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Viral haemorrhagic fevers (VHF) pose a significant threat to human health. In recent years, VHF outbreaks caused by Ebola, Marburg and Lassa viruses have caused substantial morbidity and mortality in West and Central Africa. In 2022, an Ebola disease outbreak in Uganda caused by Sudan virus resulted in 164 cases with 55 deaths. In 2023, a Marburg disease outbreak was confirmed in Equatorial Guinea and Tanzania resulting in over 49 confirmed or suspected cases; 41 of which were fatal. There are no clearly defined correlates of protection against these VHF, impeding targeted vaccine development. Any vaccine developed should therefore induce strong and preferably long-lasting humoral and cellular immunity against these viruses. Ideally this immunity should also cross-protect against viral variants, which are known to circulate in animal reservoirs and cause human disease. We have utilized two viral vectored vaccine platforms, an adenovirus (ChAdOx1) and Modified Vaccinia Ankara (MVA), to develop a multi-pathogen vaccine regime against three filoviruses (Ebola virus, Sudan virus, Marburg virus) and an arenavirus (Lassa virus). These platform technologies have consistently demonstrated the capability to induce robust cellular and humoral antigen-specific immunity in humans, most recently in the rollout of the licensed ChAdOx1-nCoV19/AZD1222. Here, we show that our multi-pathogen vaccines elicit strong cellular and humoral immunity, induce a diverse range of chemokines and cytokines, and most importantly, confers protection after lethal Ebola virus, Sudan virus and Marburg virus challenges in a small animal model.
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Ebolavirus , Fiebre Hemorrágica Ebola , Fiebre de Lassa , Virus Lassa , Enfermedad del Virus de Marburg , Marburgvirus , Animales , Ratones , Ebolavirus/inmunología , Virus Lassa/inmunología , Marburgvirus/inmunología , Fiebre Hemorrágica Ebola/prevención & control , Fiebre Hemorrágica Ebola/inmunología , Fiebre de Lassa/inmunología , Fiebre de Lassa/prevención & control , Enfermedad del Virus de Marburg/inmunología , Enfermedad del Virus de Marburg/prevención & control , Vacunas Virales/inmunología , Humanos , Vacunación , Femenino , Anticuerpos Antivirales/inmunología , Inmunogenicidad Vacunal , Vacunas contra el Virus del Ébola/inmunologíaRESUMEN
Despite promising progress in malaria vaccine development in recent years, an efficacious subunit vaccine against Plasmodium falciparum remains to be licensed and deployed. Cell-mediated protection from liver-stage malaria relies on a sufficient number of antigen-specific T cells reaching the liver during the time that parasites are present. A single vaccine expressing two antigens could potentially increase both the size and breadth of the antigen-specific response while halving vaccine production costs. In this study, we investigated combining two liver-stage antigens, P. falciparum LSA1 (PfLSA1) and PfLSAP2, and investigated the induction of protective efficacy by coadministration of single-antigen vectors or vaccination with dual-antigen vectors, using simian adenovirus and modified vaccinia virus Ankara vectors. The efficacy of these vaccines was assessed in mouse malaria challenge models using chimeric P. berghei parasites expressing the relevant P. falciparum antigens and challenging mice at the peak of the T cell response. Vaccination with a combination of the single-antigen vectors expressing PfLSA1 or PfLSAP2 was shown to improve protective efficacy compared to vaccination with each single-antigen vector alone. Vaccination with dual-antigen vectors expressing both PfLSA1 and PfLSAP2 resulted in responses to both antigens, particularly in outbred mice, and most importantly, the efficacy was equivalent to that of vaccination with a mixture of single-antigen vectors. Based on these promising data, dual-antigen vectors expressing PfLSA1 and PfLSAP2 will now proceed to manufacturing and clinical assessment under good manufacturing practice (GMP) guidelines.
