Search

In patients who progress from acute hepatitis B virus (HBV) infection to a chronic HBV (CHB) infection, CD8+ T cells fail to eliminate the virus and become impaired. A functional cure of CHB likely requires new and highly functional CD8+ T cell responses different from those induced by the infection. Here we report preclinical immunogenicity and efficacy of an HBV therapeutic vaccine that includes herpes simplex virus (HSV) glycoprotein D (gD), a checkpoint modifier of early T cell activation, that enhances, broadens, and prolongs CD8+ T cell responses. We developed a therapeutic HBV vaccine based on a chimpanzee adenovirus serotype 6 (AdC6) vector, called AdC6-gDHBV2, that targets conserved and highly immunogenic regions of the viral polymerase (pol) and core antigens fused into HSV gD. The vaccine was tested with, and without gD, in mice for immunogenicity and in an adeno-associated virus (AAV)8-1.3HBV vector model for antiviral efficacy. The vaccine encoding the HBV antigens within gD stimulates potent and broad CD8+ T cell responses. In a surrogate model of HBV infection, a single intramuscular (i.m.) injection of AdC6-gDHBV2 achieved significant and sustained declines of circulating HBV DNA copies (cps) and HBV surface antigen (HBsAg); both inversely correlated with HBV specific CD8+ T cell frequencies in spleens and livers. AdC6-gDHBV2 is the first therapeutic vaccine to show significant reductions in levels of HBV genome copies and HBsAg when used alone, even when vaccination was delayed for months from infection.

Available COVID-19 vaccine only provide protection for a limited time due in part to the rapid emergence of viral variants with spike protein mutations, necessitating the generation of new vaccines to combat SARS-CoV-2. Two serologically distinct replication-defective chimpanzee-origin adenovirus (Ad) vectors (AdC) called AdC6 and AdC7 expressing early SARS-CoV-2 isolate spike (S) or nucleocapsid (N) proteins, the latter expressed as a fusion protein within herpes simplex virus glycoprotein D (gD), were tested individually or as a mixture in a hamster COVID-19 SARS-CoV-2 challenge model. The S protein expressing AdC (AdC-S) vectors induced antibodies including those with neutralizing activity that in part cross-reacted with viral variants. Hamsters vaccinated with the AdC-S vectors were protected against serious disease and showed accelerated recovery upon SARS-CoV-2 challenge. Protection was enhanced if AdC-S vectors were given together with the AdC vaccines that expressed the gD N fusion protein (AdC-gDN). In contrast hamsters that just received the AdC-gDN vaccines showed only marginal lessening of symptoms compared to control animals. These results indicate that immune response to the N protein that is less variable than the S protein may potentiate and prolong protection achieved by the currently used S protein based genetic COVID-19 vaccines., Competing Interests: Declaration of competing interest HCJE holds equity in Virion Therapeutics. She serves as a Consultant to several Gene Therapy companies., (Copyright © 2022 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.)

Viral-vectored vaccines are highly amenable for respiratory mucosal delivery as a means of inducing much-needed mucosal immunity at the point of pathogen entry. Unfortunately, current monovalent viral-vectored tuberculosis (TB) vaccine candidates have failed to demonstrate satisfactory clinical protective efficacy. As such, there is a need to develop next-generation viral-vectored TB vaccine strategies which incorporate both vaccine antigen design and delivery route. In this study, we have developed a trivalent chimpanzee adenoviral-vectored vaccine to provide protective immunity against pulmonary TB through targeting antigens linked to the three different growth phases (acute/chronic/dormancy) of Mycobacterium tuberculosis (M.tb) by expressing an acute replication-associated antigen, Ag85A, a chronically expressed virulence-associated antigen, TB10.4, and a dormancy/resuscitation-associated antigen, RpfB. Single-dose respiratory mucosal immunization with our trivalent vaccine induced robust, sustained tissue-resident multifunctional CD4 + and CD8 + T-cell responses within the lung tissues and airways, which were further quantitatively and qualitatively improved following boosting of subcutaneously BCG-primed hosts. Prophylactic and therapeutic immunization with this multivalent trivalent vaccine in conventional BALB/c mice provided significant protection against not only actively replicating M.tb bacilli but also dormant, non-replicating persisters. Importantly, when used as a booster, it also provided marked protection in the highly susceptible C3HeB/FeJ mice, and a single respiratory mucosal inoculation was capable of significant protection in a humanized mouse model. Our findings indicate the great potential of this next-generation TB vaccine strategy and support its further clinical development for both prophylactic and therapeutic applications., (© 2023. The Author(s).)

