa and clade I viruses, but whether these differences contribute to virulence or transmission has not been determined. We demonstrate that the wild-derived inbred castaneous mouse provides an exceptional animal model for investigating clade differences in mpox virus virulence and show that the order is clade I > clade IIa > clade IIb.1. The greatly reduced replication of the clade IIb.1 major outbreak strain in mice and absence of lethality at 100 times the lethal dose of a closely related clade IIa virus, despite similar multiplication in cell culture, suggest that clade IIb is evolving diminished virulence or adapting to other species.
Asunto(s)
Monkeypox virus , Mpox , Humanos , Ratones , Animales , Monkeypox virus/genética , Mpox/epidemiología , Virulencia/genética , Modelos Animales , Brotes de EnfermedadesRESUMEN
Current vaccines have greatly diminished the severity of the COVID-19 pandemic, even though they do not entirely prevent infection and transmission, likely due to insufficient immunity in the upper respiratory tract. Here, we compare intramuscular and intranasal administration of a live, replication-deficient modified vaccinia virus Ankara (MVA)-based Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) spike (S) vaccine to raise protective immune responses in the K18-hACE2 mouse model. Using a recombinant MVA expressing firefly luciferase for tracking, live imaging revealed luminescence of the respiratory tract of mice within 6 h and persisting for 3 d following intranasal inoculation, whereas luminescence remained at the site of intramuscular vaccination. Intramuscular vaccination induced S-binding-Immunoglobulin G (IgG) and neutralizing antibodies in the lungs, whereas intranasal vaccination also induced Immunoglobulin A (IgA) and higher levels of antigen-specific CD3+CD8+IFN-γ+ T cells. Similarly, IgG and neutralizing antibodies were present in the blood of mice immunized intranasally and intramuscularly, but IgA was detected only after intranasal inoculation. Intranasal boosting increased IgA after intranasal or intramuscular priming. While intramuscular vaccination prevented morbidity and cleared SARS-CoV-2 from the respiratory tract within several days after challenge, intranasal vaccination was more effective as neither infectious virus nor viral messenger (m)RNAs were detected in the nasal turbinates or lungs as early as 2 d after challenge, indicating prevention or rapid elimination of SARS-CoV-2 infection. Additionally, we determined that neutralizing antibody persisted for more than 6 mo and that serum induced to the Wuhan S protein neutralized pseudoviruses expressing the S proteins of variants, although with less potency, particularly for Beta and Omicron.
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Vacunas contra la COVID-19 , COVID-19 , Inmunoglobulina A , Sistema Respiratorio , SARS-CoV-2 , Glicoproteína de la Espiga del Coronavirus , Virus Vaccinia , Administración Intranasal , Enzima Convertidora de Angiotensina 2/genética , Animales , Anticuerpos Neutralizantes/sangre , Anticuerpos Antivirales/sangre , COVID-19/prevención & control , COVID-19/transmisión , Vacunas contra la COVID-19/administración & dosificación , Vacunas contra la COVID-19/inmunología , Humanos , Inmunoglobulina A/sangre , Inmunoglobulina G/sangre , Ratones , Ratones Transgénicos , Sistema Respiratorio/inmunología , SARS-CoV-2/inmunología , Glicoproteína de la Espiga del Coronavirus/inmunología , Vacunación/métodos , Virus Vaccinia/genética , Virus Vaccinia/inmunologíaRESUMEN
Although providing long-lasting immunity, smallpox vaccination was associated with local and systemic reactions and rarely with severe complications, including progressive vaccinia and postvaccinia encephalitis. As the Dryvax smallpox vaccine consists of a population of variants, we investigated a particularly pathogenic isolate called clone 3 (CL3). Virus replication was monitored by inserting the gene encoding firefly luciferase (Luc) into the genomes of CL3 and ACAM2000, the second-generation smallpox vaccine derived from a less virulent clone. Greater luminescence occurred following intranasal or intraperitoneal inoculation of mice with CL3-Luc than ACAM2000-Luc. Previous genome sequencing of