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Adenovirus de los Simios/genética , Antígenos de Protozoos/inmunología , Portadores de Fármacos , Vacunas contra la Malaria/inmunología , Malaria/prevención & control , Proteínas Recombinantes de Fusión/inmunología , Virus Vaccinia/genética , Animales , Antígenos de Protozoos/genética , Modelos Animales de Enfermedad , Femenino , Humanos , Inmunidad Celular , Vacunas contra la Malaria/administración & dosificación , Vacunas contra la Malaria/genética , Ratones Endogámicos BALB C , Ratones Endogámicos ICR , Proteínas Recombinantes de Fusión/genética , Resultado del Tratamiento , Vacunas de Subunidad/administración & dosificación , Vacunas de Subunidad/genética , Vacunas de Subunidad/inmunología , Vacunas Sintéticas/administración & dosificación , Vacunas Sintéticas/genética , Vacunas Sintéticas/inmunologíaRESUMEN
Since the development of vaccinia virus as a vaccine vector in 1984, the utility of numerous viruses in vaccination strategies has been explored. In recent years, key improvements to existing vectors such as those based on adenovirus have led to significant improvements in immunogenicity and efficacy. Furthermore, exciting new vectors that exploit viruses such as cytomegalovirus (CMV) and vesicular stomatitis virus (VSV) have emerged. Herein, we summarize these recent developments in viral vector technologies, focusing on novel vectors based on CMV, VSV, measles and modified adenovirus. We discuss the potential utility of these exciting approaches in eliciting protection against infectious diseases.
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Control de Enfermedades Transmisibles , Enfermedades Transmisibles/inmunología , Vectores Genéticos/genética , Vacunas Virales/genética , Vacunas Virales/inmunología , Virus/genética , Inmunidad Adaptativa , Animales , Control de Enfermedades Transmisibles/métodos , Vectores Genéticos/inmunología , Humanos , Vacunación , Vacunas Sintéticas/genética , Vacunas Sintéticas/inmunología , Virus/clasificación , Virus/inmunologíaRESUMEN
Many critical events in the Plasmodium life cycle rely on the controlled release of Ca²âº from intracellular stores to activate stage-specific Ca²âº-dependent protein kinases. Using the motility of Plasmodium berghei ookinetes as a signalling paradigm, we show that the cyclic guanosine monophosphate (cGMP)-dependent protein kinase, PKG, maintains the elevated level of cytosolic Ca²âº required for gliding motility. We find that the same PKG-dependent pathway operates upstream of the Ca²âº signals that mediate activation of P. berghei gametocytes in the mosquito and egress of Plasmodium falciparum merozoites from infected human erythrocytes. Perturbations of PKG signalling in gliding ookinetes have a marked impact on the phosphoproteome, with a significant enrichment of in vivo regulated sites in multiple pathways including vesicular trafficking and phosphoinositide metabolism. A global analysis of cellular phospholipids demonstrates that in gliding ookinetes PKG controls phosphoinositide biosynthesis, possibly through the subcellular localisation or activity of lipid kinases. Similarly, phosphoinositide metabolism links PKG to egress of P. falciparum merozoites, where inhibition of PKG blocks hydrolysis of phosphatidylinostitol (4,5)-bisphosphate. In the face of an increasing complexity of signalling through multiple Ca²âº effectors, PKG emerges as a unifying factor to control multiple cellular Ca²âº signals essential for malaria parasite development and transmission.
