RESUMEN
BACKGROUND AND AIMS: A prophylactic vaccine targeting multiple HCV genotypes (gt) is urgently required to meet World Health Organization elimination targets. Neutralizing antibodies (nAbs) and CD4+ and CD8+ T cells are associated with spontaneous clearance of HCV, and each may contribute to protective immunity. However, current vaccine candidates generate either nAbs or T cells targeting genetically variable epitopes and have failed to show efficacy in human trials. We have previously shown that a simian adenovirus vector (ChAdOx1) encoding conserved sequences across gt1-6 (ChAd-Gt1-6), and separately gt-1a E2 protein with variable regions deleted (E2Δ123HMW ), generates pan-genotypic T cells and nAbs, respectively. We now aim to develop a vaccine to generate both viral-specific B- and T-cell responses concurrently. APPROACH AND RESULTS: We show that vaccinating with ChAd-Gt1-6 and E2Δ123HMW sequentially in mice generates T-cell and antibody (Ab) responses comparable to either vaccine given alone. We encoded E2Δ123 in ChAdOx1 (ChAd-E2Δ123) and show that this, given with an E2Δ123HMW protein boost, induces greater CD81-E2 inhibitory and HCV-pseudoparticle nAb titers compared to the E2Δ123HMW prime boost. We developed bivalent viral vector vaccines (ChAdOx1 and modified vaccinia Ankara [MVA]) encoding both Gt1-6 and E2Δ123 immunogens (Gt1-6-E2Δ123) generating polyfunctional CD4+ and CD8+ T cells and nAb titers in prime/boost strategies. This approach generated nAb responses comparable to monovalent E2Δ123 ChAd/MVA vaccines and superior to three doses of recombinant E2Δ123HMW protein, while also generating high-magnitude T-cell responses. CONCLUSIONS: These data are an important step forward for the development of a pan-genotype HCV vaccine to elicit T cells and nAbs for future assessment in humans.
Asunto(s)
Hepatitis C , Vacunas , Animales , Anticuerpos Neutralizantes , Anticuerpos Antivirales , Linfocitos T CD8-positivos , Epítopos , Genotipo , Hepacivirus/genética , Hepatitis C/prevención & control , Anticuerpos contra la Hepatitis C , Humanos , Ratones , Virus Vaccinia/genéticaRESUMEN
BACKGROUND AND AIMS: The lack of immunocompetent small animal models for hepatitis C virus (HCV) has greatly hindered the development of effective vaccines. Using rodent hepacivirus (RHV), a homolog of HCV that shares many characteristics of HCV infection, we report the development and application of an RHV outbred rat model for HCV vaccine development. APPROACH AND RESULTS: Simian adenovirus (ChAdOx1) encoding a genetic immune enhancer (truncated shark class II invariant chain) fused to the nonstructural (NS) proteins NS3-NS5B from RHV (ChAd-NS) was used to vaccinate Sprague-Dawley rats, resulting in high levels of cluster of differentiation 8-positive (CD8+ ) T-cell responses. Following RHV challenge (using 10 or 100 times the minimum infectious dose), 42% of vaccinated rats cleared infection within 6-8 weeks, while all mock vaccinated controls became infected with high-level viremia postchallenge. A single, 7-fold higher dose of ChAd-NS increased efficacy to 67%. Boosting with ChAd-NS or with a plasmid encoding the same NS3-NS5B antigens increased efficacy to 100% and 83%, respectively. A ChAdOx1 vector encoding structural antigens (ChAd-S) was also constructed. ChAd-S alone showed no efficacy. Strikingly, when combined with ChAd-NS, ChAD-S produced 83% efficacy. Protection was associated with a strong CD8+ interferon gamma-positive recall response against NS4. Next-generation sequencing of a putative RHV escape mutant in a vaccinated rat identified mutations in both identified immunodominant CD8+ T-cell epitopes. CONCLUSIONS: A simian adenovirus vector vaccine strategy is effective at inducing complete protective immunity in the rat RHV model. The RHV Sprague-Dawley rat challenge model enables comparative testing of vaccine platforms and antigens and identification of correlates of protection and thereby provides a small animal experimental framework to guide the development of an effective vaccine for HCV in humans.