CL3 and ACAM2000 revealed numerous differences that could affect pathogenicity. We focused on a 4.2-kbp segment, containing several open reading frames, in CL3 that is absent from ACAM2000 and determined that lower virulence of the latter was associated with a truncation of the interferon α/ß (IFN-α/ß) decoy receptor. Truncation of the decoy receptor in CL3-Luc and repair of the truncated version in ACAM2000-Luc decreased and increased virulence, respectively. Blockade of the mouse type 1 IFN receptor increased the virulence of ACAM2000-Luc to that of CL3-Luc, consistent with the role of IFN in attenuating the former. The severities of disease following intracranial inoculation of immunocompetent mice and intraperitoneal inoculation of T cell-depleted mice were also greater in viruses expressing the full-length decoy receptor. Previous evidence for the low affinity of a similarly truncated decoy receptor for IFN and the presence of a full-length decoy receptor in virus isolated from a patient with progressive vaccinia support our findings. IMPORTANCE Attenuated live viruses make effective vaccines, although concerns exist due to infrequent complications, particularly in individuals with immunological defects. Such complications occurred with smallpox vaccines, which were shown to be comprised of populations of variants. Clone 3, isolated from Dryvax, the vaccine most widely used in the United States during the smallpox eradication campaign, was particularly pathogenic in animal models. We demonstrated that the full-length IFN-α/ß decoy receptor in CL3 and a truncation of the receptor in the clone used for the second-generation smallpox vaccine ACAM2000 account for their difference in pathogenicity. Viruses expressing the full-length decoy receptor were more virulent following intranasal, intraperitoneal, or intracranial inoculation of mice than ACAM2000, and disease was exacerbated following T cell depletion. Correspondingly, the full-length decoy receptor is present in smallpox vaccines with high rates of side effects and in a Dryvax clone obtained from a lesion in a patient with progressive vaccinia.
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Vacuna contra Viruela , Viruela , Vaccinia , Animales , Anticuerpos Antivirales , Antígenos Virales , Interferón-alfa , Ratones , Viruela/prevención & control , Vacuna contra Viruela/efectos adversos , Vacuna contra Viruela/genética , Vaccinia/inducido químicamente , Vaccinia/epidemiología , Virus Vaccinia/genética , VirulenciaRESUMEN
Modified vaccinia virus Ankara (MVA) is a replication-restricted smallpox vaccine, and numerous clinical studies of recombinant MVAs (rMVAs) as vectors for prevention of other infectious diseases, including COVID-19, are in progress. Here, we characterize rMVAs expressing the S protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Modifications of full-length S individually or in combination included two proline substitutions, mutations of the furin recognition site, and deletion of the endoplasmic retrieval signal. Another rMVA in which the receptor binding domain (RBD) is flanked by the signal peptide and transmembrane domains of S was also constructed. Each modified S protein was displayed on the surface of rMVA-infected cells and was recognized by anti-RBD antibody and soluble hACE2 receptor. Intramuscular injection of mice with the rMVAs induced antibodies, which neutralized a pseudovirus in vitro and, upon passive transfer, protected hACE2 transgenic mice from lethal infection with SARS-CoV-2, as well as S-specific CD3+CD8+IFNγ+ T cells. Antibody boosting occurred following a second rMVA or adjuvanted purified RBD protein. Immunity conferred by a single vaccination of hACE2 mice prevented morbidity and weight loss upon intranasal infection with SARS-CoV-2 3 wk or 7 wk later. One or two rMVA vaccinations also prevented detection of infectious SARS-CoV-2 and subgenomic viral mRNAs in the lungs and greatly reduced induction of cytokine and chemokine mRNAs. A low amount of virus was found in the nasal turbinates of only one of eight rMVA-vaccinated mice on day 2 and none later. Detection of low levels of subgenomic mRNAs in turbinates indicated that replication was aborted in immunized animals.
