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The aim of this study was to determine the reliability of lactic acid bacteria (LAB) as heterologous hosts for the expression of MPB70 and MPB83, two Mycobacterium bovis antigens that possess diagnostics and immunogenic properties, respectively. We therefore generated recombinant cells of Lactococcus lactis and Lactobacillus plantarum that carried hybrid genes encoding MPB70 and MPB83 fused to signal peptides that are specifically recognized by LAB. Only L. lactis was able to secrete MPB70 using the L. lactis signal peptide Usp45, and to produce MPB83 as an immunogenic membrane protein following its expression with the signal peptide of the L. plantarum lipoprotein prsA. Inactivated cells of MPB83-expressing L. lactis cultures enhanced NF-κB activation in macrophages. Our results show that L. lactis is a reliable host for the secretion and functional expression of antigens that are naturally produced by M. bovis, the causative agent of bovine tuberculosis (bTB). This represents the first step on a long process to establishing whether recombinant LAB could serve as a food-grade platform for potential diagnostic tools and/or vaccine interventions for use against bTB, a chronic disease that primarily affects cattle but also humans and a wide range of domestic and wild animals., (Copyright © 2019. Published by Elsevier Ltd.)

African swine fever (ASF) is a lethal haemorrhagic disease of domestic pigs for which there is no vaccine. Strains of the virus with reduced virulence can provide protection against related virulent strains of ASFV, but protection is not 100% and there are concerns about the safety profile of such viruses. However, they provide a useful tool for understanding the immune response to ASFV and previous studies using the low virulent isolate OUR T88/3 have shown that CD8+ cells are crucial for protection. In order to develop a vaccine that stimulates an effective anti-ASFV T-cell response we need to know which of the >150 viral proteins are recognized by the cellular immune response. Therefore, we used a gamma interferon ELIspot assay to screen for viral proteins recognized by lymphocytes from ASF-immune pigs using peptides corresponding to 133 proteins predicted to be encoded by OUR T88/3. Eighteen antigens that were recognized by ASFV-specific lymphocytes were then incorporated into adenovirus and MVA vectors, which were used in immunization and challenge experiments in pigs. We present a systematic characterization of the cellular immune response to this devastating disease and identify proteins capable of inducing ASFV-specific cellular and humoral immune responses in pigs. Pools of viral vectors expressing these genes did not protect animals from severe disease, but did reduce viremia in a proportion of pigs following ASFV challenge.

Immune responses to therapeutic transgenes are a potential hurdle to treat monogenic muscle disorders. These responses result from the neutralizing activity of transgene-specific B cells and cytotoxic T cells recruited upon gene transfer. We explored here how dual muscle-liver expression of a foreign transgene allows muscle transgene engraftment after adenoassociated viral vector delivery. We found in particular that induction of transgene-specific tolerance is imposed by concurrent muscle and liver targeting, resulting in the absence of CD8+ T cell responses to the transgene. This tolerance can be temporally decoupled, because transgene engraftment can be achieved in muscle weeks after liver transduction. Importantly, transgene-specific CD8+ T cell tolerance can be established despite preexisting immunity to the transgene. Whenever preexisting, transgene-specific CD4+ and CD8+ memory T cell responses are present, dual muscle-liver transduction turns polyclonal, transgene-specific CD8+ T cells into typically exhausted T cells with high programmed cell death 1 (PD-1) expression and lack of IFN-γ production. Our results demonstrate that successful transduction of muscle tissue can be achieved through liver-mediated control of humoral and cytotoxic T cell responses, even in the presence of preexisting immunity to the muscle-associated transgene.

Foot and Mouth Disease is a highly contagious and economically important disease of livestock. While vaccination is often effective at controlling viral spread, failures can occur due to strain mismatch or viral mutation. Foot and Mouth Disease Virus (FMDV) possesses a hypervariable region within the G-H Loop of VP1, a capsid protein commonly associated with virus neutralization. Here, we investigate the effect of replacement of the G-H loop hypervariable epitope with a xenoepitope from PRRS virus on the immunogenicity and efficacy of an adenovirus vectored FMDV vaccine (Ad5-FMD). Pigs were vaccinated with Ad5-FMD, the modified Ad5-FMD xeno , or PBS, followed by intradermal challenge with FDMV strain O 1 Manisa at 21 days post-vaccination. While overall serum antibody titers were significantly higher in Ad5-FMD xeno vaccinated animals, neutralizing antibody titers were decreased in pigs that received Ad5-FMD xeno , when compared to those vaccinated with Ad5-FMD, prior to viral challenge, indicative of immune redirection away from VP1 towards non-neutralizing epitopes. As expected, animals vaccinated with unmodified Ad5-FMD were protected from lesions, fever, and viremia. In contrast, animals vaccinated with Ad5-FMD xeno developed clinical signs and viremia, but at lower levels than that observed in PBS-treated controls. No significant difference was found in nasal shedding of virions between the two Ad5-FMD vaccinated groups. This data suggests that the hypervariable epitope of the VP1 G-H loop contributes to protective immunity conferred by Ad5 vector-delivered FMD vaccines in swine, and cannot be substituted without a loss of immunogenicity., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Many mRNA-based vaccines have been investigated for their specific potential to activate dendritic cells (DCs), the highly-specialized antigen-presenting cells of the immune system that play a key role in inducing effective CD4+ and CD8+ T-cell responses. In this paper we report a new vaccine/gene delivery platform that demonstrates the benefits of using a self-amplifying ("replicon") mRNA that is protected in a viral-protein capsid. Purified capsid protein from the plant virus Cowpea Chlorotic Mottle Virus (CCMV) is used to in vitro assemble monodisperse virus-like particles (VLPs) containing reporter proteins (e.g., Luciferase or eYFP) or the tandem-repeat model antigen SIINFEKL in RNA gene form, coupled to the RNA-dependent RNA polymerase from the Nodamura insect virus. Incubation of immature DCs with these VLPs results in increased activation of maturation markers - CD80, CD86 and MHC-II - and enhanced RNA replication levels, relative to incubation with unpackaged replicon mRNA. Higher RNA uptake/replication and enhanced DC activation were detected in a dose-dependent manner when the CCMV-VLPs were pre-incubated with anti-CCMV antibodies. In all experiments the expression of maturation markers correlates with the RNA levels of the DCs. Overall, these studies demonstrate that: VLP protection enhances mRNA uptake by DCs; coupling replicons to the gene of interest increases RNA and protein levels in the cell; and the presence of anti-VLP antibodies enhances mRNA levels and activation of DCs in vitro. Finally, preliminary in vivo experiments involving mouse vaccinations with SIINFEKL-replicon VLPs indicate a small but significant increase in antigen-specific T cells that are doubly positive for IFN and TFN induction., Competing Interests: The authors have declared that no competing interests exist.

Recombinant vectors based on a nonpathogenic parvovirus, the adeno-associated virus (AAV), have taken center stage in the past decade. The safety of AAV vectors in clinical trials and clinical efficacy in several human diseases are now well documented. Despite these achievements, it is increasingly clear that the full potential of AAV vectors composed of the naturally occurring capsids is unlikely to be realized. This article describes advances that have been made and challenges that remain in the optimal use of AAV vectors in human gene therapy applications., (Copyright © 2019 Elsevier Inc. All rights reserved.)

The prevalence of adeno-associated virus (AAV) has been investigated in bat populations, but little is known about the biological properties of this virus. In this study, four full-length bat AAV capsid genes were isolated in China, with their amino acid sequences sharing 61% identity with those of AAV2 on average. These capsid genes could package AAV particles in combination with AAV2 rep and ITRs, albeit at a lower efficiency. Bat AAVs could only slightly infect mouse liver but could transduce mouse muscle to some extent after systemic administration with a higher muscle/liver ratio than that of primate AAVs. Bat AAV 10HB showed moderate muscle transduction, similar to that of AAV2, during direct intramuscular injection and, compared with other AAV serotypes, was also relatively efficient in resisting human antibody neutralization after intramuscular injection. Evolutionary analysis revealed a number of codons in bat AAV capsid genes subject to positive selection, with sites corresponding to V259 and N691 in 10HB capsids being localized on the surface of the AAV2 capsid. Mutagenesis studies indicated that the positive selection in bat AAV capsids is driven by their tropism evolution in host species. Taken together, the results of this study indicate that bat AAV 10HB vector has the possible applications for muscular gene therapy, especially in the presence of human AAV neutralizing antibodies.