Asunto(s)
Hepacivirus/inmunología , Vacunación , Vacunas contra Hepatitis Viral/inmunología , Adenoviridae/genética , Adenoviridae/inmunología , Animales , Linfocitos T CD8-positivos/inmunología , Modelos Animales de Enfermedad , Epítopos de Linfocito T , Interferón gamma/sangre , Masculino , Ratas , Ratas Sprague-Dawley , Vacunas Sintéticas/inmunología , Proteínas no Estructurales Virales/inmunologíaRESUMEN
BACKGROUND: Respiratory syncytial virus (RSV) disease is a major cause of infant morbidity and mortality. This Phase I, randomized, observer-blind, placebo- and active-controlled study evaluated an investigational vaccine against RSV (ChAd155-RSV) using the viral vector chimpanzee-adenovirus-155, encoding RSV fusion (F), nucleocapsid, and transcription antitermination proteins. METHODS: Healthy 18-45-year-old adults received ChAd155-RSV, a placebo, or an active control (Bexsero) at Days (D) 0 and 30. An escalation from a low dose (5 × 109 viral particles) to a high dose (5 × 1010 viral particles) occurred after the first 16 participants. Endpoints were solicited/unsolicited and serious adverse events (SAEs), biochemical/hematological parameters, cell-mediated immunogenicity by enzyme-linked immunospot, functional neutralizing antibodies, anti RSV-F immunoglobin (Ig) G, and ChAd155 neutralizing antibodies. RESULTS: There were 7 participants who received the ChAd155-RSV low dose, 31 who received the ChAd155-RSV high dose, 19 who received the placebo, and 15 who received the active control. No dose-related toxicity or attributable SAEs at the 1-year follow-up were observed. The RSV-A neutralizing antibodies geometric mean titer ratios (post/pre-immunization) following a high dose were 2.6 (D30) and 2.3 (D60). The ratio of the fold-rise (D0 to D30) in anti-F IgG over the fold-rise in RSV-A-neutralizing antibodies was 1.01. At D7 after the high dose of the study vaccine, the median frequencies of circulating B-cells secreting anti-F antibodies were 133.3/106 (IgG) and 16.7/106 (IgA) in peripheral blood mononuclear cells (PBMCs). The median frequency of RSV-F-specific interferon γ-secreting T-cells after a ChAd155-RSV high dose was 108.3/106 PBMCs at D30, with no increase after the second dose. CONCLUSIONS: In adults previously naturally exposed to RSV, ChAd155-RSV generated increases in specific humoral and cellular immune responses without raising significant safety concerns. CLINICAL TRIALS REGISTRATION: NCT02491463.
Asunto(s)
Infecciones por Virus Sincitial Respiratorio , Vacunas contra Virus Sincitial Respiratorio , Adenoviridae , Adolescente , Adulto , Animales , Anticuerpos Neutralizantes , Anticuerpos Antivirales , Humanos , Leucocitos Mononucleares , Persona de Mediana Edad , Nucleocápside , Pan troglodytes , Infecciones por Virus Sincitial Respiratorio/prevención & control , Proteínas Virales , Virión , Adulto JovenRESUMEN
Adenoviral vectors induce robust epitope-specific CD8+ T cell responses. Within the repertoire of responses generated both conventional memory evolution and the phenomenon of memory inflation are seen. The rules governing which epitopes inflate are not fully known, but may include a role for both antigen processing and competition. To investigate this, we looked at memory generated from vectors targeting the Gp33-41 (KAVYNFATC/K9C) epitope from the gp of lymphocytic choriomeningitis virus (LCMV) in mice. This well-described epitope has both the Gp33-41 and Gp34-41 epitopes embedded within it. Vaccination with a full-length gp or a minigene Ad-Gp33/K9C vector-induced conventional memory responses against the immunodominant Gp33/K9C epitope but a strong inflationary response against the Gp34/A8C epitope. These responses showed sustained in vivo function, with complete protection against LCMV infectious challenge. Given the unexpected competition between epitopes seen in the minigene model, we further tested epitope competition using the full-length Ad-LacZ (ß-galactosidase) model. Generation of an Ad-LacZ vector with a single amino acid disruption of the inflationary ß-gal96-103 /D8V epitope transformed the ß-gal497-504 /I8V epitope from conventional to inflationary memory. This work collectively demonstrates the importance of epitope competition within adenoviral vector inserts and is of relevance to future studies using adenoviral vectored immunogens.