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Señalización del Calcio , Proteínas Quinasas Dependientes de GMP Cíclico/fisiología , Fosfatidilinositoles/metabolismo , Plasmodium falciparum/fisiología , Animales , Culicidae/parasitología , Proteínas Quinasas Dependientes de GMP Cíclico/metabolismo , Interacciones Huésped-Parásitos , Humanos , Estadios del Ciclo de Vida , Malaria/parasitología , Modelos Biológicos , Plasmodium falciparum/crecimiento & desarrollo , Plasmodium falciparum/metabolismoRESUMEN
PURPOSE: Sexual and gender minority and racialized populations experienced heightened vulnerability during the Covid-19 pandemic. Marginalization due to structural homophobia, transphobia and racism, and resulting adverse social determinants of health that contribute to health disparities among these populations, were exacerbated by the Covid-19 pandemic and public health measures to control it. We developed and tested a tailored online intervention (#SafeHandsSafeHearts) to support racialized lesbian, gay, bisexual, transgender, queer, and other persons outside of heteronormative and cisgender identities (LGBTQ+) in Toronto, Canada during the pandemic. METHODS: We used a quasi-experimental pre-test post-test design to evaluate the effectiveness of a 3-session, peer-delivered eHealth intervention in reducing psychological distress and increasing Covid-19 knowledge and protective behaviors. Individuals ≥18-years-old, resident in Toronto, and self-identified as sexual or gender minority were recruited online. Depressive and anxiety symptoms, and Covid-19 knowledge and protective behaviors were assessed at baseline, 2-weeks postintervention, and 2-months follow-up. We used generalized estimating equations and zero-truncated Poisson models to evaluate the effectiveness of the intervention on the four primary outcomes. RESULTS: From March to November 2021, 202 participants (median age, 27 years [Interquartile range: 23-32]) were enrolled in #SafeHandsSafeHearts. Over half (54.5%, n = 110) identified as cisgender lesbian or bisexual women or women who have sex with women, 26.2% (n = 53) cisgender gay or bisexual men or men who have sex with men, and 19.3% (n = 39) transgender or nonbinary individuals. The majority (75.7%, n = 143) were Black and other racialized individuals. The intervention led to statistically significant reductions in the prevalence of clinically significant depressive (25.4% reduction, p < .01) and anxiety symptoms (16.6% reduction, p < .05), and increases in Covid-19 protective behaviors (4.9% increase, p < .05), from baseline to postintervention. CONCLUSION: We demonstrated the effectiveness of a brief, peer-delivered eHealth intervention for racialized LGBTQ+ communities in reducing psychological distress and increasing protective behaviors amid the Covid-19 pandemic. Implementation through community-based organizations by trained peer counselors supports feasibility, acceptability, and the importance of engaging racialized LGBTQ+ communities in pandemic response preparedness. This trial is registered with ClinicalTrials.gov, number NCT04870723.
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COVID-19 , Distrés Psicológico , Minorías Sexuales y de Género , Telemedicina , Humanos , Masculino , COVID-19/prevención & control , COVID-19/epidemiología , COVID-19/psicología , Femenino , Minorías Sexuales y de Género/psicología , Adulto , Telemedicina/métodos , Persona de Mediana Edad , SARS-CoV-2 , Adulto Joven , Conocimientos, Actitudes y Práctica en Salud , Depresión/prevención & control , Pandemias/prevención & control , Canadá/epidemiologíaRESUMEN
There are varying data concerning the effect of prior anti-vector immunity on the T-cell response induced by immunisation with an identical vectored vaccine containing a heterologous antigen insert. To determine whether prior exposure to ChAdOx1-SARS-CoV2 immunisation (Vaxzevria®) impacts magnitudes of antigen-specific T-cell responses elicited by subsequent administration of the same viral vector (encoding HBV antigens, ChAdOx1-HBV), healthy volunteers that had received Vaxzevria® (n = 15) or the Pfizer or Moderna mRNA COVID-19 vaccine (n = 11) between 10 and 18 weeks prior were recruited to receive a single intramuscular injection of ChAdOx1-HBV. Anti-ChAdOx1-neutralising antibody titers were determined, and vector or insert-specific T-cell responses were measured by a gamma-interferon ELISpot and intracellular cytokine staining (ICS) assay using multiparameter flow cytometry. Participants were followed for three months after the ChAdOx1-HBV injection, which was well-tolerated, and no dropouts occurred. The baseline ChAdOx1 neutralisation titers were higher in the Vaxzevria® cohort (median of 848) than in the mRNA cohort (median of 25). T-cell responses to HBV antigens, measured by ELISpot, were higher on day 28 in the mRNA group (p = 0.013) but were similar between groups on day 84 (p = 0.441). By ICS, these differences persisted at the last time point. There was no clear correlation between the baseline responses to the adenoviral hexon and the subsequent ELISpot responses. As vaccination within 3 months using the same viral vector backbone affected the insert-specific T-cell responses, a greater interval after prior adenoviral immunisation using heterologous antigens may be warranted in settings in which these cells play critical roles.