This study was conducted to determine whether exposure to particulate matter 2.5 (PM 2.5 ) affects the immune tolerance of neonatal mice via the regulation of PD-L1 expression. One-week-old BALB/c mice were exposed to PM 2.5 for 8 days. From day 8 to day 18, the mice were treated with 5 μg house dust mite (HDM) (i. n.) every two days. Adenovirus-carried PD-L1 overexpression vectors were infected into mice via nasal inhalation 6 days after exposure to PM 2.5 . Airway hyperresponsiveness (AHR) was examined in mice 19 days after exposure to PM 2.5 , and the related parameters of airway inflammation were studied on day 22. Co-exposure to PM 2.5 and HDM reduced PD-L1 expression but greatly increased infiltration of inflammatory cells, which was reversed by PD-L1 overexpression. Co-exposure to PM 2.5 and HDM also elevated serum IL-4, IL-5 and IL-13 levels and reduced TGF-β level. Exposure to PM 2.5 alone slightly increased the numbers of dendritic cells (DCs) but reduced the numbers of antigen-presenting cells expressing PD-L1 and Treg cells. Therefore, early exposure to PM 2.5 reduced PD-L1 expression in the lungs of neonatal mice, which interfered with immune tolerance establishment and subsequently resulted in allergic airway inflammation.

Modified vaccinia virus Ankara (MVA) is the leading poxvirus vector for development of vaccines against diverse infectious diseases. This distinction is based on high expression of proteins and good immunogenicity despite an inability to assemble infectious progeny in human cells, which together promote efficacy and safety. Nevertheless, the basis for the host-range restriction is unknown despite past systematic attempts to identify the relevant missing viral gene(s). The search for host-range factors is exacerbated by the large number of deletions, truncations and mutations that occurred during the long passage history of MVA in chicken embryo fibroblasts. By whole genome sequencing of a panel of recombinant host-range extended (HRE) MVAs generated by marker rescue with 40 kbp segments of vaccinia virus DNA, we identified serine protease inhibitor 1 (SPI-1) as one of several candidate host-range factors present in those viruses that gained the ability to replicate in human cells. Electron microscopy revealed that the interruption of morphogenesis in human cells infected with MVA occurred at a similar stage as that of a vaccinia virus strain WR SPI-1 deletion mutant. Moreover, the introduction of the SPI-1 gene into the MVA genome led to more than a 2-log enhancement of virus spread in human diploid MRC-5 cells, whereas deletion of the gene diminished the spread of HRE viruses by similar extents. Furthermore, MRC-5 cells stably expressing SPI-1 also enhanced replication of MVA. A role for additional host range genes was suggested by the restoration of MVA replication to a lower level relative to HRE viruses, particularly in other human cell lines. Although multiple sequence alignments revealed genetic changes in addition to SPI-1 common to the HRE MVAs, no evidence for their host-range function was found by analysis thus far. Our finding that SPI-1 is host range factor for MVA should simplify use of high throughput RNAi or CRISPR/Cas single gene methods to identify additional viral and human restriction elements., Competing Interests: The authors have declared that no competing interests exist.

Host immune responses play an important role in the outcome of infection with hepatitis C virus (HCV). They can lead to viral clearance and a positive outcome, or progression and severity of chronic disease. Extensive research in the past >25 years into understanding the immune responses against HCV have still resulted in many unanswered questions implicating a role for unknown factors and events. In our earlier studies, we made a surprising discovery that peptides derived from structural and non-structural proteins of HCV have substantial amino acid sequence homologies with various proteins of adenoviruses and that immunizing mice with a non-replicating, non-recombinant adenovirus vector leads to induction of a robust cross-reactive cellular and humoral response against various HCV antigens. In this work, we further demonstrate antibody cross-reactivity between Ad and HCV in vivo. We also extend this observation to show that recombinant adenoviruses containing antigens from unrelated pathogens also possess the ability to induce cross-reactive immune responses against HCV antigens along with the induction of transgene antigen-specific immunity. This cross-reactive immunity can (a) accommodate the making of dual-pathogen vaccines, (b) play an important role in the natural course of HCV infection and (c) provide a plausible answer to many unexplained questions regarding immunity to HCV.

HIV-1-infected individuals are treated with lifelong antiretroviral drugs to control the infection. A means to strengthen the antiviral T cell response might allow them to control viral loads without antiretroviral drugs. We report the development of a lentiviral vector-based dendritic cell (DC) vaccine in which HIV-1 antigen is co-expressed with CD40 ligand (CD40L) and a soluble, high-affinity programmed cell death 1 (PD-1) dimer. CD40L activates the DCs, whereas PD-1 binds programmed death ligand 1 (PD-L1) to prevent checkpoint activation and strengthen the cytotoxic T lymphocyte (CTL) response. The injection of humanized mice with DCs transduced with vector expressing CD40L and the HIV-1 SL9 epitope induced antigen-specific T cell proliferation and memory differentiation. Upon HIV-1 challenge of vaccinated mice, viral load was suppressed by 2 logs for 6 weeks. Introduction of the soluble PD-1 dimer into a vector that expressed full-length HIV-1 proteins accelerated the antiviral response. The results support development of this approach as a therapeutic vaccine that might allow HIV-1-infected individuals to control virus replication without antiretroviral therapy., (Copyright © 2019 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.)

Background: The colorectal cancer antigen GUCY2C exhibits unique split tolerance, evoking antigen-specific CD8 + , but not CD4 + , T-cell responses that deliver anti-tumor immunity without autoimmunity in mice. Here, the cancer vaccine Ad5-GUCY2C-PADRE was evaluated in a first-in-man phase I clinical study of patients with early-stage colorectal cancer to assess its safety and immunological efficacy., Methods: Ten patients with surgically-resected stage I or stage II (pN0) colon cancer received a single intramuscular injection of 10 11 viral particles (vp) of Ad5-GUCY2C-PADRE. Safety assessment and immunomonitoring were carried out for 6 months following immunization. This trial employed continual monitoring of both efficacy and toxicity of subjects as joint primary outcomes., Results: All patients receiving Ad5-GUCY2C-PADRE completed the study and none developed adverse events greater than grade 1. Antibody responses to GUCY2C were detected in 10% of patients, while 40% exhibited GUCY2C-specific T-cell responses. GUCY2C-specific responses were exclusively CD8 + cytotoxic T cells, mimicking pre-clinical studies in mice in which GUCY2C-specific CD4 + T cells are eliminated by self-tolerance, while CD8 + T cells escape tolerance and mediate antitumor immunity. Moreover, pre-existing neutralizing antibodies (NAbs) to the Ad5 vector were associated with poor vaccine-induced responses, suggesting that Ad5 NAbs oppose GUCY2C immune responses to the vaccine in patients and supported by mouse studies., Conclusions: Split tolerance to GUCY2C in cancer patients can be exploited to safely generate antigen-specific cytotoxic CD8 + , but not autoimmune CD4 + , T cells by Ad5-GUCY2C-PADRE in the absence of pre-existing NAbs to the viral vector., Trial Registration: This trial (NCT01972737) was registered at ClinicalTrials.gov on October 30th, 2013. https://clinicaltrials.gov/ct2/show/NCT01972737.