Asunto(s)
Epítopos de Linfocito T/inmunología , Vectores Genéticos/inmunología , Epítopos Inmunodominantes/inmunología , Memoria Inmunológica/inmunología , Adenoviridae/inmunología , Animales , Antígenos Virales/inmunología , Linfocitos T CD8-positivos/inmunología , Femenino , Coriomeningitis Linfocítica/inmunología , Virus de la Coriomeningitis Linfocítica/inmunología , Ratones , Ratones Endogámicos C57BL , Proteínas Virales/inmunologíaRESUMEN
The efficacies of many new T cell vaccines rely on generating large populations of long-lived pathogen-specific effector memory CD8 T cells. However, it is now increasingly recognized that prior infection history impacts on the host immune response. Additionally, the order in which these infections are acquired could have a major effect. Exploiting the ability to generate large sustained effector memory (i.e. inflationary) T cell populations from murine cytomegalovirus (MCMV) and human Adenovirus-subtype (AdHu5) 5-beta-galactosidase (Ad-lacZ) vector, the impact of new infections on pre-existing memory and the capacity of the host's memory compartment to accommodate multiple inflationary populations from unrelated pathogens was investigated in a murine model. Simultaneous and sequential infections, first with MCMV followed by Ad-lacZ, generated inflationary populations towards both viruses with similar kinetics and magnitude to mono-infected groups. However, in Ad-lacZ immune mice, subsequent acute MCMV infection led to a rapid decline of the pre-existing Ad-LacZ-specific inflating population, associated with bystander activation of Fas-dependent apoptotic pathways. However, responses were maintained long-term and boosting with Ad-lacZ led to rapid re-expansion of the inflating population. These data indicate firstly that multiple specificities of inflating memory cells can be acquired at different times and stably co-exist. Some acute infections may also deplete pre-existing memory populations, thus revealing the importance of the order of infection acquisition. Importantly, immunization with an AdHu5 vector did not alter the size of the pre-existing memory. These phenomena are relevant to the development of adenoviral vectors as novel vaccination strategies for diverse infections and cancers. (241 words).
Asunto(s)
Adenovirus Humanos/inmunología , Linfocitos T CD8-positivos/inmunología , Memoria Inmunológica , Muromegalovirus/inmunología , Vacunas Virales/inmunología , Infecciones por Adenovirus Humanos/inmunología , Infecciones por Adenovirus Humanos/prevención & control , Adenovirus Humanos/genética , Adenovirus Humanos/patogenicidad , Animales , Coinfección/inmunología , Coinfección/prevención & control , Epítopos de Linfocito T/genética , Epítopos de Linfocito T/inmunología , Infecciones por Herpesviridae/inmunología , Infecciones por Herpesviridae/prevención & control , Interacciones Huésped-Patógeno/inmunología , Humanos , Operón Lac , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Modelos Inmunológicos , Muromegalovirus/genética , Muromegalovirus/patogenicidad , Receptores de Interleucina-18/deficiencia , Receptores de Interleucina-18/genética , Receptores de Interleucina-18/inmunología , Vacunas Virales/genéticaRESUMEN
Tuberculosis remains a global health problem so that a more effective vaccine than bacillus Calmette-Guérin is urgently needed. Cytomegaloviruses persist lifelong in vivo and induce powerful immune and increasing ("inflationary") responses, making them attractive vaccine vectors. We have used an m1-m16-deleted recombinant murine CMV (MCMV) expressing Mycobacterium tuberculosis Ag 85A to show that infection of mice with this recombinant significantly reduces the mycobacterial load after challenge with M. tuberculosis, whereas control empty virus has a lesser effect. Both viruses induce immune responses to H-2(d)-restricted epitopes of MCMV pp89 and M18 Ags characteristic of infection with other MCMVs. A low frequency of 85A-specific memory cells could be revealed by in vivo or in vitro boosting or after challenge with M. tuberculosis. Kinetic analysis of M. tuberculosis growth in the lungs of CMV-infected mice shows early inhibition of M. tuberculosis growth abolished by treatment with NK-depleting anti-asialo ganglio-N-tetraosylceramide Ab. Microarray analysis of the lungs of naive and CMV-infected mice shows increased IL-21 mRNA in infected mice, whereas in vitro NK assays indicate increased levels of NK activity. These data indicate that activation of NK cells by MCMV provides early nonspecific protection against M. tuberculosis, potentiated by a weak 85A-specific T cell response, and they reinforce the view that the innate immune system plays an important role in both natural and vaccine-induced protection against M. tuberculosis.
Asunto(s)
Epítopos/inmunología , Vectores Genéticos , Muromegalovirus , Mycobacterium tuberculosis/inmunología , Vacunas contra la Tuberculosis/inmunología , Tuberculosis Pulmonar/prevención & control , Animales , Epítopos/genética , Femenino , Antígeno de Histocompatibilidad H-2D/genética , Antígeno de Histocompatibilidad H-2D/inmunología , Interleucinas/genética , Interleucinas/inmunología , Células Asesinas Naturales/inmunología , Células Asesinas Naturales/patología , Ratones , Ratones Endogámicos BALB C , Mycobacterium tuberculosis/genética , Vacunas contra la Tuberculosis/genética , Tuberculosis Pulmonar/genética , Tuberculosis Pulmonar/inmunología , Tuberculosis Pulmonar/patologíaRESUMEN
BACKGROUND: Vaccine development in human Plasmodium falciparum malaria has been hampered by the exceptionally high levels of CD8(+) T cells required for efficacy. Use of potently immunogenic human adenoviruses as vaccine vectors could overcome this problem, but these are limited by preexisting immunity to human adenoviruses. METHODS: From 2007 to 2010, we undertook a phase I dose and route finding study of a new malaria vaccine, a replication-incompetent chimpanzee adenovirus 63 (ChAd63) encoding the preerythrocytic insert multiple epitope thrombospondin-related adhesion protein (ME-TRAP; n = 54 vaccinees) administered alone (n = 28) or with a modified vaccinia virus Ankara (MVA) ME-TRAP booster immunization 8 weeks later (n = 26). We observed an excellent safety profile. High levels of TRAP antigen-specific CD8(+) and CD4(+) T cells, as detected by interferon γ enzyme-linked immunospot assay and flow cytometry, were induced by intramuscular ChAd63 ME-TRAP immunization at doses of 5 × 10(10) viral particles and above. Subsequent administration of MVA ME-TRAP boosted responses to exceptionally high levels, and responses were maintained for up to 30 months postvaccination. CONCLUSIONS: The ChAd63 chimpanzee adenovirus vector appears safe and highly immunogenic, providing a viable alternative to human adenoviruses as vaccine vectors for human use. CLINICAL TRIALS REGISTRATION: NCT00890019.