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Although licensed vaccines against influenza virus have been successful in reducing pathogen-mediated disease, they have been less effective at preventing viral infection of the airways and current seasonal updates to influenza vaccines do not always successfully accommodate viral drift. Most licensed influenza and recently licensed RSV vaccines are administered via the intramuscular route. Alternative immunisation strategies, such as intranasal vaccinations, and "prime-pull" regimens, may deliver a more sterilising form of protection against respiratory viruses. A bivalent ChAdOx1-based vaccine (ChAdOx1-NP + M1-RSVF) encoding conserved nucleoprotein and matrix 1 proteins from influenza A virus and a modified pre-fusion stabilised RSV A F protein, was designed, developed and tested in preclinical animal models. The aim was to induce broad, cross-protective tissue-resident T cells against heterotypic influenza viruses and neutralising antibodies against RSV in the respiratory mucosa and systemically. When administered via an intramuscular prime-intranasal boost (IM-IN) regimen in mice, superior protection was generated against challenge with either RSV A, Influenza A H3N2 or H1N1. These results support further clinical development of a pan influenza & RSV vaccine administered in a prime-pull regimen.
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Herpes zoster (HZ) results from waning immunity following childhood infection with varicella zoster virus (VZV) but is preventable by vaccination with recombinant HZ vaccine or live HZ vaccine (two doses or one dose, respectively). Vaccine efficacy declines with age, live HZ vaccine is contraindicated in immunosuppressed individuals, and severe local reactogenicity of recombinant HZ vaccine is seen in up to 20% of older adults, indicating a potential need for new vaccines. Nonreplicating chimpanzee adenovirus (ChAd) vectors combine potent immunogenicity with well-established reactogenicity and safety profiles. We evaluated the cellular and humoral immunogenicity of ChAdOx1 encoding VZV envelope glycoprotein E (ChAdOx1-VZVgE) in mice using IFN-γ ELISpot, flow cytometry with intracellular cytokine staining, and ELISA. In outbred CD-1 mice, one dose of ChAdOx1-VZVgE (1 × 107 infectious units) elicited higher gE-specific T cell responses than two doses of recombinant HZ vaccine (1 µg) or one dose of live HZ vaccine (1.3 × 103 plaque-forming units). Antibody responses were higher with two doses of recombinant HZ vaccine than with two doses of ChAdOx1-VZVgE or one dose of live HZ vaccine. ChAdOx1-VZVgE boosted T cell and antibody responses following live HZ vaccine priming. The frequencies of polyfunctional CD4+ and CD8+ T cells expressing more than one cytokine (IFN-γ, TNF-α and IL-2) were higher with ChAdOx1-VZVgE than with the conventional vaccines. Results were similar in young and aged BALB/c mice. These findings support the clinical development of ChAdOx1-VZVgE for prevention of HZ in adults aged 50 years or over, including those who have already received conventional vaccines.
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Vacunas contra el Adenovirus , Vacuna contra el Herpes Zóster , Herpes Zóster , Animales , Ratones , Herpesvirus Humano 3 , Adenoviridae/genética , Anticuerpos Antivirales , Herpes Zóster/prevención & control , Vacunación/métodos , Citocinas , Inmunogenicidad VacunalRESUMEN
Respiratory syncytial virus (RSV) infection and shingles are two viral diseases that affect older adults, and a combined vaccine to protect against both could be beneficial. RSV infection causes hospitalisations and significant morbidity in both children and adults and can be fatal in the elderly. The RSV fusion (F) envelope glycoprotein induces a strong RSV-neutralising antibody response and is the target of protective immunity in the first RSV vaccine for older adults, recently approved by the FDA. An initial childhood infection with the varicella zoster virus (VZV) results in chickenpox disease, but reactivation in older adults can cause shingles. This reactivation in sensory and autonomic neurons is characterized by a skin-blistering rash that can be accompanied by prolonged pain. The approved protein-in-adjuvant shingles vaccine induces VZV glycoprotein E (gE)-fspecific antibody and CD4+ T cell responses and is highly effective. Here we report the evaluation of RSV/shingles combination vaccine candidates based on non-replicating chimpanzee adenovirus (ChAd) vectors. We confirmed the cellular and humoral immunogenicity of the vaccine vectors in mice using T cell and antibody assays. We also carried out an RSV challenge study in cotton rats which demonstrated protective efficacy following a homologous prime-boost regimen with our preferred vaccine candidate.