Induction of an antibody response capable of recognizing highly diverse strains is a major obstacle to the development of vaccines for viruses such as HIV and influenza. Here, we report the dynamics of B cell expansion and evolution at the single-cell level after vaccination with a replication-competent adenovirus type 4 recombinant virus expressing influenza H5 hemagglutinin. Fluorescent H1 or H5 probes were used to quantitate and isolate peripheral blood B cells and their antigen receptors. We observed increases in H5-specific antibody somatic hypermutation and potency for several months beyond the period of active viral replication that was not detectable at the serum level. Individual broad and potent antibodies could be isolated, including one stem-specific antibody that is part of a new multidonor class. These results demonstrate prolonged evolution of the B cell response for months after vaccination and should be considered in efforts to evaluate or boost vaccine-induced immunity., (Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Inducing strong mucosal immune responses by vaccination is important for providing protection against simian immunodeficiency virus (SIV). A replicating adenovirus type 5 host range mutant vector (Ad5hr) expressing SIV proteins induced mucosal immune responses in rectal tissue associated with delayed SIV acquisition in female rhesus macaques, but the initial mechanisms leading to the induced immunity have not been elucidated. As dendritic cells (DCs) are known to orchestrate both innate and adaptive effector immune cell responses, we investigated their role here. Rhesus macaques were immunized twice mucosally with a replicating Ad5hr expressing SIV Env, Gag, and Nef (Ad-SIV) or empty Ad5hr vector (Ad-Empty). DC subsets and their activation were examined in rectal tissue, blood, and LNs at 3 timepoints after each immunization. Plasmacytoid DCs, myeloid DCs, and Langerhans cells were significantly increased in the rectal mucosa, but only myeloid DCs were significantly increased in blood post-immunizations. All rectal DC subsets showed increased frequencies of cells expressing activation markers and cytokines post-immunization, blood DCs showed mixed results, and LN DCs showed few changes. Rectal DCs responded strongly to the vector rather than expressed SIV antigens, but rectal DC frequencies positively correlated with induced rectal antigen-specific memory T and B cells. These correlations were confirmed by in vitro co-cultures showing that rectal Ad-SIV DCs induced proliferation and antigen-specific cytokine production by autologous naïve T cells. Our results highlight the rapid response of DCs to Ad immunization and their role in mucosal immune activation and identify initial cellular mechanisms of the replicating Ad-SIV vaccine in the rhesus macaque model.

Chikungunya virus (CHIKV) has caused extensive outbreaks in several countries within the Americas, Asia, Oceanic/Pacific Islands, and Europe. In humans, CHIKV infections cause a debilitating disease with acute febrile illness and long-term polyarthralgia. Acute and chronic symptoms impose a major economic burden to health systems and contribute to poverty in affected countries. An efficacious vaccine would be an important step towards decreasing the disease burden caused by CHIKV infection. Despite no licensed vaccine is yet available for CHIKV, there is strong evidence of effective asymptomatic viral clearance due to neutralising antibodies against the viral structural proteins. We have designed viral-vectored vaccines to express the structural proteins of CHIKV, using the replication-deficient chimpanzee adenoviral platform, ChAdOx1. Expression of the CHIKV antigens results in the formation of chikungunya virus-like particles. Our vaccines induce high frequencies of anti-chikungunya specific T-cell responses as well as high titres of anti-CHIKV E2 antibodies with high capacity for in vitro neutralisation. Our results indicate the potential for further clinical development of the ChAdOx1 vaccine platform in CHIKV vaccinology.

Vaccination is the most effective and least expensive technique used for human diseases prevention and eradication. The need for more vaccine doses and the rapid establishment of facilities for the development of new vaccines are stimulating significate changes in the vaccine industry, which is gradually moving towards cell culture production. One approach is the third generation of vaccines, which are based on the use of plasmid DNA (pDNA) containing transgenes that encode an antigen capable of mimicking intracellular pathogenic infection and triggering both humoral and cellular immune responses. Plasmid DNA vaccination has distinct advantages over other vaccine technologies in terms of safety, ease of fabrication and stability. The effectiveness of pDNA vaccines against viruses, bacteria, parasites and cancer cells has been demonstrated in preclinical and clinical assays. Furthermore, currently there are a few veterinary pDNA vaccines in the market. The application of a simple formulation of naked pDNA as a vaccine is attractive, but a low transfection efficiency is often obtained. The use of nanoparticles to increase transfection efficiency is an approach that has been tested clinically. This review provides a summary of vaccine production, advances and major challenges associated with pDNA lipid and polymeric nanovaccines applications.

Hepatitis C is a liver disease caused by the hepatitis C virus (HCV) affecting 71 million people worldwide with no licensed vaccines that prevent infection. Here, we have generated four novel alphavirus-based DNA-launched self-amplifying RNA replicon (DREP) vaccines expressing either structural core-E1-E2 or nonstructural p7-NS2-NS3 HCV proteins of genotype 1a placed under the control of an alphavirus promoter, with or without an alphaviral translational enhancer (grouped as DREP-HCV or DREP-e-HCV, respectively). DREP vectors are known to induce cross-priming and further stimulation of immune responses through apoptosis, and here we demonstrate that they efficiently trigger apoptosis-related proteins in transfected cells. Immunization of mice with the DREP vaccines as the priming immunization followed by a heterologous boost with a recombinant modified vaccinia virus Ankara (MVA) vector expressing the nearly full-length genome of HCV (MVA-HCV) induced potent and long-lasting HCV-specific CD4 + and CD8 + T cell immune responses that were significantly stronger than those of a homologous MVA-HCV prime/boost immunization, with the DREP-e-HCV/MVA-HCV combination the most immunogenic regimen. HCV-specific CD4 + and CD8 + T cell responses were highly polyfunctional, had an effector memory phenotype, and were mainly directed against E1-E2 and NS2-NS3, respectively. Additionally, DREP/MVA-HCV immunization regimens induced higher antibody levels against HCV E2 protein than homologous MVA-HCV immunization. Collectively, these results provided an immunization protocol against HCV by inducing high levels of HCV-specific T cell responses as well as humoral responses. These findings reinforce the combined use of DREP-based vectors and MVA-HCV as promising prophylactic and therapeutic vaccines against HCV. IMPORTANCE HCV represents a global health problem as more than 71 million people are chronically infected worldwide. Direct-acting antiviral agents can cure HCV infection in most patients, but due to the high cost of these agents and the emergence of resistant mutants, they do not represent a feasible and affordable strategy to eradicate the virus. Therefore, a vaccine is an urgent goal that requires efforts to understand the correlates of protection for HCV clearance. Here, we describe for the first time the generation of novel vaccines against HCV based on alphavirus DNA replicons expressing HCV antigens. We demonstrate that potent T cell immune responses, as well as humoral immune responses, against HCV can be achieved in mice by using a combined heterologous prime/boost immunization protocol consisting of the administration of alphavirus replicon DNA vectors as the priming immunization followed by a boost with a recombinant modified vaccinia virus Ankara vector expressing HCV antigens., (Copyright © 2019 American Society for Microbiology.)

Adeno-associated virus (AAV) vectors represent promising candidates for gene therapy; however, pre-existing neutralizing antibodies (NAb) may reduce AAV vector delivery efficiency. In this study, the presence of AAV NAb was investigated in cats, which serve as a larger and outbred animal model for the prediction of gene therapy outcomes in humans but also in cats.Serum/plasma samples from 230 client-owned Swiss cats and 20 specified pathogen-free cats were investigated for NAb to AAV1, AAV2, AAV5, AAV6, AAV7, AAV8 and AAV9 using in vitro transduction inhibition and a beta-galactosidase assay. NAb to all tested AAV serotypes were found. Of the client-owned cats, 53% had NAb to one or more of the AAV serotypes. NAb (≥1:10) were found at frequencies of 5% (AAV6) to 28% (AAV7). The highest titers were found against AAV7 (≥1:160). The NAb prevalence to AAV2, AAV7 and AAV9 differed geographically. Regarding titers ≥1:10 against single AAV serotypes, age, breed and sex of the cats were not associated with the NAb prevalence. Cats with titers ≥1:20 against AAV2 and titers ≥1:40 against AAV7 were significantly younger than cats with low/no titers, and purebred cats were significantly more likely than non-purebred cats to have NAb to AAV2 (≥1:40). Additionally, regarding NAb to all AAV combined, female cats were significantly more likely than male cats to have NAb titers ≥1:40. Preliminary data using AAV-DJ indicated that less pre-existing NAb to the hybrid AAV-DJ can be expected compared to the wild-type AAV serotypes. AAV NAb will need to be taken into account for future in vivo gene therapy studies in cats., Competing Interests: The authors have declared that no competing interests exist.