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Vacunas contra la COVID-19/inmunología , COVID-19/prevención & control , Vectores Genéticos/genética , SARS-CoV-2/inmunología , Vacunas de ADN/inmunología , Virus Vaccinia/genética , Enzima Convertidora de Angiotensina 2/genética , Animales , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , Especificidad de Anticuerpos/inmunología , Vacunas contra la COVID-19/administración & dosificación , Vacunas contra la COVID-19/genética , Modelos Animales de Enfermedad , Expresión Génica , Humanos , Inmunización , Inmunización Pasiva , Inmunoglobulina G/inmunología , Ratones , Ratones Transgénicos , Glicoproteína de la Espiga del Coronavirus/inmunología , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismo , Vacunas de ADN/administración & dosificación , Vacunas de ADN/genéticaRESUMEN
Replication-restricted modified vaccinia virus Ankara (MVA) is a licensed smallpox vaccine and numerous clinical studies investigating recombinant MVAs (rMVAs) as vectors for prevention of other infectious diseases have been completed or are in progress. Two rMVA COVID-19 vaccine trials are at an initial stage, though no animal protection studies have been reported. Here, we characterize rMVAs expressing the S protein of CoV-2. Modifications of full length S individually or in combination included two proline substitutions, mutations of the furin recognition site and deletion of the endoplasmic retrieval signal. Another rMVA in which the receptor binding domain (RBD) flanked by the signal peptide and transmembrane domains of S was also constructed. Each modified S protein was displayed on the surface of rMVA-infected human cells and was recognized by anti-RBD antibody and by soluble hACE2 receptor. Intramuscular injection of mice with the rMVAs induced S-binding and pseudovirus-neutralizing antibodies. Boosting occurred following a second homologous rMVA but was higher with adjuvanted purified RBD protein. Weight loss and lethality following intranasal infection of transgenic hACE2 mice with CoV-2 was prevented by one or two immunizations with rMVAs or by passive transfer of serum from vaccinated mice. One or two rMVA vaccinations also prevented recovery of infectious CoV-2 from the lungs. A low amount of virus was detected in the nasal turbinates of only one of eight rMVA-vaccinated mice on day 2 and none later. Detection of subgenomic mRNA in turbinates on day 2 only indicated that replication was abortive in immunized animals.
RESUMEN
The wild-derived inbred CAST/EiJ mouse, one of eight founder strains in the Collaborative Cross panel, is an exceptional model for studying monkeypox virus (MPXV), an emerging human pathogen, and other orthopoxviruses including vaccinia virus (VACV). Previous studies suggested that the extreme susceptibility of the CAST mouse to orthopoxviruses is due to an insufficient innate immune response. Here, we focused on the low number of natural killer (NK) cells in the naïve CAST mouse as a contributing factor to this condition. Administration of IL-15 to CAST mice transiently increased NK and CD8+ T cells that could express IFN-γ, indicating that the progenitor cells were capable of responding to cytokines. However, the number of NK cells rapidly declined indicating a defect in their homeostasis. Furthermore, IL-15-treated mice were protected from an otherwise lethal challenge with VACV or MPXV. IL-15 decreased virus spread and delayed death even when CD4+/CD8+ T cells were depleted with antibody, supporting an early protective role of the expanded NK cells. Purified splenic NK cells from CAST mice proliferated in vitro in response to IL-15 and could be activated with IL-12/IL-18 to secrete interferon-γ. Passive transfer of non-activated or activated CAST NK cells reduced VACV spread but only the latter completely prevented death at the virus dose used. Moreover, antibodies to interferon-γ abrogated the protection by activated NK cells. Thus, the inherent susceptibility of CAST mice to orthopoxviruses can be explained by a low level of NK cells and this vulnerability can be overcome either by expanding their NK cells in vivo with IL-15 or by passive transfer of purified NK cells that were expanded and activated in vitro.