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While historically viewed as an insulin insensitive organ, it is now accepted that insulin has a role in brain physiology. Changes in brain insulin and IGF1 signaling have been associated with neurological diseases, however the molecular factors regulating brain insulin sensitivity remain uncertain. In this study, we proposed that a recently described protein, termed Inceptor, may play a role in brain insulin and IGF1 resistance. We studied Inceptor in healthy and diseased nervous tissue to understand the distribution of the protein and examine how it may change in states of insulin resistance. We found that Inceptor is in fact present in cerebellum, hippocampus, hypothalamus, and cortex of the brain in neurons, with higher levels in cortex of female compared to male mice. We also confirmed that Inceptor colocalized with IR and IGF1R in brain. We saw little difference in insulin receptor signaling following Inceptor knockdown in neuron cultures, or in Inceptor levels with high-fat diet in mouse or Alzheimer's disease in mouse or human tissue. These results all provide significant advancements to our understanding of Inceptor in the brain. PROTOCOL REGISTRATION: The Stage 1 registered report manuscript was accepted-in-principle on 9 August 2022. This manuscript was registered through Open Science Forum (OSF) on 24 August 2022 and is available here: https://osf.io/9q8sw .
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Enfermedad de Alzheimer , Resistencia a la Insulina , Masculino , Femenino , Ratones , Humanos , Animales , Encéfalo/metabolismo , Insulina/metabolismo , Hipocampo/metabolismo , Enfermedad de Alzheimer/metabolismo , Receptor de Insulina/metabolismoRESUMEN
Background & Aims: Millions of people worldwide are infected chronically with HBV, which results in significant morbidity and mortality. Therapeutic vaccination is a strategy that aims to induce functional cure by restoring cellular immunity to HBV. Previously we have shown the candidate HBV immunotherapeutic vaccine ChAdOx1-HBV, encoding all major HBV antigens and a genetic adjuvant (shark invariant chain), is highly immunogenic in mice. Methods: Here we report the results of HBV001, a first-in-human, phase I, non-randomised, dose-escalation trial of ChAdOx1-HBV assessed in healthy volunteers and patients with chronic HBV (CHB). Results: Vaccination with a single dose of ChAdOx1-HBV was safe and well tolerated in both healthy and CHB cohorts. Vaccination induced high magnitude HBV-specific T cell responses against all major HBV antigens (core, polymerase, and surface) in healthy volunteers. Responses were detected but lower in patients with CHB. T cells generated by vaccination were cross-reactive between HBV C and D genotypes. Conclusions: ChAdOx1-HBV is safe and immunogenic in healthy volunteers and patients with CHB. In further studies, ChAdOx1-HBV will be used in combination with other therapeutic strategies with an aim to overcome the attenuated immunogenicity in patients with CHB. Impact and implications: Therapeutic vaccine ChAdOx1-HBV, a novel treatment for chronic hepatitis B infection (CHB), has been shown to be immunogenic in preclinical studies. In HBV001, a first-in-human phase I study, we show vaccination with ChAdOx1-HBV is safe and generates high magnitude T cell responses in healthy volunteers and lower levels of responses in patients with CHB. This is an important first step in the development of ChAdOx1-HBV as part of a wider therapeutic strategy to induce hepatitis B functional cure, and is of great interest to patients CHB and clinicians treating the condition. Clinical Trials Registration: This study is registered at ClinicalTrials.gov (NCT04297917).
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BACKGROUND: The use of immunotherapeutic vaccination in prostate cancer is a promising approach that likely requires the induction of functional, cytotoxic T cells . The experimental approach described here uses a well-studied adenovirus-poxvirus heterologous prime-boost regimen, in which the vectors encode a combination of prostate cancer antigens, with the booster dose delivered by either the intravenous or intramuscular (IM) route. This prime-boost regimen was investigated for antigen-specific CD8+ T cell induction. METHODS: The coding sequences for four antigens expressed in prostate cancer, 5T4, PSA, PAP, and STEAP1, were inserted into replication-incompetent chimpanzee adenovirus Oxford 1 (ChAdOx1) and into replication-deficient modified vaccinia Ankara (MVA). In four strains of mice, ChAdOx1 prime was delivered intramuscularly, with an MVA boost delivered by either IM or intravenous routes. Immune responses were measured in splenocytes using ELISpot, multiparameter flow cytometry, and a targeted in vivo killing assay. RESULTS: The prime-boost regimen was highly immunogenic, with intravenous administration of the boost resulting in a sixfold increase in the magnitude of antigen-specific T cells induced and increased in vivo killing relative to the intramuscular boosting route. Prostate-specific antigen (PSA)-specific responses were dominant in all mouse strains studied (C57BL/6, BALBc, CD-1 and HLA-A2 transgenic). CONCLUSION: This quadrivalent immunotherapeutic approach using four antigens expressed in prostate cancer induced high magnitude, functional CD8+ T cells in murine models. The data suggest that comparing the intravenous versus intramuscular boosting routes is worthy of investigation in humans.