Human respiratory syncytial virus (RSV) is a major pediatric respiratory pathogen. The attachment (G) and fusion (F) glycoproteins are major neutralization and protective antigens. RSV G is expressed as membrane-anchored (mG) and -secreted (sG) forms, both containing a central fractalkine-like CX3C motif. The CX3C motif and sG are thought to interfere with host immune responses and have been suggested to be omitted from a vaccine. We used a chimeric bovine/human parainfluenza virus type 3 (rB/HPIV3) vector to express RSV wild-type (wt) G and modified forms, including sG alone, mG alone, mutants with ablated CX3C, and G with enhanced packaging into vector virions. In hamsters, these viruses replicated to similar titers. When assayed with a complement-enhanced neutralization assay in Vero cells, sG did not reduce the serum RSV- or PIV3-neutralizing antibody (NAb) responses, whereas ablating CX3C drastically reduced the RSV NAb response. Protective efficacy against RSV challenge was not reduced by sG but was strongly dependent on the CX3C motif. In ciliated human airway epithelial (HAE) cells, NAbs induced by wt G, but not by wt F, completely blocked RSV infection in the absence of added complement. This activity was dependent on the integrity of the CX3C motif. In hamsters, the rB/HPIV3 expressing wt G conferred better protection against RSV challenge than that expressing wt F. Codon optimization of the wt G further increased its immunogenicity and protective efficacy. This study showed that ablation of the CX3C motif or sG in an RSV vaccine, as has been suggested previously, would be ill advised. IMPORTANCE Human RSV is the leading viral cause of severe pediatric respiratory illness. An RSV vaccine is not yet available. The RSV attachment protein G is an important protective and neutralization antigen. G contains a conserved fractalkine-like CX3C motif and is expressed in mG and sG forms. sG and the CX3C motif are thought to interfere with host immune responses, but this remains poorly characterized. Here, we used an attenuated chimeric bovine/human parainfluenza virus type 3 (rB/HPIV3) vector to express various modified forms of RSV G. We demonstrated that strong antibody and protective responses could be induced by G alone, and that this was highly dependent on the integrity of the CX3C motif. There was no evidence that sG or the CX3C motif impaired immune responses against RSV G or the rB/HPIV3 vector. rB/HPIV3 expressing wt RSV G provides a bivalent vaccine against RSV and HPIV3., (Copyright © 2019 American Society for Microbiology.)

AAV vectors poorly transduce Dendritic cells (DC), a feature invoked to explain AAV's low immunogenicity. However, the reason for this non-permissiveness remained elusive. Here, we performed an in-depth analysis using human monocyte-derived immature DC (iDC) as model. iDC internalized AAV vectors of various serotypes, but even the most efficient serotype failed to transduce iDC above background. Since AAV vectors reached the cell nucleus, we hypothesized that AAV's intracellular processing occurs suboptimal. On this basis, we screened an AAV peptide display library for capsid variants more suitable for DC transduction and identified the I/VSS family which transduced DC with efficiencies of up to 38%. This property correlated with an improved vector uncoating. To determine the consequence of this novel feature for AAV's in vivo performance, we engineered one of the lead candidates to express a cytoplasmic form of ovalbumin, a highly immunogenic model antigen, and assayed transduction efficiency as well as immunogenicity. The capsid variant clearly outperformed the parental serotype in muscle transduction and in inducing antigen-specific humoral and T cell responses as well as anti-capsid CD8 + T cells. Hence, vector uncoating represents a major barrier hampering AAV vector-mediated transduction of DC and impacts on its use as vaccine platform.

Vaccinia virus (VACV) is a notorious virus for a number of scientific reasons; however, most of its notoriety comes from the fact that it was used as a vaccine against smallpox, being ultimately responsible for the eradication of that disease. Nonetheless, many different vaccinia virus strains have been obtained over the years; some are suitable to be used as vaccines, whereas others are virulent and unsuitable for this purpose. Interestingly, different vaccinia virus strains elicit different immune responses in vivo , and this is a direct result of the genomic differences among strains. In order to evaluate the net result of virus-encoded immune evasion strategies of vaccinia viruses, we compared antiviral immune responses in mice intranasally infected by the highly attenuated and nonreplicative MVA strain, the attenuated and replicative Lister strain, or the virulent WR strain. Overall, cell responses elicited upon WR infections are downmodulated compared to those elicited by MVA and Lister infections, especially in determined cell compartments such as macrophages/monocytes and CD4 + T cells. CD4 + T cells are not only diminished in WR-infected mice but also less activated, as evaluated by the expression of costimulatory molecules such as CD25, CD212, and CD28 and by the production of cytokines, including tumor necrosis factor alpha (TNF-α), gamma interferon (IFN-γ), interleukin-4 (IL-4), and IL-10. On the other hand, MVA infections are able to induce strong T-cell responses in mice, whereas Lister infections consistently induced responses that were intermediary between those induced by WR and MVA. Together, our results support a model in which the virulence of a VACV strain is proportional to its potential to downmodulate the host's immune responses. IMPORTANCE Vaccinia virus was used as vaccine against smallpox and was instrumental in the successful eradication of that disease. Although smallpox vaccination is no longer in place in the overall population, the use of vaccinia virus in the development of viral vector-based vaccines has become popular. Nonetheless, different vaccinia virus strains are known and induce different immune responses. To look into this, we compared immune responses triggered by mouse infections with the nonreplicative MVA strain, the attenuated Lister strain, or the virulent WR strain. We observed that the WR strain was capable of downmodulating mouse cell responses, whereas the highly attenuated MVA strain induced high levels of cell-mediated immunity. Infections by the intermediately attenuated Lister strain induced cell responses that were intermediary between those induced by WR and MVA. We propose that the virulence of a vaccinia virus strain is directly proportional to its ability to downmodulate specific compartments of antiviral cell responses., (Copyright © 2019 American Society for Microbiology.)

Purpose of Review: CMV-vectored vaccines expressing SIV antigens have mediated unprecedented levels of virus control following SIV challenge in rhesus macaques. Remarkably, protection was dependent on nonclassically restricted CD8 T cells. Here, we review the latest research in CMV-vectored vaccines in both humans and nonhuman primates as well as recent advances in the understanding nonclassically restricted T cells, particularly MHC-E-restricted CD8 T cells., Recent Findings: Recent studies have investigated human translation of CMV-vectored vaccines including studies to ensure vaccine vector safety. Other work has focused on testing of animal models to investigate the relative contribution of MHC diversity and CMV strain on T-cell induction. Lastly, several groups have investigated MHC-E peptide binding, including HLA-E, have found that MHC-E can accommodate different peptide motifs, consistent with the original observations in CMV-vaccinated macaques., Summary: CMV remains a promising vaccine vector with the potential to be protective against multiple diseases, including HIV. However, CMV is highly species-specific and in humans, congenital infection can lead to serious birth defects. To ensure safe translation to humans, further clinical and animal studies are needed to better understand CMV-vectored immunity as well as more basic immunological questions relating to the induction of classical vs. nonclassical T cells.