Asunto(s)
Adenovirus de los Simios/inmunología , Vacunas contra la Malaria/administración & dosificación , Vacunas contra la Malaria/inmunología , Malaria Falciparum/inmunología , Proteínas Protozoarias/inmunología , Vacunas de ADN/administración & dosificación , Vacunas de ADN/inmunología , Adenovirus de los Simios/genética , Animales , Anticuerpos Neutralizantes/sangre , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD8-positivos/metabolismo , Epítopos , Citometría de Flujo , Humanos , Interferón gamma/metabolismo , Interleucina-2/metabolismo , Vacunas contra la Malaria/efectos adversos , Factor de Necrosis Tumoral alfa/metabolismo , Vacunas de ADN/efectos adversosRESUMEN
Protein acetylation plays a key role in regulating cellular processes and is subject to aberrant control in diverse pathologies. Although histone deacetylase (HDAC) inhibitors are approved drugs for certain cancers, it is not known whether they can be deployed in other therapeutic contexts. We have explored the clinical HDAC inhibitor, zabadinostat/CXD101, and found that it is a stand-alone regulator of the adaptive immune response. Zabadinostat treatment increased expression of MHC class I and II genes in a variety of cells, including dendritic cells (DCs) and healthy tissue. Remarkably, zabadinostat enhanced the activity of DCs, and CD4 and CD8 T lymphocytes. Using an antigenic peptide presented to the immune system by MHC class I, zabadinostat caused an increase in antigen-specific CD8 T lymphocytes. Further, mice immunised with covid19 spike protein and treated with zabadinostat exhibit enhanced covid19 neutralising antibodies and an increased level of T lymphocytes. The enhanced humoral response reflected increased activity of T follicular helper (Tfh) cells and germinal centre (GC) B cells. Our results argue strongly that zabadinostat has potential to augment diverse therapeutic agents that act through the immune system.
Asunto(s)
COVID-19 , Inmunidad Humoral , Ratones , Animales , Linfocitos T Colaboradores-Inductores , Inhibidores de Histona Desacetilasas/farmacología , Inmunidad Adaptativa , AntígenosRESUMEN
Protein arginine methyltransferase (PRMT) 5 is over-expressed in a variety of cancers and the master transcription regulator E2F1 is an important methylation target. We have explored the role of PRMT5 and E2F1 in regulating the non-coding genome and report here a striking effect on long non-coding (lnc) RNA gene expression. Moreover, many MHC class I protein-associated peptides were derived from small open reading frames in the lncRNA genes. Pharmacological inhibition of PRMT5 or adjusting E2F1 levels qualitatively altered the repertoire of lncRNA-derived peptide antigens displayed by tumour cells. When presented to the immune system as either ex vivo-loaded dendritic cells or expressed from a viral vector, lncRNA-derived peptides drove a potent antigen-specific CD8 T lymphocyte response, which translated into a significant delay in tumour growth. Thus, lncRNA genes encode immunogenic peptides that can be deployed as a cancer vaccine.