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Antígeno Prostático Específico , Neoplasias de la Próstata , Masculino , Humanos , Ratones , Animales , Ratones Endogámicos C57BL , Linfocitos T CD8-positivos , Virus Vaccinia , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/terapia , Administración Intravenosa , Antígenos de Neoplasias , OxidorreductasasRESUMEN
BACKGROUND: The TRIM5 proteins are cellular restriction factors that prevent retroviral infection in a species-specific manner. Multiple experiments indicate that restriction activity requires accessory host factors, including E2-enzymes. To better understand the mechanism of restriction, we conducted yeast-two hybrid screens to identify proteins that bind to two TRIM5 orthologues. RESULTS: The only cDNAs that scored on repeat testing with both TRIM5 orthologues were the proteasome subunit PSMC2 and ubiquitin. Using co-immunoprecipitation assays, we demonstrated an interaction between TRIM5α and PSMC2, as well as numerous other proteasome subunits. Fluorescence microscopy revealed co-localization of proteasomes and TRIM5α cytoplasmic bodies. Forster resonance energy transfer (FRET) analysis indicated that the interaction between TRIM5 and PSMC2 was direct. Previous imaging experiments demonstrated that, when cells are challenged with fluorescently-labeled HIV-1 virions, restrictive TRIM5α orthologues assemble cytoplasmic bodies around incoming virion particles. Following virus challenge, we observed localization of proteasome subunits to rhTRIM5α cytoplasmic bodies that contained fluorescently labeled HIV-1 virions. CONCLUSIONS: Taken together, the results presented here suggest that localization of the proteasome to TRIM5α cytoplasmic bodies makes an important contribution to TRIM5α-mediated restriction.
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Proteínas Portadoras/metabolismo , VIH-1/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , ATPasas Asociadas con Actividades Celulares Diversas , Factores de Restricción Antivirales , Proteínas Portadoras/genética , Citoplasma/genética , Citoplasma/metabolismo , Citoplasma/virología , ADN Recombinante/genética , ADN Recombinante/metabolismo , Transferencia Resonante de Energía de Fluorescencia , Biblioteca de Genes , Células HEK293 , Infecciones por VIH/metabolismo , Infecciones por VIH/virología , VIH-1/patogenicidad , Humanos , Inmunoprecipitación , Microscopía Fluorescente , Complejo de la Endopetidasa Proteasomal/genética , Unión Proteica , Mapeo de Interacción de Proteínas/métodos , Especificidad de la Especie , Transfección , Proteínas de Motivos Tripartitos , Técnicas del Sistema de Dos Híbridos , Ubiquitina/genética , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligasas , UbiquitinaciónRESUMEN
Lassa virus (LASV) infects hundreds of thousands of individuals each year, highlighting the need for the accelerated development of preventive, diagnostic, and therapeutic interventions. To date, no vaccine has been licensed for LASV. ChAdOx1-Lassa-GPC is a chimpanzee adenovirus-vectored vaccine encoding the Josiah strain LASV glycoprotein precursor (GPC) gene. In the following study, we show that ChAdOx1-Lassa-GPC is immunogenic, inducing robust T-cell and antibody responses in mice. Furthermore, a single dose of ChAdOx1-Lassa-GPC fully protects Hartley guinea pigs against morbidity and mortality following lethal challenge with a guinea pig-adapted LASV (strain Josiah). By contrast, control vaccinated animals reached euthanasia criteria 10-12 days after infection. Limited amounts of LASV RNA were detected in the tissues of vaccinated animals. Viable LASV was detected in only one animal receiving a single dose of the vaccine. A prime-boost regimen of ChAdOx1-Lassa-GPC in guinea pigs significantly increased antigen-specific antibody titers and cleared viable LASV from the tissues. These data support further development of ChAdOx1-Lassa-GPC and testing in non-human primate models of infection.