Oncolytic viruses are genetically engineered viruses designed for the treatment of solid tumors, and are often coupled with the antitumor immunity of the host. The challenge of using oncolytic herpes simplex virus (oHSV) as an efficacious oncolytic agent is the potential host tissue damage caused by the production of a range of cytokines following intratumoral oHSV injection. An HSV‑suppressor of cytokine signaling 4 (SOCS4) recombinant virus was created to investigate whether it inhibits cytokine storm. Recombinant HSV‑SOCS4 and HSV‑1(F) were used to infect mice, and levels of several representative cytokines, including monocyte chemoattractant protein‑1, interleukin (IL)‑1β, tumor necrosis factor‑α, IL‑6 and interferon γ, in serum and bronchoalveolar lavage fluid (BALF) of infected mice were determined, and immune cells in BALF and spleen were enumerated. Lung damage, virus titers in the lung, body weight and survival rates of infected mice were also determined and compared between the two groups. The cytokine concentration of HSV‑SOCS4‑infected mice was significantly decreased compared with that of HSV‑1(F)‑infected mice in BALF and serum, and a smaller number of cluster of differentiation (CD)11b+ cells of BALF, and CD8+CD62L+ T cells and CD4+CD62L+ T cells of the spleen were also identified in HSV‑SOCS4‑infected mice. HSV‑SOCS4‑infected mice exhibited slight lung damage, a decrease in body weight loss and a 100% survival rate. The results of the present study indicated that SOCS4 protein may be a useful regulator to inhibit cytokine overproduction, and that HSV‑SOCS4 may provide a possible solution to control cytokine storm and its consequences following induction by oncolytic virus treatment.

A new modality of targeting therapeutic drugs based on the use of bacteriophage (virus), as an emerging tool for specific targeting and for vaccine development, has been an area of interest for genetic and cancer research. The approach is based on genetic manipulation and modification in the chemical structure of a filamentous bacteriophage that facilitates its application not only for in vivo imaging but also for therapeutic purpose, as a gene delivery vehicle, as drug carriers, and also as an immunomodulatory agent. Filamentous bacteriophage on account of its high surface holding ability with adaptable genetic engineering properties can effectively be used in loading of chemical and genetic drugs specifically on to the targeted lesion location. Moreover, the specific peptides/proteins exhibited on the phage surface can be applied directly as self-navigating drug delivery nanovehicles. The present review article has been framed with an objective to summarize the importance of bacteriophage in phage cancer therapy and to understand the possible future prospective of this approach in developing new tools for biotechnological and genetic research, especially in phage -mediated cancer therapy. Importantly, the peptides or proteins emerging from the surface of a nano carrier will make the expense of such peptides economically more effective as compared to other immunological tools, and this seems to be a potential approach for developing a new nanodrug carrier platform., Competing Interests: None

Antibodies and cytotoxic T cells represent 2 arms of host defense against pathogens. We hypothesized that vaccines that induce both high-magnitude CD8+ T cell responses and antibody responses might confer enhanced protection against HIV. To test this hypothesis, we immunized 3 groups of nonhuman primates: (a) Group 1, which includes sequential immunization regimen involving heterologous viral vectors (HVVs) comprising vesicular stomatitis virus, vaccinia virus, and adenovirus serotype 5-expressing SIVmac239 Gag; (b) Group 2, which includes immunization with a clade C HIV-1 envelope (Env) gp140 protein adjuvanted with nanoparticles containing a TLR7/8 agonist (3M-052); and (c) Group 3, which includes a combination of both regimens. Immunization with HVVs induced very high-magnitude Gag-specific CD8+ T cell responses in blood and tissue-resident CD8+ memory T cells in vaginal mucosa. Immunization with 3M-052 adjuvanted Env protein induced robust and persistent antibody responses and long-lasting innate responses. Despite similar antibody titers in Groups 2 and 3, there was enhanced protection in the younger animals in Group 3, against intravaginal infection with a heterologous SHIV strain. This protection correlated with the magnitude of the serum and vaginal Env-specific antibody titers on the day of challenge. Thus, vaccination strategies that induce both CD8+ T cell and antibody responses can confer enhanced protection against infection.

Infectious bronchitis virus (IBV) causes a major disease problem for the poultry industry worldwide. The currently used live-attenuated vaccines have the tendency to mutate and/or recombine with circulating field strains resulting in the emergence of vaccine-derived variant viruses. In order to circumvent these issues, and to develop a vaccine that is more relevant to Egypt and its neighboring countries, a recombinant avirulent Newcastle disease virus (rNDV) strain LaSota was constructed to express the codon-optimized S glycoprotein of the Egyptian IBV variant strain IBV/Ck/EG/CU/4/2014 belonging to GI-23 lineage, that is prevalent in Egypt and in the Middle East. A wild type and two modified versions of the IBV S protein were expressed individually by rNDV. A high level of S protein expression was detected in vitro by Western blot and immunofluorescence analyses. All rNDV-vectored IBV vaccine candidates were genetically stable, slightly attenuated and showed growth patterns comparable to that of parental rLaSota virus. Single-dose vaccination of 1-day-old SPF White Leghorn chicks with the rNDVs expressing IBV S protein provided significant protection against clinical disease after IBV challenge but did not show reduction in tracheal viral shedding. Single-dose vaccination also provided complete protection against virulent NDV challenge. However, prime-boost vaccination using rNDV expressing the wild type IBV S protein provided better protection, after IBV challenge, against clinical signs and significantly reduced tracheal viral shedding. These results indicate that the NDV-vectored IBV vaccines are promising bivalent vaccine candidates to control both infectious bronchitis and Newcastle disease in Egypt.

Hepatitis C virus (HCV) infection remains a major public health issue despite the introduction of several direct‑acting antiviral agents (DAAs), with some 185 million individuals infected with HCV worldwide. There is an urgent need for an effective prophylactic HCV vaccine. In the present study, we constructed genetic vaccines based on novel recombinant adeno‑associated viral (rAAV) vectors (AAV2/8 or AAV2/rh32.33) that express the envelope glycoprotein E2 from the HCV genotype 1b. Expression of HCV E2 protein in 293 cells was confirmed by western blot analysis. rAAV2/8.HCV E2 vaccine or rAAV2/rh32.33.HCV E2 vaccine was intramuscularly injected into C57BL/6 mice. HCV E2‑specific antigen was produced, and long‑lasting specific antibody responses remained detectable XVI weeks following immunization. In addition, the rAAV2/rh32.33 vaccine induced higher antigen‑specific antibody levels than the rAAV2/8 vaccine or AAV plasmid. Moreover, both AAV vaccines induced neutralizing antibodies against HCV genotypes 1a and 1b. Finally, it is worth mentioning that neutralizing antibody levels directed against AAV2/rh32.33 were lower than those against AAV2/8 in both mouse and human serum. These results demonstrate that AAV vectors, especially the AAVrh32.33, have particularly favorable immunogenicity for development into an effective HCV vaccine.

Dengue fever has become a global threat, causing millions of infections every year. An effective vaccine against all four serotypes of dengue virus (DENV) has not been developed yet. Among the different vaccination strategies available today, DNA vaccines are safe and practical, but currently induce relatively weak immune responses in humans. In order to improve immunogenicity, antigens may be targeted to dendritic cells (DCs), the main antigen presenting cells and orchestrators of the adaptive immune response, inducing T and B cell activation. It was previously shown that a DNA vaccine encoding a fusion protein comprised of an antigen and a single-chain Fv antibody (scFv) specific for the DC endocytic receptor DEC205 induced strong immune responses to the targeted antigen. In this work, we evaluate this strategy to improve the immunogenicity of dengue virus (DENV) proteins. Plasmids encoding the scFv αDEC205, or an isotype control (scFv ISO), fused to the DENV2 envelope protein domain III (EDIII) were generated, and EDIII specific immune responses were evaluated in immunized mice. BALB/c mice were intramuscularly (i.m.) immunized three times with plasmid DNAs encoding either scDEC-EDIII or scISO-EDIII followed by electroporation. Analyses of the antibody responses indicated that EDIII fusion with scFv targeting the DEC205 receptor significantly enhanced serum anti-EDIII IgG titers that inhibited DENV2 infection. Similarly, mice immunized with the scDEC-EDIII plasmid developed a robust CD4 + T cell response to the targeted antigen, allowing the identification of two linear epitopes recognized by the BALB/c haplotype. Taken together, these results indicate that targeting DENV2 EDIII protein to DCs using a DNA vaccine encoding the scFv αDEC205 improves both antibody and CD4 + T cell responses. This strategy opens perspectives for the use of DNA vaccines that encode antigens targeted to DCs as a strategy to increase immunogenicity.