Asunto(s)
Interleucina-15/farmacología , Células Asesinas Naturales/inmunología , Orthopoxvirus/inmunología , Infecciones por Poxviridae/inmunología , Animales , Linfocitos T CD8-positivos/efectos de los fármacos , Linfocitos T CD8-positivos/inmunología , Citocinas/inmunología , Femenino , Inmunidad Innata/efectos de los fármacos , Interferón gamma/inmunología , Interleucina-15/inmunología , Células Asesinas Naturales/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos , Orthopoxvirus/efectos de los fármacos , Orthopoxvirus/patogenicidad , Infecciones por Poxviridae/tratamiento farmacológico , Transducción de Señal/efectos de los fármacos , Bazo/efectos de los fármacos , Bazo/patología , Bazo/virología , Virus Vaccinia/inmunologíaRESUMEN
DNA-based vaccines able to induce efficient cytotoxic T-cell responses targeting conserved elements (CE) of human immunodeficiency virus type 1 (HIV-1) Gag have been developed. These CE were selected by stringent conservation, the ability to induce T-cell responses with broad human leukocyte antigen coverage, and the association between recognition of CE epitopes and viral control in HIV-infected individuals. Based on homology to HIV, a simian immunodeficiency virus p27gag CE DNA vaccine has also been developed. This study reports on the durability of the CE-specific T-cell responses induced by HIV and simian immunodeficiency virus CE DNA-based prime/boost vaccine regimens in rhesus macaques, and shows that the initially primed CE-specific T-cell responses were efficiently boosted by a single CE DNA vaccination after the long rest period (up to 2 years). In another cohort of animals, the study shows that a single inoculation with non-replicating recombinant Modified Vaccinia Ankara (rMVA62B) also potently boosted CE-specific responses after around 1.5 years of rest. Both CE DNA and rMVA62B booster vaccinations increased the magnitude and cytotoxicity of the CE-specific responses while maintaining the breadth of CE recognition. Env produced by rMVA62B did not negatively interfere with the recall of the Gag CE responses. rMVA62B could be beneficial to further boosting the immune response to Gag in humans. Vaccine regimens that employ CE DNA as a priming immunogen hold promise for application in HIV prevention and therapy.
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Vacunas contra el SIDA/administración & dosificación , ADN/inmunología , Infecciones por VIH/inmunología , Vacunas de ADN/administración & dosificación , Vacunas contra el SIDA/inmunología , Animales , Linfocitos T CD4-Positivos/inmunología , Citotoxicidad Inmunológica/efectos de los fármacos , Infecciones por VIH/prevención & control , Infecciones por VIH/virología , VIH-1/inmunología , VIH-1/patogenicidad , Humanos , Inmunización Secundaria , Macaca mulatta , Virus de la Inmunodeficiencia de los Simios/inmunología , Virus de la Inmunodeficiencia de los Simios/patogenicidad , Linfocitos T Citotóxicos/inmunología , Vacunas de ADN/inmunología , Vaccinia/inmunología , Virus Vaccinia/genética , Virus Vaccinia/inmunologíaRESUMEN
Background: Mosaic immunogens are bioinformatically engineered human immunodeficiency virus type 1 (HIV-1) sequences designed to elicit clade-independent coverage against globally circulating HIV-1 strains. Methods: This phase 1, double-blinded, randomized, placebo-controlled trial enrolled healthy HIV-uninfected adults who received 2 doses of a modified vaccinia Ankara (MVA)-vectored HIV-1 bivalent mosaic immunogen vaccine or placebo on days 0 and 84. Two groups were enrolled: those who were HIV-1 vaccine naive (n = 15) and those who had received an HIV-1 vaccine (Ad26.ENVA.01) 4-6 years earlier (n = 10). We performed prespecified blinded cellular and humoral immunogenicity analyses at days 0, 14, 28, 84, 98, 112, 168, 270, and 365. Results: All 50 planned vaccinations were administered. Vaccination was safe and generally well tolerated. No vaccine-related serious adverse events occurred. Both cellular and humoral cross-clade immune responses were elicited after 1 or 2 vaccinations in all participants in the HIV-1 vaccine-naive group. Env-specific responses were induced after a single immunization in nearly all subjects who had previously received the prototype Ad26.ENVA.01 vaccine. Conclusions: No safety concerns were identified, and multiclade HIV-1-specific immune responses were elicited. Clinical Trials Registration: NCT02218125.