Asunto(s)
Neoplasias , ARN Largo no Codificante , Humanos , ARN Largo no Codificante/genética , Neoplasias/genética , Neoplasias/terapia , Antígenos de Histocompatibilidad Clase I/genética , Antígenos de Histocompatibilidad Clase I/metabolismo , Péptidos/genética , Linfocitos T CD8-positivos , Proteína-Arginina N-MetiltransferasasRESUMEN
Infection of mice with sporozoites of Plasmodium berghei or Plasmodium yoelii has been used extensively to evaluate liver-stage protection by candidate preerythrocytic malaria vaccines. Unfortunately, repeated success of such vaccines in mice has not translated readily to effective malaria vaccines in humans. Thus, mice may be used better as models to dissect basic parameters required for immunity to Plasmodium-infection than as preclinical vaccine models. In turn, this basic information may aid in the rational design of malaria vaccines. Here, we describe a model of circumsporozoite-specific memory CD8 T cell generation that protects mice against multiple P. berghei sporozoite challenges for at least 19 months. Using this model we defined a threshold frequency of memory CD8 T cells in the blood that predicts long-term sterilizing immunity against liver-stage infection. Importantly, the number of Plasmodium-specific memory CD8 T cells required for immunity greatly exceeds the number required for resistance to other pathogens. In addition, this model allowed us to identify readily individual immunized mice that exceed or fall below the protective threshold before infection, information that should greatly facilitate studies to dissect basic mechanisms of protective CD8 T cell memory against liver-stage Plasmodium infection. Furthermore, the extremely large threshold in memory CD8 T cell frequencies required for long-term protection in mice may have important implications for development of effective malaria vaccines.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Memoria Inmunológica/inmunología , Malaria/inmunología , Animales , Células Dendríticas/inmunología , Malaria/parasitología , Ratones , Ratones Endogámicos BALB C , Plasmodium berghei/inmunología , Plasmodium berghei/patogenicidad , Proteínas Protozoarias/inmunología , Especificidad por Sustrato , Factores de TiempoRESUMEN
Mucosal-associated invariant T (MAIT) cells are innate sensors of viruses and can augment early immune responses and contribute to protection. We hypothesized that MAIT cells may have inherent adjuvant activity in vaccine platforms that use replication-incompetent adenovirus vectors. In mice and humans, ChAdOx1 (chimpanzee adenovirus Ox1) immunization robustly activated MAIT cells. Activation required plasmacytoid dendritic cell (pDC)-derived interferon (IFN)-α and monocyte-derived interleukin-18. IFN-α-induced, monocyte-derived tumor necrosis factor was also identified as a key secondary signal. All three cytokines were required in vitro and in vivo. Activation of MAIT cells positively correlated with vaccine-induced T cell responses in human volunteers and MAIT cell-deficient mice displayed impaired CD8+ T cell responses to multiple vaccine-encoded antigens. Thus, MAIT cells contribute to the immunogenicity of adenovirus vectors, with implications for vaccine design.
Asunto(s)
Adenoviridae/inmunología , Inmunogenicidad Vacunal , Células T Invariantes Asociadas a Mucosa/inmunología , Vacunas Virales/inmunología , Animales , Linfocitos T CD8-positivos/inmunología , Células Dendríticas/inmunología , Vectores Genéticos/inmunología , Humanos , Interferón-alfa/metabolismo , Interleucina-18/metabolismo , Activación de Linfocitos , Ratones , Ratones Endogámicos C57BL , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Protective immunity generated following malaria infection may be comprised of Ab or T cells against malaria Ag of different stages; however, the short-lived immunity that is observed suggests deficiency in immune memory or regulatory activity. In this study, cellular immune responses were investigated in individuals receiving Plasmodium falciparum sporozoite challenge by the natural (mosquito bite) route as part of a malaria vaccine efficacy trial. Parasitemia, monitored by blood film microscopy and PCR, was subsequently cleared with drugs. All individuals demonstrated stable IFN-gamma, IL-2 and IL-4 ex vivo ELISPOT effector responses against P. falciparum-infected RBC (iRBC) Ag, 28 and 90 days after challenge. However, infected RBC-specific central memory responses, as measured by IFN-gamma cultured ELISPOT, were low and unstable over time, despite CD4(+) T cells being highly proliferative by CFSE dilution, and showed an inverse relationship to parasite density. In support of the observation of poor memory, co-culture experiments showed reduced responses to common recall Ag, indicating malaria-specific regulatory activity. This activity could not be accounted for by the expression of IL-10, TGF-beta, FOXP3 or CTLA-4, but proliferating T cells expressed high levels of CD95, indicating a pro-apoptotic phenotype. Lastly, there was an inverse relationship between FOXP3 expression, when measured 10 days after challenge, and ex vivo IFN-gamma measured more than 100 days later. This study shows that malaria infection elicits specific Th1 and Th2 effector cells, but concomitant weak central memory and regulatory activity, which may help to explain the short-lived immunity observed.