Decades ago, Friedmann and Roblin postulated several barriers to gene therapy, including tissue targeting, delivery across the blood⁻brain barrier (BBB), and host immune responses. These issues remain pertinent till today. Since then, several advances have been made in elucidating structures of adeno-associated virus (AAV) serotypes, antibody epitopes, and ways to modify antibody-binding sites. AAVs capsid has also been engineered to re-direct tissue tropism, reduce ubiquitination, and promote passage across the BBB. Furthermore, the use of high(er) dose recombinant AAV (rAAV) has been accompanied by a better understanding of immune responses in both experimental animals and early clinical trials, and novel work is being performed to modulate the immune response. While the immune responses to rAAV remains a major challenge in translating experimental drugs to approved medicine, and will likely require more than a single solution, we now better understand the hurdles to formulate and test experimental solutions to surmount them., Competing Interests: The authors declare a conflict of interest. R. Jude Samulski is the founder and a shareholder at Asklepios BioPharmaceutical and Bamboo Therapeutics, Inc. He holds patents that have been licensed by UNC to Asklepios Biopharmaceutical, for which he receives royalties. He has consulted for Baxter Healthcare and has received payment for speaking.

Background: Nearly 3 million people worldwide are coinfected with HIV and HCV. Affordable strategies for prevention are needed. We developed a novel vaccination regimen involving replication-defective and serologically distinct chimpanzee adenovirus (ChAd3, ChAd63) vector priming followed by modified vaccinia Ankara (MVA) boosts, for simultaneous delivery of HCV non-structural (NSmut) and HIV-1 conserved (HIVconsv) region immunogens. Methods: We conducted a phase I trial in which 33 healthy volunteers were sequentially enrolled and vaccinated via the intramuscular route as follows: 9 received ChAd3-NSmut [2.5 × 10 10 vp] and MVA-NSmut [2 × 10 8 pfu] at weeks 0 and 8, respectively; 8 received ChAdV63.HIVconsv [5 × 10 10 vp] and MVA.HIVconsv [2 × 10 8 pfu] at the same interval; 16 were co-primed with ChAd3-NSmut [2.5 × 10 10 vp] and ChAdV63.HIVconsv [5 × 10 10 vp] followed at week 8 by MVA-NSmut and MVA.HIVconsv [both 1 × 10 8 pfu]. Immunogenicity was assessed using peptide pools in ex vivo ELISpot and intracellular cytokine assays. Vaccine-induced whole blood transcriptome changes were assessed by microarray analysis. Results: All vaccines were well tolerated and no vaccine-related serious adverse events occurred. Co-administration of the prime-boost vaccine regimens induced high magnitude and broad T cell responses that were similar to those observed following immunization with either regimen alone. Median (interquartile range, IQR) peak responses to NSmut were 3,480 (2,728-4,464) and 3,405 (2,307-7,804) spot-forming cells (SFC)/10 6 PBMC for single and combined HCV vaccinations, respectively ( p = 0.8). Median (IQR) peak responses to HIVconsv were 1,305 (1,095-4,967) and 1,005 (169-2,482) SFC/10 6 PBMC for single and combined HIV-1 vaccinations, respectively ( p = 0.5). Responses were maintained above baseline to 34 weeks post-vaccination. Intracellular cytokine analysis indicated that the responding populations comprised polyfunctional CD4 + and CD8 + T cells. Canonical pathway analysis showed that in the single and combined vaccination groups, pathways associated with antiviral and innate immune responses were enriched for upregulated interferon-stimulated genes 24 h after priming and boosting vaccinations. Conclusions: Serologically distinct adenoviral vectors encoding HCV and HIV-1 immunogens can be safely co-administered without reducing the immunogenicity of either vaccine. This provides a novel strategy for targeting these viruses simultaneously and for other pathogens that affect the same populations. Clinical trial registration: https://clinicaltrials.gov, identifier: NCT02362217.

Natural killer (NK) cells are innate immune cells that can be activated rapidly to target abnormal and virus-infected cells without prior sensitization. With significant advancements in cell biology technologies, many NK cell lines have been established. Among these cell lines, NK-92 cells are not only the most widely used but have also been approved for clinical applications. Additionally, chimeric antigen receptor-modified NK-92 cells (CAR-NK-92 cells) have shown strong antitumor effects. In this review, we summarize established human NK cell lines and their biological characteristics, and highlight the applications of NK-92 cells and CAR-NK-92 cells in tumor immunotherapy.

In the last few decades, Ebola virus (EBOV) has emerged periodically and infected people in Africa, resulting in an extremely high mortality rate. With no available prophylaxis or cure so far, a highly effective Ebola vaccine is urgently needed. In this study, we developed a novel chimpanzee adenovirus-based prime-boost vaccine by exploiting two recombinant replication-deficient chimpanzee adenoviral vectors, AdC7 and AdC68, which express glycoproteins (GP) of the EBOV strain identified in the 2014 outbreak. Our results indicated that a single immunization using AdC7 or AdC68 could stimulate potent EBOV-specific antibody responses, whereas the AdC7 prime-AdC68 boost regimen induced much stronger and sustained humoral and cellular immune responses in both mice and rhesus monkeys, compared with AdC7 or AdC68 single vaccination or the AdC68 prime-AdC7 boost regimen. This prime-boost vaccine could also protect mice from the simulated infection with EBOV-like particle (EBOVLP) in biosafety level 2 (BSL-2) laboratories, and antibodies from the prime-boost immunized rhesus macaques could passively provide protection against EBOVLP infection. Altogether, our results show that the AdC7 prime-AdC68 boost vaccine is a promising candidate for further development to combat EBOV infections.

Zika virus (ZIKV) is a major public health concern due to the risk of congenital Zika syndrome in developing fetuses and Guillain-Barre syndrome in adults. Currently, there are no approved vaccines available to protect against infection. Adenoviruses are safe and highly immunogenic vaccine vectors capable of inducing lasting humoral and cellular immune responses. Here, we developed two Adenovirus (Ad) vectored Zika virus vaccines by inserting a ZIKV prM-E gene expression cassette into human Ad types 4 (Ad4-prM-E) and 5 (Ad5-prM-E). Immune correlates indicate that Ad5-prM-E vaccination induces both an anti-ZIKV antibody and T-cell responses whereas Ad4-prM-E vaccination only induces a T-cell response. In a highly lethal challenge in an interferon α/β receptor knockout mice, 80% of Ad5 vaccinated animals and 33% of Ad4 vaccinated animals survived a lethal ZIKV challenge, whereas no animals in the sham vaccinated group survived. In an infection model utilizing immunocompetent C57BL/6 mice that were immunized and then treated with a blocking anti-IFNAR-1 antibody immediately before ZIKV challenge, 100% of Ad4-prM-E and Ad5-prM-E vaccinated mice survived. This indicates that Ad4-prM-E vaccination is protective without the development of detectable anti-ZIKV antibodies. The protection seen in these highly lethal mouse models demonstrate the efficacy of Ad vectored vaccines for use against ZIKV.

Interferon (IFN) inhibits HIV replication by inducing antiviral effectors. To comprehensively identify IFN-induced HIV restriction factors, we assembled a CRISPR sgRNA library of Interferon Stimulated Genes (ISGs) into a modified lentiviral vector that allows for packaging of sgRNA-encoding genomes in trans into budding HIV-1 particles. We observed that knockout of Zinc Antiviral Protein (ZAP) improved the performance of the screen due to ZAP-mediated inhibition of the vector. A small panel of IFN-induced HIV restriction factors, including MxB, IFITM1, Tetherin/BST2 and TRIM5alpha together explain the inhibitory effects of IFN on the CXCR4-tropic HIV-1 strain, HIV-1 LAI , in THP-1 cells. A second screen with a CCR5-tropic primary strain, HIV-1 Q23.BG505 , described an overlapping, but non-identical, panel of restriction factors. Further, this screen also identifies HIV dependency factors. The ability of IFN-induced restriction factors to inhibit HIV strains to replicate in human cells suggests that these human restriction factors are incompletely antagonized., Editorial Note: This article has been through an editorial process in which the authors decide how to respond to the issues raised during peer review. The Reviewing Editor's assessment is that all the issues have been addressed (see decision letter)., Competing Interests: MO, LH, JV, FR, DH, RB, JD, JO, ME No competing interests declared, (© 2018, OhAinle et al.)