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TRIM5alpha is a retrovirus restriction factor in the host cell cytoplasm that blocks infection before provirus establishment. Restriction activity requires capsid (CA)-specific recognition by the PRYSPRY domain of TRIM5alpha. To better understand the restriction mechanism, nine charge-cluster-to-triple-alanine mutants in the TRIM5alpha PRYSPRY domain were assessed for CA-specific restriction activity. Five mutants distributed along the TRIM5alpha PRYSPRY primary sequence disrupted restriction activity against N-tropic murine leukemia virus and equine infectious anemia virus. Modeling of the TRIM5alpha PRYSPRY domain based on the crystal structures of PRYSPRY-19q13.4.1, GUSTAVUS, and TRIM21 identified a surface patch where disruptive mutants clustered. All mutants in this patch retained CA-binding activity, a reticular distribution in the cytoplasm, and steady-state protein levels comparable to those of the wild type. Residues in the essential patch are conserved in TRIM5alpha orthologues and in closely related paralogues. The same surface patch in the TRIM18 and TRIM20 PRYSPRY domains is the site of mutants causing Opitz syndrome and familial Mediterranean fever. These results indicate that, in addition to CA-specific binding, the PRYSPRY domain possesses a second function, possibly binding of a cofactor, that is essential for retroviral restriction activity by TRIM5alpha.
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Proteínas de la Cápside/metabolismo , Proteínas Portadoras/metabolismo , Virus de la Anemia Infecciosa Equina/inmunología , Virus de la Leucemia Murina/inmunología , Secuencia de Aminoácidos , Sustitución de Aminoácidos/genética , Factores de Restricción Antivirales , Sitios de Unión , Proteínas Portadoras/genética , Línea Celular , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Mutación Missense , Unión Proteica , Conformación Proteica , Estructura Terciaria de Proteína , Proteínas de Motivos Tripartitos , Ubiquitina-Proteína LigasasRESUMEN
Chronic hepatitis B virus (HBV) infection affects 257 million people globally. Current therapies suppress HBV but viral rebound occurs on cessation of therapy; novel therapeutic strategies are urgently required. To develop a therapeutic HBV vaccine that can induce high magnitude T cells to all major HBV antigens, we have developed a novel HBV vaccine using chimpanzee adenovirus (ChAd) and modified vaccinia Ankara (MVA) viral vectors encoding multiple HBV antigens. ChAd vaccine alone generated very high magnitude HBV specific T cell responses to all HBV major antigens. The inclusion of a shark Invariant (SIi) chain genetic adjuvant significantly enhanced the magnitude of T-cells against HBV antigens. Compared to ChAd alone vaccination, ChAd-prime followed by MVA-boost vaccination further enhanced the magnitude and breadth of the vaccine induced T cell response. Intra-cellular cytokine staining study showed that HBV specific CD8+ and CD4+ T cells were polyfunctional, producing combinations of IFNγ, TNF-α, and IL-2. In summary, we have generated genetically adjuvanted ChAd and MVA vectored HBV vaccines with the potential to induce high-magnitude T cell responses through a prime-boost therapeutic vaccination approach. These pre-clinical studies pave the way for new studies of HBV therapeutic vaccination in humans with chronic hepatitis B infection.
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In the infectious diseases field, protective immunity against individual virus species or strains does not always confer cross-reactive immunity to closely related viruses, leaving individuals susceptible to disease after exposure to related virus species. This is a significant hurdle in the field of vaccine development, in which broadly protective vaccines represent an unmet need. This is particularly evident for filoviruses, as there are multiple family members that can cause lethal haemorrhagic fever, including Zaire ebolavirus, Sudan ebolavirus, and Marburg virus. In an attempt to address this need, both pre-clinical and clinical studies previously used mixed or co-administered monovalent vaccines to prevent filovirus mediated disease. However, these multi-vaccine and multi-dose vaccination regimens do not represent a practical immunisation scheme when considering the target endemic areas. We describe here the development of a single multi-pathogen filovirus vaccine candidate based on a replication-deficient simian adenoviral vector. Our vaccine candidate encodes three different filovirus glycoproteins in one vector and induces strong cellular and humoral immunity to all three viral glycoproteins after a single vaccination. Crucially, it was found to be protective in a stringent Zaire ebolavirus challenge in guinea pigs in a one-shot vaccination regimen. This trivalent filovirus vaccine offers a tenable vaccine product that could be rapidly translated to the clinic to prevent filovirus-mediated viral haemorrhagic fever.