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Vacunas contra el SIDA/administración & dosificación , Vacunas contra el SIDA/inmunología , Infecciones por VIH/prevención & control , VIH-1/inmunología , Vacunas contra el SIDA/efectos adversos , Vacunas contra el SIDA/genética , Adulto , Método Doble Ciego , Portadores de Fármacos , Efectos Colaterales y Reacciones Adversas Relacionados con Medicamentos/epidemiología , Efectos Colaterales y Reacciones Adversas Relacionados con Medicamentos/patología , Femenino , Vectores Genéticos , Humanos , Inmunidad Celular , Inmunidad Humoral , Estudios Longitudinales , Masculino , Persona de Mediana Edad , Placebos/administración & dosificación , Resultado del Tratamiento , Vacunas de Subunidad/administración & dosificación , Vacunas de Subunidad/efectos adversos , Vacunas de Subunidad/genética , Vacunas de Subunidad/inmunología , Vacunas Sintéticas/administración & dosificación , Vacunas Sintéticas/efectos adversos , Vacunas Sintéticas/genética , Vacunas Sintéticas/inmunología , Virus Vaccinia/genética , Adulto JovenRESUMEN
We assessed the safety and immunogenicity of HIV-DNA priming using Zetajet™, a needle-free device intradermally followed by intramuscular HIV-MVA boosts, in 24 healthy Mozambicans. Volunteers were randomized to receive three immunizations of 600 µg (n = 10; 2 × 0.1 ml) or 1,200 µg (n = 10; 2 × 0.2 ml) of HIV-DNA (3 mg/ml), followed by two boosts of 108 pfu HIV-MVA. Four subjects received placebo saline injections. Vaccines and injections were safe and well tolerated with no difference between the two priming groups. After three HIV-DNA immunizations, IFN-γ ELISpot responses to Gag were detected in 9/17 (53%) vaccinees, while none responded to Envelope (Env). After the first HIV-MVA, the overall response rate to Gag and/or Env increased to 14/15 (93%); 14/15 (93%) to Gag and 13/15 (87%) to Env. There were no significant differences between the immunization groups in frequency of response to Gag and Env or magnitude of Gag responses. Env responses were significantly higher in the higher dose group (median 420 vs. 157.5 SFC/million peripheral blood mononuclear cell, p = .014). HIV-specific antibodies to subtype C gp140 and subtype B gp160 were elicited in all vaccinees after the second HIV-MVA, without differences in titers between the groups. Neutralizing antibody responses were not detected. Two (13%) of 16 vaccinees, one in each of the priming groups, exhibited antibodies mediating antibody-dependent cellular cytotoxicity to CRF01_AE. In conclusion, HIV-DNA vaccine delivered intradermally in volumes of 0.1-0.2 ml using Zetajet was safe and well tolerated. Priming with the 1,200 µg dose of HIV-DNA generated higher magnitudes of ELISpot responses to Env.
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Vacunas contra el SIDA/administración & dosificación , Vacunas contra el SIDA/inmunología , VIH-1/inmunología , Esquemas de Inmunización , Vacunas de ADN/inmunología , Vacunas contra el SIDA/efectos adversos , Administración Cutánea , Adolescente , Adulto , Anticuerpos Neutralizantes/sangre , Método Doble Ciego , Efectos Colaterales y Reacciones Adversas Relacionados con Medicamentos/epidemiología , Efectos Colaterales y Reacciones Adversas Relacionados con Medicamentos/patología , Ensayo de Immunospot Ligado a Enzimas , Femenino , Anticuerpos Anti-VIH/sangre , VIH-1/genética , Humanos , Inyecciones Intramusculares , Interferón gamma/análisis , Leucocitos Mononucleares/inmunología , Masculino , Mozambique , Placebos/administración & dosificación , Resultado del Tratamiento , Vacunas de ADN/administración & dosificación , Vacunas de ADN/efectos adversos , Vacunas Sintéticas/administración & dosificación , Vacunas Sintéticas/inmunología , Voluntarios , Adulto Joven , Productos del Gen env del Virus de la Inmunodeficiencia Humana/inmunología , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/inmunologíaRESUMEN
Droplet digital polymerase chain reaction (ddPCR) was adapted for quantifying the number of orthopoxviral genomes in purified virus samples, infected cell lysates and tissues of infected animals. In contrast to the more commonly used qPCR, the newer ddPCR provides absolute numbers of DNA copies in samples without need for standard curves and has the ability to detect rare mutants in a population. The genome/infectious unit ratio for several sucrose gradient-purified orthopoxviruses varied from 5 to 10, which correlated well with values obtained using the Virocyt, a dedicated fluorescence flow cytometer. By employing a nuclease step to digest unencapsulated DNA, the genome/infectious unit ratios of virus in crude cell lysates approached that of purified virus particles. The speed, accuracy, sensitivity, and dynamic range of less than one to millions of infectious units in a sample make this semi-automated method well suited to a variety of laboratory, animal and clinical studies.