Asunto(s)
Memoria Inmunológica/inmunología , Vacunas contra la Malaria/inmunología , Malaria Falciparum/inmunología , Subgrupos de Linfocitos T/inmunología , Linfocitos T/inmunología , Adolescente , Adulto , Separación Celular , Ensayos Clínicos como Asunto , Citometría de Flujo , Humanos , Interferón gamma/biosíntesis , Interferón gamma/inmunología , Persona de Mediana Edad , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Adulto JovenRESUMEN
BACKGROUND: Viral genetic variability presents a major challenge to the development of a prophylactic hepatitis C virus (HCV) vaccine. A promising HCV vaccine using chimpanzee adenoviral vectors (ChAd) encoding a genotype (gt) 1b non-structural protein (ChAd-Gt1b-NS) generated high magnitude T cell responses. However, these T cells showed reduced cross-recognition of dominant epitope variants and the vaccine has recently been shown to be ineffective at preventing chronic HCV. To address the challenge of viral diversity, we developed ChAd vaccines encoding HCV genomic sequences that are conserved between all major HCV genotypes and adjuvanted by truncated shark invariant chain (sIitr). METHODS: Age-matched female mice were immunised intramuscularly with ChAd (108 infectious units) encoding gt-1 and -3 (ChAd-Gt1/3) or gt-1 to -6 (ChAd-Gt1-6) conserved segments spanning the HCV proteome, or gt-1b (ChAd-Gt1b-NS control), with immunogenicity assessed 14-days post-vaccination. RESULTS: Conserved segment vaccines, ChAd-Gt1/3 and ChAd-Gt1-6, generated high-magnitude, broad, and functional CD4+ and CD8+ T cell responses. Compared to the ChAd-Gt1b-NS vaccine, these vaccines generated significantly greater responses against conserved non-gt-1 antigens, including conserved subdominant epitopes that were not targeted by ChAd-Gt1b-NS. Epitopes targeted by the conserved segment HCV vaccine induced T cells, displayed 96.6% mean sequence homology between all HCV subtypes (100% sequence homology for the majority of genotype-1, -2, -4 sequences and 94% sequence homology for gt-3, -6, -7, and -8) in contrast to 85.1% mean sequence homology for epitopes targeted by ChAd-Gt1b-NS induced T cells. The addition of truncated shark invariant chain (sIitr) increased the magnitude, breadth, and cross-reactivity of the T cell response. CONCLUSIONS: We have demonstrated that genetically adjuvanted ChAd vectored HCV T cell vaccines encoding genetic sequences conserved between genotypes are immunogenic, activating T cells that target subdominant conserved HCV epitopes. These pre-clinical studies support the use of conserved segment HCV T cell vaccines in human clinical trials.
Asunto(s)
Hepatitis C , Vacunas contra Hepatitis Viral , Vacunas Virales , Animales , Epítopos , Epítopos de Linfocito T/genética , Femenino , Genotipo , Hepacivirus/genética , Ratones , Linfocitos T , Vacunas contra Hepatitis Viral/genética , Vacunas Virales/genéticaRESUMEN
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.
RESUMEN
Background: Persistent viruses such as murine cytomegalovirus (MCMV) and adenovirus-based vaccines induce strong, sustained CD8 + T-cell responses, described as memory "inflation". These retain functionality, home to peripheral organs and are associated with a distinct transcriptional program. Methods: To further define the nature of the transcriptional mechanisms underpinning memory inflation at different sites we used single-cell RNA sequencing of tetramer-sorted cells from MCMV-infected mice, analyzing transcriptional networks in virus-specific populations in the spleen and gut intra-epithelial lymphocytes (IEL). Results: We provide a transcriptional map of T-cell memory and define a module of gene expression, which distinguishes memory inflation in spleen from resident memory T-cells (T RM) in the gut. Conclusions: These data indicate that CD8 + T-cell memory in the gut epithelium induced by persistent viruses and vaccines has a distinct quality from both conventional memory and "inflationary" memory which may be relevant to protection against mucosal infections.
RESUMEN
OBJECTIVES: Respiratory syncytial virus (RSV) causes respiratory infection across the world, with infants and the elderly at particular risk of developing severe disease and death. The replication-defective chimpanzee adenovirus (PanAd3-RSV) and modified vaccinia virus Ankara (MVA-RSV) vaccines were shown to be safe and immunogenic in young healthy adults. Here we report an extension to this first-in-man vaccine trial to include healthy older adults aged 60-75 years. METHODS: We evaluated the safety and immunogenicity of a single dose of MVA-RSV given by intra-muscular (IM) injection (nâ¯=â¯6), two doses of IM PanAd3-RSV given 4-weeks apart (nâ¯=â¯6), IM PanAd3-RSV prime and IM MVA-RSV boost 8-weeks later (nâ¯=â¯6), intra-nasal (IN) spray of PanAd3-RSV prime and IM MVA-RSV boost 8-weeks later (nâ¯=â¯6), or no vaccine (nâ¯=â¯6). Safety measures included all adverse events within one week of vaccination and blood monitoring. Immunogenicity measures included serum antibody responses (RSV- and PanAd3-neutralising antibody titres measured by plaque-reduction neutralisation and SEAP assays, respectively), peripheral B-cell immune responses (frequencies of F-specific IgG and IgA antibody secreting cells and memory B-cells by ex vivo and cultured dual-colour ELISpot assays respectively), and peripheral RSV-specific T-cell immune responses (frequencies of IFNγ-producing T-cells by ex vivo ELISpot and CD4+/CD8+/Tfh-like cell frequencies by ICS/FACS assay). RESULTS: The vaccines were safe and well tolerated. Compared with each individual baseline immunity the mean fold-changes in serum RSV-neutralising antibody, appearance and magnitude of F-specific IgG and IgA ASCs and expansion of CD4+/CD8+ IFNγ-producing T-cells in peripheral circulation were comparable to the results seen from younger healthy adults who received the same vaccine combination and dose. There were little/no IgA memory B-cell responses in younger and older adults. Expansion of IFNγ-producing T-cells was most marked in older adults following IM prime, with balanced CD4+ and CD8+ T cell responses. The RSV-specific immune responses to vaccination did not appear to be attenuated in the presence of PanAd3 (vector) neutralising antibody. CONCLUSIONS: PanAd3-RSV and MVA-RSV was safe and immunogenic in older adults and the parallel induction of RSV-specific humoral and cellular immunity merits further assessment in providing protection from severe disease.