Recombinant adeno-associated virus (AAV) vectors have been broadly adopted as a gene delivery tool in clinical trials, owing to their high efficiency of transduction of several host tissues and their low immunogenicity. However, a considerable proportion of the population is naturally exposed to the WT virus from which AAV vectors are derived, which leads to the acquisition of immunological memory that can directly determine the outcome of gene transfer. Here, we show that prior exposure to AAV drives distinct capsid immunity profiles in healthy subjects. In peripheral blood mononuclear cells (PBMCs) isolated from AAV-seropositive donors, recombinant AAV triggered TNF-α secretion in memory CD8+ T cells, B cell differentiation into antibody-secreting cells, and anti-capsid antibody production. Conversely, PBMCs isolated from AAV-seronegative individuals appeared to carry a population of NK cells reactive to AAV. Further, we demonstrated that the AAV capsid activates IL-1β and IL-6 cytokine secretion in monocyte-related dendritic cells (moDCs). IL-1β and IL-6 blockade inhibited the anti-capsid humoral response in vitro and in vivo. These results provide insights into immune responses to AAV in humans, define a possible role for moDCs and NK cells in capsid immunity, and open new avenues for the modulation of vector immunogenicity.

The adenovirus (Ad) is widely used as a vaccine because of its ability to induce a cellular and humoral immune response. In addition, human clinical trials have validated the safety and efficacy of Ad as a vaccine vector. The traditional approach for employing the adenovirus as vaccine is to configure the antigen genes into the expression cassette of the Ad genome. An alternative method for inducing an immune response is the "capsid-incorporation" strategy. This strategy is based upon the incorporation of proteins or peptides into the capsid proteins. This review will focus on the established uses of this approach as well as highlighting the new developments regarding the capsid-incorporation strategy.

The immunomodulatory effect of IL-10 as an immunosuppressive and anti-inflammatory cytokine is well known. Taking advantage of our established mouse model of autoimmune cholangitis using 2-octynoic acid conjugated ovalbumin (2-OA-OVA) induction, we compared liver pathology, immune cell populations and antimitochondrial antibodies between IL-10 knockout and wild type mice immunized with 2-OA-OVA. At 10 weeks post immunization, portal inflammation and fibrosis were more severe in 2-OA-OVA immunized IL-10 knockout mice than in wild type mice. This was accompanied by significant higher levels of collagen I and III expression, T, NK and NKT subsets in liver and IgG anti-mitochondrial autoantibodies (AMAs) compared to 2-OA-OVA immunized wild type mice, suggesting that endogenous IL-10 is necessary for the maintenance of immune tolerance in primary biliary cholangitis (PBC). Further, we investigated whether administration of exogenous IL-10 could prevent PBC by administration of IL-10 expressing recombinant adeno-associated virus (AAV-IL-10) either 3 days before or 3 weeks after the establishment of liver pathology. Interestingly, administration of AAV-IL-10 resulted in increased liver inflammation and fibrosis, accompanied by increases in IFN-γ in liver CD4 + T cell, granzyme B, FasL, and CD107a in liver CD8 + T and NKT cells, and granzyme B and FasL in liver NK cells of AAV-IL-10 administered mice compared with control mice. Furthermore, administration of AAV-IL-10 significantly increased levels of proinflammatory cytokines and chemokines (IFN-γ, TNF-α, CXCL9 and CXCL10) and collagen I and III production in naïve mice, together with increase in immune cell infiltration and collagen deposition in the liver, suggesting a role of IL-10 in fibrosis. In conclusion, our data demonstrate that endogenous IL-10 is critical in the maintenance of immune tolerance but exogenous administration of IL-10 exacerbates liver inflammation and fibrosis. Furthermore, the distinctive presence of inflammatory immune cell populations and collagen expression in AAV-IL-10 treated naïve mice cautions against the clinical use of exogenous IL-10 in patients with autoimmune cholangitis., (Copyright © 2018. Published by Elsevier Ltd.)

Introduction: Conventional hemophilia treatment is based on repeated infusion of the missing clotting factor. This therapy is lifelong, expensive and can result in the formation of neutralizing antibodies, thus causing failure of the treatment and requiring higher doses of the replacement drug. Areas covered: Gene and cell therapies offer the advantage of providing a definitive and long-lasting correction of the mutated gene, promoting its physiological expression and preventing neutralizing antibody development. This review focuses on the most recent approaches that have been shown to prevent and even eradicate immune response toward the replaced factor. Expert commentary: Despite the encouraging data demonstrated by ongoing clinical trials and pre-clinical studies, more extensive investigations are necessary to establish the long-term safety and efficacy of gene therapy treatments in maintaining immune tolerance.

Upon viral infection, antigen-specific CD8 + cytotoxic T cells (CTLs) arise and contribute to the elimination of infected cells to prevent the spread of pathogens. Therefore, the frequency of antigen-specific CTLs is indicative of the strength of the T cell response against a specific antigen. Such analysis is important in basic immunology, vaccine development, cancer immunobiology and the adaptive immunology. In the vaccine field, the CTL response directed against components of a viral vector co-determines how effective the generation of antigen-specific cells against the antigen of interest (i.e., transgene) is. Antigen-specific CTLs can either be detected by stimulation with specific peptides followed by intracellular cytokine staining or by the direct staining of antigen-specific T cell receptors (TCRs) and analysis by flow cytometry. The first method is rather time-consuming since it requires sacrificing of animals to isolate cells from organs. Also, it requires isolation of blood from small animals which is difficult to perform. The latter method is rather fast, can be easily done with small amounts of blood and is not dependent on specific effector functions, such as cytolytic activity. MHC tetramers are an ideal tool to detect antigen-specific TCRs. Here, we describe a protocol to simultaneously detect antigen-specific CTLs for the immunodominant peptides of the viral vector VSV-GP (LCMV-GP, VSV-NP) and transgenes (OVA, HPV 16 E7, eGFP) by MHC I tetramer staining and flow cytometry. Staining is possible either directly from blood or from single cell suspensions of organs, such as spleen. Blood or single cell suspensions of organs are incubated with tetramers. After staining with antibodies against CD3 and CD8, antigen-specific CTLs are quantified by flow cytometry. Optionally, antibodies against CD43, CD44, CD62L or others can be included to determine the activation status of antigen-specific CD8 + T cells and to discriminate between naïve and effector cells.

Zika virus (ZIKV) is a re-emerging mosquito-borne flavivirus that affects humans and can cause severe neurological complications, including Guillain-Barré syndrome and microcephaly. Since 2007 there have been three large outbreaks; the last and larger spread in the Americas in 2015. Actually, ZIKV is circulating in the Americas, Southeast Asia, and the Pacific Islands, and represents a potential pandemic threat. Given the rapid ZIKV dissemination and the severe neurological and teratogenic sequelae associated with ZIKV infection, the development of a safe and efficacious vaccine is critical. In this study, we have developed and characterized the immunogenicity and efficacy of a novel ZIKV vaccine based on the highly attenuated poxvirus vector modified vaccinia virus Ankara (MVA) expressing the ZIKV prM and E structural genes (termed MVA-ZIKV). MVA-ZIKV expressed efficiently the ZIKV structural proteins, assembled in virus-like particles (VLPs) and was genetically stable upon nine passages in cell culture. Immunization of mice with MVA-ZIKV elicited antibodies that were able to neutralize ZIKV and induced potent and polyfunctional ZIKV-specific CD8 + T cell responses that were mainly of an effector memory phenotype. Moreover, a single dose of MVA-ZIKV reduced significantly the viremia in susceptible immunocompromised mice challenged with live ZIKV. These findings support the use of MVA-ZIKV as a potential vaccine against ZIKV.