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Orthopoxvirus/aislamiento & purificación , Reacción en Cadena de la Polimerasa/métodos , Carga Viral/métodos , Animales , Automatización de Laboratorios , Orthopoxvirus/genética , Sensibilidad y Especificidad , Factores de TiempoRESUMEN
This unit describes how to infect cells with vaccinia virus and then transfect them with a plasmid-transfer vector or PCR fragment to generate a recombinant virus. Selection and screening methods used to isolate recombinant viruses and a method for the amplification of recombinant viruses are described. Finally, a method for live immunostaining that has been used primarily for detection of recombinant modified vaccinia virus Ankara (MVA) is presented. © 2017 by John Wiley & Sons, Inc.
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Virus Vaccinia/genética , Animales , Línea Celular , Vectores Genéticos , Recombinación Homóloga , Humanos , Plásmidos , Reacción en Cadena de la Polimerasa , Transfección , Virus Vaccinia/aislamiento & purificación , Cultivo de VirusRESUMEN
The culturing of cell lines used with vaccinia virus, both as monolayer and in suspension, is described. The preparation of chick embryo fibroblasts (CEF) is presented for use in the production of the highly attenuated and host range-restricted modified vaccinia virus Ankara (MVA) strain of vaccinia virus. Protocols for the preparation, titration, and trypsinization of vaccinia virus stocks, as well as viral DNA preparation and virus purification methods are also included. © 2017 by John Wiley & Sons, Inc.
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Técnicas de Cultivo de Célula/métodos , Virus Vaccinia/aislamiento & purificación , Cultivo de Virus/métodos , Animales , Línea Celular , Humanos , Virus Vaccinia/crecimiento & desarrolloRESUMEN
The castaneous (CAST) mouse, a wild-derived inbred strain, is highly susceptible to orthopoxvirus infection by intranasal and systemic routes. The 50% lethal intraperitoneal dose of vaccinia virus (VACV) was 3 PFU for CAST mice, whereas BALB/c mice survived 106 PFU. At all times and in all organs analyzed, virus titers were higher in CAST than in BALB/c mice. In individual CAST mice, luciferase-expressing VACV was seen to replicate rapidly leading to death, whereas virus levels increased for a few days and then declined in BALB/c mice. Increases in gamma interferon (IFN-γ) and tumor necrosis factor alpha (TNF-α) were delayed and low in CAST mice compared to BALB/c mice following VACV infection or poly(I-C) inoculation, consistent with differences in innate immune responses. In addition, naive CAST mice had considerably lower numbers of NK and T cells than BALB/c mice. The percentage of IFN-γ-producing CD4+ and CD8+ T cells increased following infection of CAST mice only after considerable virus spread, and the absolute cell numbers remained low. Administration of exogenous IFN-γ or -α to CAST mice before or during the first days of infection suppressed virus replication and prolonged survival, allowing the mice to make adaptive CD4+ and CD8+ T cell responses that were necessary to clear the virus after cessation of interferon treatment. Thus, insufficient innate cytokine and cellular immune responses contribute to the unique susceptibility of CAST mice to VACV, whereas the adaptive immune response can be protective only if virus replication is suppressed during the first several days of infection.IMPORTANCE Most inbred mouse strains are relatively resistant to orthopoxviruses. The castaneous (CAST) mouse is a notable exception, exhibiting extreme vulnerability to monkeypox virus, cowpox virus, and vaccinia virus and thus providing a unique model for studying pathogenicity, immunity, vaccines, and antiviral drugs. To fully utilize the CAST mouse for such purposes, it is necessary to understand the basis for virus susceptibility. We showed that naive CAST mice make low IFN-γ and TNF-α responses and have low levels of NK cells and CD4+ and CD8+ T cells compared to a resistant classical inbred mouse strain. Attenuating virus replication with one or more doses of exogenous IFN-α or -γ before or during the first few days of infection enabled the development of adaptive cellular immunity and clearance of virus. Further genetic studies may reveal the basis for the low innate immunity.