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Nipah virus (NiV) is a highly pathogenic re-emerging virus that causes outbreaks in South East Asia. Currently, no approved and licensed vaccine or antivirals exist. Here, we investigated the efficacy of ChAdOx1 NiVB, a simian adenovirus-based vaccine encoding NiV glycoprotein (G) Bangladesh, in Syrian hamsters. Prime-only as well as prime-boost vaccination resulted in uniform protection against a lethal challenge with NiV Bangladesh: all animals survived challenge and we were unable to find infectious virus either in oral swabs, lung or brain tissue. Furthermore, no pathological lung damage was observed. A single-dose of ChAdOx1 NiVB also prevented disease and lethality from heterologous challenge with NiV Malaysia. While we were unable to detect infectious virus in swabs or tissue of animals challenged with the heterologous strain, a very limited amount of viral RNA could be found in lung tissue by in situ hybridization. A single dose of ChAdOx1 NiVB also provided partial protection against Hendra virus and passive transfer of antibodies elicited by ChAdOx1 NiVB vaccination partially protected Syrian hamsters against NiV Bangladesh. From these data, we conclude that ChAdOx1 NiVB is a suitable candidate for further NiV vaccine pre-clinical development.
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Adenovirus de los Simios/genética , Portadores de Fármacos , Infecciones por Henipavirus/prevención & control , Virus Nipah/inmunología , Vacunas Virales/inmunología , Estructuras Animales/virología , Animales , Modelos Animales de Enfermedad , Femenino , Infecciones por Henipavirus/inmunología , Mesocricetus , Virus Nipah/genética , Análisis de Supervivencia , Vacunas Sintéticas/administración & dosificación , Vacunas Sintéticas/genética , Vacunas Sintéticas/inmunología , Vacunas Virales/administración & dosificación , Vacunas Virales/genéticaRESUMEN
Influenza-virus-mediated disease can be associated with high levels of morbidity and mortality, particularly in younger children and older adults. Vaccination is the primary intervention used to curb influenza virus infection, and the WHO recommends immunization for at-risk individuals to mitigate disease. Unfortunately, influenza vaccine composition needs to be updated annually due to antigenic shift and drift in the viral immunogen hemagglutinin (HA). There are a number of alternate vaccination strategies in current development which may circumvent the need for annual re-vaccination, including new platform technologies such as viral-vectored vaccines. We discuss the different vectored vaccines that have been or are currently in clinical trials, with a forward-looking focus on immunogens that may be protective against seasonal and pandemic influenza infection, in the context of viral-vectored vaccines. We also discuss future perspectives and limitations in the field that will need to be addressed before new vaccines can significantly impact disease levels.
RESUMEN
Despite promising progress in malaria vaccine development, an efficacious subunit vaccine against P. falciparum remains to be licensed and deployed. This study aimed to improve on the immunogenicity of the leading liver-stage vaccine candidate (ChAd63-MVA ME-TRAP), known to confer protection by eliciting high levels of antigen-specific CD8+ T cells. We previously showed fusion of ME-TRAP to the human MHC class II invariant chain (Ii) could enhance CD8+ T cell responses in non-human primates, but did not progress to clinical testing due to potential risk of auto-immunity by vaccination of humans with a self-antigen. Initial immunogenicity analyses of ME-TRAP fused to subdomains of the Ii showed that the Ii transmembrane domain alone can enhance CD8+ T cell responses. Subsequently, truncated Ii sequences with low homology to human Ii were developed and shown to enhance CD8+ T cell responses. By systematically mutating the TM domain sequence, multimerization of the Ii chain was shown to be important for immune enhancement. We subsequently identified several proteins from a variety of microbial pathogens with similar characteristics, that also enhance the CD8+ T cell response and could therefore be used in viral vector vaccines when potent cell mediated immunity is required.