The tetracycline regulatory system has been widely used to control the transgene expression. With this powerful tool, it might be possible to effectively control the functional activity of chimeric antigen receptor (CAR) T cells and manage the severe side effects after infusion. In this study, we developed novel inducible CD19CAR (iCAR19) T cells by incorporating a one-vector Tet-on system into the CD19CAR construct. The iCAR19 T cells showed dox-dependent cell proliferation, cytokine production, CAR expression, and strong CD19-specific cytotoxicity. After 48 h of dox induction, the relative CAR expression of induced cells was five times greater than that of uninduced cells. Twenty-four hours after dox removal, CAR expression significantly decreased by more than 60%. In cytotoxicity assays, dox-treated cells induced significantly higher specific lysis against target cells. These results suggested that the activity of iCAR19 T cells was successfully controlled by our Tet-on system, offering an enhanced safety profile while maintaining a robust anti-tumor effect. Besides, all manufacture processes of the lentiviral vectors and the T cells were conducted according to the Good Manufacturing Practice (GMP) standards for subsequent clinical translation.

Duck circovirus (DuCV) is an immunosuppressive pathogen that causes a huge economic loss in the avian industry. Efficient vaccination has become a necessary strategy for preventing DuCV infection in the breeding industry. Three DNA vaccines encoding the Capsid (Cap) protein of DuCV were developed in this study, which were based on the eukaryotic vector pcDNA3.1 containing (i) the full length of Cap gene, pcDNA3.1-Cap, (ii) the Cap gene with a deletion of its nuclear localization signal (NLS) peptide encoding sequence, pcDNA3.1-CapΔNLS, and (iii) the Cap gene without NLS but harboring a fragment encoding the secretory signal peptide of tissue plasminogen activator (tPA), pcDNA3.1-tPA-CapΔNLS. Production of Cap protein-derived antigens from these three DNA vaccines was confirmed in vitro. The deletion of the NLS coding sequence of the Cap gene changed the subcellular location of the Capsid protein from the nucleus to the cytoplasm. Secretion of the Cap protein was observed in pcDNA3.1-tPA-CapΔNLS-transfected cells. The immunogenicity of these three DNA vaccines was assessed in vivo by measuring Cap-specific antibody and related cytokine levels. The results demonstrated that all these vaccines could induce a significant, specific immune response to protect ducks from DuCV challenge. Notably, higher titers of Cap-specific antibody were produced in ducks vaccinated with pcDNA3.1-tPA-CapΔNLS, which provided the highest protective efficacy at a rate of 90% in the challenge experiment. Taken together, DNA vaccines expressing the DuCV Cap protein show promising immunogenicity, which can be enhanced by replacing the NLS of the Cap protein with a secretory signal peptide of tPA., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Eukaryotic cells constitutively produce nanovesicles of 50-150 nm of diameter, referred to as exosomes, upon release of the contents of multivesicular bodies (MVBs). We recently characterized a novel, exosome-based way to induce cytotoxic T lymphocyte (CTL) immunization against full-length antigens. It is based on DNA vectors expressing products of fusion between the exosome-anchoring protein Nef mutant (Nef mut ) with the antigen of interest. The strong efficiency of Nef mut to accumulate in MVBs results in the production of exosomes incorporating huge amounts of the desired antigen. When translated in animals, the injection of Nef mut -based DNA vectors generates engineered exosomes whose internalization in antigen-presenting cells induces cross-priming and antigen-specific CTL immunity. Here, we describe the molecular strategies we followed to produce DNA vectors aimed at generating immunogenic exosomes potentially useful to elicit a CTL immune response against antigens expressed by the etiologic agents of major chronic viral infections, i.e., HIV-1, HBV, and the novel tumor-associated antigen HOXB7. Unique methods intended to counteract intrinsic RNA instability and nuclear localization of the antigens have been developed. The success we met with the production of these engineered exosomes opens the way towards pre-clinic experimentations devoted to the optimization of new vaccine candidates against major infectious and tumor pathologies.

Hepatitis B virus (HBV) infection is a global public health problem. T helper (Th)1‑associated cytokines are involved in HBV clearance during acute and persistent infection. In our previous study, it was demonstrated that lentiviral vectors encoding ubiquitinated hepatitis B core antigen (LV‑Ub‑HBcAg) effectively transduced dendritic cells (DCs) to induce maturation, which promoted T cell polarization to Th1 and generated HBcAg‑specific cytotoxic T lymphocytes (CTLs) ex vivo. In the present study, HBV transgenic mice were immunized with LV‑Ub‑HBcAg‑transduced DCs and HBcAg‑specific immune responses were evaluated. Cytokine expression was analyzed by ELISA. T lymphocyte proliferation was detected with a Cell Counting Kit‑8 assay and HBcAg‑specific CTL activity was determined using a lactate dehydrogenase release assay. The expression levels of p38‑mitogen‑activated protein kinase (p38‑MAPK), phosphorylated (p)‑p38MAPK, c‑Jun N‑terminal kinase (JNK) and p‑JNK were detected by western blot analysis. The results demonstrated that LV‑Ub‑HBcAg‑transduced DCs significantly increased the Th1/Th2 cytokine ratio, and effectively reduced the levels of serum hepatitis B surface antigen (HBsAg), HBV DNA, and liver HBsAg and HBcAg. Furthermore, the LV‑Ub‑HBcAg‑transduced DCs upregulated the expression of p‑P38‑MAPK and p‑JNK in T lymphocytes. In conclusion, the present study indicated that LV‑Ub‑HBcAg‑transduced DCs generated predominant Th1 responses and enhanced CTL activity in HBV transgenic mice. Activation of the P38‑MAPK/JNK signaling pathway may be involved in this induction.

Gene therapy mediated by recombinant adeno-associated virus (AAV) vectors is a promising treatment for systemic monogenic diseases. However, vector immunogenicity represents a major limitation to gene transfer with AAV vectors, particularly for vector re-administration. Here, we demonstrate that synthetic vaccine particles encapsulating rapamycin (SVP[Rapa]), co-administered with AAV vectors, prevents the induction of anti-capsid humoral and cell-mediated responses. This allows successful vector re-administration in mice and nonhuman primates. SVP[Rapa] dosed with AAV vectors reduces B and T cell activation in an antigen-selective manner, inhibits CD8 + T cell infiltration in the liver, and efficiently blocks memory T cell responses. SVP[Rapa] immunomodulatory effects can be transferred from treated to naive mice by adoptive transfer of splenocytes, and is inhibited by depletion of CD25 + T cells, suggesting a role for regulatory T cells. Co-administration of SVP[Rapa] with AAV vector represents a powerful strategy to modulate vector immunogenicity and enable effective vector re-administration.

T cells that are genetically modified to express chimeric antigen receptors (CARs) specific for CD19 show great promise for the treatment of relapsed/refractory acute lymphoblastic leukemia (ALL). The first U.S. Food and Drug Administration approval of a cellular cancer therapy in 2017, Novartis's CD19-targeting CAR T-cell product Kymriah™ within the context of relapsed/refractory pediatric ALL, followed rapidly by approval of Kite's Yescarta™ and, more recently, Kymriah™ for diffuse large B-cell indications in adults, highlights the pace of progress made in this field. In this review, we will consider the latest evidence from CAR T-cell therapy for B-lineage ALL. We discuss the barriers to CAR T-cell therapy for ALL patients and give a perspective on the strategy we have taken to date to widen access to CAR T-cell therapy for UK pediatric patients with high-risk ALL., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Lentiviral vectors (LVs) transduce quiescent cells and provide stable integration to maintain transgene expression. Several approaches have been adopted to optimize LV safety profiles. Similarly, LV targeting has been tailored through strategies including the modification of envelope components, the use of specific regulatory elements, and the selection of appropriate administration routes. Models of aortic disease based on a single injection of pleiotropic LVs have been developed that efficiently transduce the three aorta layers in wild type mice. This approach allows the dissection of pathways involved in aortic aneurysm formation and the identification of targets for gene therapy in aortic diseases. LVs provide a fast, efficient, and affordable alternative to genetically modified mice to study disease mechanisms and develop therapeutic tools., (Copyright © 2018 Elsevier Ltd. All rights reserved.)