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Inmunidad Innata/inmunología , Interferón gamma/metabolismo , Células Asesinas Naturales/inmunología , Infecciones por Poxviridae/inmunología , Factor de Necrosis Tumoral alfa/metabolismo , Virus Vaccinia/inmunología , Animales , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Línea Celular , Chlorocebus aethiops , Femenino , Interferón gamma/uso terapéutico , Recuento de Linfocitos , Ratones , Ratones Endogámicos BALB C , Infecciones por Poxviridae/virología , Factor de Necrosis Tumoral alfa/uso terapéutico , Replicación Viral/inmunologíaRESUMEN
Viruses are used as expression vectors for protein synthesis, immunology research, vaccines, and therapeutics. Advantages of poxvirus vectors include the accommodation of large amounts of heterologous DNA, the presence of a cytoplasmic site of transcription, and high expression levels. On the other hand, competition of approximately 200 viral genes with the target gene for expression and immune recognition may be disadvantageous. We describe a vaccinia virus (VACV) vector that uses an early promoter to express the bacteriophage T7 RNA polymerase; has the A23R intermediate transcription factor gene deleted, thereby restricting virus replication to complementing cells; and has a heterologous gene regulated by a T7 promoter. In noncomplementing cells, viral early gene expression and DNA replication occurred normally but synthesis of intermediate and late proteins was prevented. Nevertheless, the progeny viral DNA provided templates for abundant expression of heterologous genes regulated by a T7 promoter. Selective expression of the Escherichia coli lac repressor gene from an intermediate promoter reduced transcription of the heterologous gene specifically in complementing cells, where large amounts might adversely impact VACV replication. Expression of heterologous proteins mediated by the A23R deletion vector equaled that of a replicating VACV, was higher than that of a nonreplicating modified vaccinia virus Ankara (MVA) vector used for candidate vaccines in vitro and in vivo, and was similarly immunogenic in mice. Unlike the MVA vector, the A23R deletion vector still expresses numerous early genes that can restrict immunogenicity as demonstrated here by the failure of the prototype vector to induce interferon alpha. By deleting immunomodulatory genes, we anticipate further improvements in the system.IMPORTANCE Vaccines provide an efficient and effective way of preventing infectious diseases. Nevertheless, new and better vaccines are needed. Vaccinia virus, which was used successfully as a live vaccine to eradicate smallpox, has been further attenuated and adapted as a recombinant vector for immunization against other pathogens. However, since the initial description of this vector system, only incremental improvements largely related to safety have been implemented. Here we described novel modifications of the platform that increased expression of the heterologous target gene and decreased expression of endogenous vaccinia virus genes while providing safety by preventing replication of the candidate vaccine except in complementing cells used for vector propagation.
Asunto(s)
Expresión Génica , Vectores Genéticos , Virus Vaccinia/fisiología , Proteínas Virales/metabolismo , Replicación Viral , Animales , Replicación del ADN , ADN Viral , ARN Polimerasas Dirigidas por ADN/genética , Genes Virales , Represoras Lac/genética , Ratones , Orthomyxoviridae/genética , Poxviridae/genética , Regiones Promotoras Genéticas , Virus Vaccinia/inmunología , Proteínas Virales/genéticaRESUMEN
This unit describes how to infect cells with vaccinia virus and then transfect them with a plasmid-transfer vector or PCR fragment to generate a recombinant virus. Selection and screening methods used to isolate recombinant viruses and a method for the amplification of recombinant viruses are described. Finally, a method for live immunostaining that has been used primarily for detection of recombinant modified vaccinia virus Ankara (MVA) is presented. © 2017 by John Wiley & Sons, Inc.