Asunto(s)
Portadores de Fármacos , Inmunidad Celular , Inmunidad Humoral , Infecciones por Virus Sincitial Respiratorio/prevención & control , Vacunas contra Virus Sincitial Respiratorio/efectos adversos , Vacunas contra Virus Sincitial Respiratorio/inmunología , Virus Sincitial Respiratorio Humano/inmunología , Administración Intranasal , Adolescente , Adulto , Anciano , Anticuerpos Neutralizantes/sangre , Anticuerpos Antivirales/sangre , Células Productoras de Anticuerpos/inmunología , Linfocitos B/inmunología , Efectos Colaterales y Reacciones Adversas Relacionados con Medicamentos/epidemiología , Femenino , Voluntarios Sanos , Humanos , Esquemas de Inmunización , Inyecciones Intramusculares , Masculino , Mastadenovirus/genética , Persona de Mediana Edad , Vacunas contra Virus Sincitial Respiratorio/administración & dosificación , Vacunas contra Virus Sincitial Respiratorio/genética , Virus Sincitial Respiratorio Humano/genética , Linfocitos T/inmunología , Vacunas Sintéticas/administración & dosificación , Vacunas Sintéticas/efectos adversos , Vacunas Sintéticas/genética , Vacunas Sintéticas/inmunología , Virus Vaccinia/genética , Adulto JovenRESUMEN
Cytomegalovirus (CMV) and non-replicating adenoviral vectors can induce expanded, sustained effector-memory CD8+ T-cell responses, termed "memory inflation". During murine CMV (MCMV) infection, CD4+ Tcells maintain inflationary virus-specific CD8+ T-cell responses via IL-2 but not IL-21. Adenovirus vector vaccination can induce phenotypically, functionally and transcriptionally similar inflationary responses, but it is not known how IL-21 influences the inflating memory response to adenoviral vaccination. Here, we show that IL-21 is an absolute requirement for induction and maintenance of vaccine-derived inflationary CD8+ T-cell responses. These data indicate that the induction pathway of inflationary Ad-LacZ T-cells is distinct from inflationary MCMV-specific T-cells and is highly dependent on IL-21. Our observations highlight a fundamental difference in the mechanism by which adenovirus vectors and MCMV drive inflationary T-cell responses.
Asunto(s)
Vacunas contra el Adenovirus/administración & dosificación , Vacunas contra el Adenovirus/inmunología , Linfocitos T CD8-positivos/inmunología , Memoria Inmunológica , Interleucinas/metabolismo , Animales , Interleucinas/genética , Ratones Endogámicos C57BL , Ratones TransgénicosRESUMEN
BACKGROUND: Hepatitis C virus (HCV) genomic variability is a major challenge to the generation of a prophylactic vaccine. We have previously shown that HCV specific T-cell responses induced by a potent T-cell vaccine encoding a single strain subtype-1b immunogen target epitopes dominant in natural infection. However, corresponding viral regions are highly variable at a population level, with a reduction in T-cell reactivity to these variants. We therefore designed and manufactured second generation simian adenovirus vaccines encoding genomic segments, conserved between viral genotypes and assessed these for immunogenicity. METHODS: We developed a computer algorithm to identify HCV genomic regions that were conserved between viral subtypes. Conserved segments below a pre-defined diversity threshold spanning the entire HCV genome were combined to create novel immunogens (1000-1500 amino-acids), covering variation in HCV subtypes 1a and 1b, genotypes 1 and 3, and genotypes 1-6 inclusive. Simian adenoviral vaccine vectors (ChAdOx) encoding HCV conserved immunogens were constructed. Immunogenicity was evaluated in C57BL6 mice using panels of genotype-specific peptide pools in ex-vivo IFN-Ï ELISpot and intracellular cytokine assays. RESULTS: ChAdOx1 conserved segment HCV vaccines primed high-magnitude, broad, cross-reactive T-cell responses; the mean magnitude of total HCV specific T-cell responses was 1174 SFU/106 splenocytes for ChAdOx1-GT1-6 in C57BL6 mice targeting multiple genomic regions, with mean responses of 935, 1474 and 1112 SFU/106 against genotype 1a, 1b and 3a peptide panels, respectively. Functional assays demonstrated IFNg and TNFa production by vaccine-induced CD4 and CD8 T-cells. In silico analysis shows that conserved immunogens contain multiple epitopes, with many described in natural HCV infection, predicting immunogenicity in humans. CONCLUSIONS: Simian adenoviral vectored vaccines encoding genetic segments that are conserved between all major HCV genotypes contain multiple T-cell epitopes and are highly immunogenic in pre-clinical models. These studies pave the way for the assessment of multi-genotypic HCV T-cell vaccines in humans.