Asunto(s)
Vectores Genéticos/genética , Recombinación Homóloga , Plásmidos/genética , Transfección , Virus Vaccinia/genética , Animales , Línea Celular , Humanos , Inmunohistoquímica/métodos , Reacción en Cadena de la Polimerasa/métodosRESUMEN
The culturing of cell lines used with vaccinia virus, both as monolayer and in suspension, is described. The preparation of chick embryo fibroblasts (CEF) is presented for use in the production of the highly attenuated and host range-restricted modified vaccinia virus Ankara (MVA) strain of vaccinia virus. Protocols for the preparation, titration, and trypsinization of vaccinia virus stocks, as well as viral DNA preparation and virus purification methods are also included. © 2017 by John Wiley & Sons, Inc.
Asunto(s)
Técnicas de Cultivo de Célula/métodos , Virus Vaccinia/crecimiento & desarrollo , Virus Vaccinia/aislamiento & purificación , Animales , Línea Celular , Células Cultivadas , Embrión de Pollo , ADN Viral/aislamiento & purificación , Fibroblastos/virología , HumanosRESUMEN
Most poxviruses encode a homolog of a ~200,000-kDa membrane protein originally identified in variola virus. We investigated the importance of the ectromelia virus (ECTV) homolog C15 in a natural infection model. In cultured mouse cells, the replication of a mutant virus with stop codons near the N-terminus (ECTV-C15Stop) was indistinguishable from a control virus (ECTV-C15Rev). However, for a range of doses injected into the footpads of BALB/c mice there was less mortality with the mutant. Similar virus loads were present at the site of infection with mutant or control virus whereas there was less ECTV-C15Stop in popliteal and inguinal lymph nodes, spleen and liver indicating decreased virus spread and replication. The latter results were supported by immunohistochemical analyses. Decreased spread was evidently due to immune modulatory activity of C15, rather than to an intrinsic viral function, as the survival of infected mice depended on CD4+ and CD8+ T cells.
Asunto(s)
Virus de la Ectromelia/metabolismo , Virus de la Ectromelia/patogenicidad , Ectromelia Infecciosa/metabolismo , Ectromelia Infecciosa/virología , Proteínas de la Membrana/metabolismo , Proteínas Virales/metabolismo , Animales , Modelos Animales de Enfermedad , Virus de la Ectromelia/genética , Ectromelia Infecciosa/genética , Ectromelia Infecciosa/patología , Femenino , Humanos , Hígado/patología , Hígado/virología , Proteínas de la Membrana/genética , Ratones , Ratones Endogámicos BALB C , Bazo/patología , Bazo/virología , Virus de la Viruela/genética , Virus de la Viruela/metabolismo , Proteínas Virales/genética , VirulenciaRESUMEN
We explored the duration of immune responses and the effect of a late third HIV-modified vaccinia virus Ankara (MVA) boost in HIV-DNA primed and HIV-MVA boosted Tanzanian volunteers. Twenty volunteers who had previously received three HIV-DNA and two HIV-MVA immunizations were given a third HIV-MVA immunization 3 years after the second HIV-MVA boost. At the time of the third HIV-MVA, 90% of the vaccinees had antibodies to HIV-1 subtype C gp140 (median titer 200) and 85% to subtype B gp160 (median titer 100). The majority of vaccinees had detectable antibody-dependent cellular cytotoxicity (ADCC)-mediating antibodies, 70% against CRF01_AE virus-infected cells (median titer 239) and 84% against CRF01_AE gp120-coated cells (median titer 499). A high proportion (74%) of vaccinees had IFN-γ ELISpot responses, 63% to Gag and 42% to Env, 3 years after the second HIV-MVA boost. After the third HIV-MVA, there was an increase in Env-binding antibodies and ADCC-mediating antibodies relative to the response seen at the time of the third HIV-MVA vaccination, p < .0001 and p < .05, respectively. The frequency of IFN-γ ELISpot responses increased to 95% against Gag or Env and 90% to both Gag and Env, p = .064 and p = .002, respectively. In conclusion, the HIV-DNA prime/HIV-MVA boost regimen elicited potent antibody and cellular immune responses with remarkable durability, and a third HIV-MVA immunization significantly boosted both antibody and cellular immune responses relative to the levels detected at the time of the third HIV-MVA, but not to higher levels than after the second HIV-MVA.