Asunto(s)
Adenovirus de los Simios/genética , Portadores de Fármacos , Hepacivirus/inmunología , Hepatitis C/prevención & control , Leucocitos Mononucleares/inmunología , Vacunas Virales/inmunología , Animales , Secuencia Conservada , Citocinas/análisis , Ensayo de Immunospot Ligado a Enzimas , Femenino , Genotipo , Hepacivirus/clasificación , Hepacivirus/genética , Masculino , Ratones Endogámicos C57BL , Vacunas Sintéticas/genética , Vacunas Sintéticas/inmunología , Vacunas Virales/genéticaRESUMEN
The induction and maintenance of T cell memory is critical to the success of vaccines. A recently described subset of memory CD8+ T cells defined by intermediate expression of the chemokine receptor CX3CR1 was shown to have self-renewal, proliferative, and tissue-surveillance properties relevant to vaccine-induced memory. We tracked these cells when memory is sustained at high levels: memory inflation induced by cytomegalovirus (CMV) and adenovirus-vectored vaccines. In mice, both CMV and vaccine-induced inflationary T cells showed sustained high levels of CX3R1int cells exhibiting an effector-memory phenotype, characteristic of inflationary pools, in early memory. In humans, CX3CR1int CD8+ T cells were strongly induced following adenovirus-vectored vaccination for hepatitis C virus (HCV) (ChAd3-NSmut) and during natural CMV infection and were associated with a memory phenotype similar to that in mice. These data indicate that CX3CR1int cells form an important component of the memory pool in response to persistent viruses and vaccines in both mice and humans.
Asunto(s)
Adenoviridae/inmunología , Linfocitos T CD8-positivos/metabolismo , Receptor 1 de Quimiocinas CX3C/metabolismo , Citomegalovirus/inmunología , Memoria Inmunológica , Vacunas Virales/inmunología , Adenoviridae/genética , Adenoviridae/patogenicidad , Adulto , Animales , Linfocitos T CD8-positivos/inmunología , Receptor 1 de Quimiocinas CX3C/genética , Citomegalovirus/patogenicidad , Vectores Genéticos/genética , Vectores Genéticos/metabolismo , Hepacivirus/inmunología , Hepacivirus/metabolismo , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Persona de Mediana Edad , Miembro 7 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/metabolismo , Vacunas Sintéticas/inmunología , Proteínas no Estructurales Virales/genética , Proteínas no Estructurales Virales/inmunología , Proteínas no Estructurales Virales/metabolismo , Adulto JovenRESUMEN
The search for an efficacious vaccine against malaria is ongoing, and it is now widely believed that to confer protection a vaccine must induce very strong cellular and humoral immunity concurrently. We studied the immune response in mice immunized with the recombinant viral vaccines fowlpox strain FP9 and modified virus Ankara (MVA), a protein vaccine (CV-1866), or a combination of the two; all vaccines express parts of the same preerythrocytic malaria antigen, the Plasmodium berghei circumsporozoite protein (CSP). Mice were then challenged with P. berghei sporozoites to determine the protective efficacies of different vaccine regimens. Two immunizations with the protein vaccine CV-1866, based on the hepatitis B core antigen particle, induced strong humoral immunity to the repeat region of CSP that was weakly protective against sporozoite challenge. Prime-boost with the viral vector vaccines, FP9 followed by MVA, induced strong T-cell immunity to the CD8+ epitope Pb9 and partially protected animals from challenge. Physically mixing CV-1866 with FP9 or MVA and then immunizing with the resultant combinations in a prime-boost regimen induced both cellular and humoral immunity and afforded substantially higher levels of protection (combination, 90%) than either vaccine alone (CV-1866, 12%; FP9/MVA, 37%). For diseases such as malaria in which different potent immune responses are required to protect against different stages, using combinations of partially effective vaccines may offer a more rapid route to achieving deployable levels of efficacy than individual vaccine strategies.