Antibody responses to vaccinations or infections decline upon aging. In this study we tested if metabolic changes in B cells may contribute to attenuation of responses to influenza vaccination in aged humans. Our data show that aging affects mitochondrial functions in B cells leading to increases in mitochondrial reactive oxygen species (MROS) and mitochondrial mass (MM) in some aged B cell subsets and decreases in expression levels of Sirtuin 1 (SIRT1), Forkhead box protein (FOX)O1 and carnitine palmitoyltransferase 1 (CPT-1). Seahorse analyses showed minor defects in glycolysis in the aged B cells after activation but a strong reduction in oxidative phosphorylation. The analyses of the transcriptome revealed further pronounced defects in one-carbon metabolism, a pathway that is essential for amino acid and nucleotide metabolism. Overall our data support the notion that the declining ability of aged B cells to increase their metabolism following activation contributes to the weakened antibody responses of the elderly.

Influenza is a leading cause of death in the elderly, and the vaccine protects only a fraction of this population. A key aspect of antibody-mediated anti-influenza virus immunity is adaptation to antigenically distinct epitopes on emerging strains. We examined factors contributing to reduced influenza vaccine efficacy in the elderly and uncovered a dramatic reduction in the accumulation of de novo immunoglobulin gene somatic mutations upon vaccination. This reduction is associated with a significant decrease in the capacity of antibodies to target the viral glycoprotein, hemagglutinin (HA), and critical protective epitopes surrounding the HA receptor-binding domain. Immune escape by antigenic drift, in which viruses generate mutations in key antigenic epitopes, becomes highly exaggerated. Because of this reduced adaptability, most B cells activated in the elderly cohort target highly conserved but less potent epitopes. Given these findings, vaccines driving immunoglobulin gene somatic hypermutation should be a priority to protect elderly individuals., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Background: Estimates of current global rabies mortality range from 26,000 to 59,000 deaths per annum. Although pre-exposure prophylaxis using inactivated rabies virus vaccines (IRVs) is effective, it requires two to three doses and is regarded as being too expensive and impractical for inclusion in routine childhood immunization programmes., Methodology/ Principal Findings: Here we report the development of a simian-adenovirus-vectored rabies vaccine intended to enable cost-effective population-wide pre-exposure prophylaxis against rabies. ChAdOx2 RabG uses the chimpanzee adenovirus serotype 68 (AdC68) backbone previously shown to achieve pre-exposure protection against rabies in non-human primates. ChAdOx2 differs from AdC68 in that it contains the human adenovirus serotype 5 (AdHu5) E4 orf6/7 region in place of the AdC68 equivalents, enhancing ease of manufacturing in cell lines which provide AdHu5 E1 proteins in trans. We show that immunogenicity of ChAdOx2 RabG in mice is comparable to that of AdC68 RabG and other adenovirus serotypes expressing rabies virus glycoprotein. High titers of rabies virus neutralizing antibody (VNA) are elicited after a single dose. The relationship between levels of VNA activity and rabies virus glycoprotein monomer-binding antibody differs after immunization with adenovirus-vectored vaccines and IRV vaccines, suggesting routes to further enhancement of the efficacy of the adenovirus-vectored candidates. We also demonstrate that ChAdOx2 RabG can be thermostabilised using a low-cost method suitable for clinical bio-manufacture and ambient-temperature distribution in tropical climates. Finally, we show that a dose-sparing effect can be achieved by formulating ChAdOx2 RabG with a simple chemical adjuvant. This approach could lower the cost of ChAdOx2 RabG and other adenovirus-vectored vaccines., Conclusions/ Significance: ChAdOx2 RabG may prove to be a useful tool to reduce the human rabies death toll. We have secured funding for Good Manufacturing Practice- compliant bio-manufacture and Phase I clinical trial of this candidate., Competing Interests: AVSH and ADD are named inventors on a patent application relating to the development of the ChAdOx2 vector. AVSH and SJD are named inventors on a patent relating to the use of the intron-containing promoter used in ChAdOx2 RabG. The University of Oxford and the Wistar Institute have entered into a partnership to share any future revenue from development of the ChAdOx2 RabG vaccine.

Competing Interests: CONFLICTS OF INTEREST The authors declare no potential conflicts of interest.

Background: Influenza vaccination aims to prevent infection by influenza virus and reduce associated morbidity and mortality; however, vaccine effectiveness (VE) can be modest, especially for subtype A(H3N2). Low VE has been attributed to mismatches between the vaccine and circulating influenza strains and to the vaccine's elicitation of protective immunity in only a subset of the population. The low H3N2 VE in the 2012-2013 season was attributed to egg-adaptive mutations that created antigenic mismatch between the actual vaccine strain (IVR-165) and both the intended vaccine strain (A/Victoria/361/2011) and the predominant circulating strains (clades 3C.2 and 3C.3)., Methods: We investigated the basis of low VE in 2012-2013 by determining whether vaccinated and unvaccinated individuals were infected by different viral strains and by assessing the serologic responses to IVR-165, A/Victoria/361/2011, and 3C.2 and 3C.3 strains in an adult cohort before and after vaccination., Results: We found no significant genetic differences between the strains that infected vaccinated and unvaccinated individuals. Vaccination increased titers to A/Victoria/361/2011 and 3C.2 and 3C.3 representative strains as much as to IVR-165. These results are consistent with the hypothesis that vaccination boosted cross-reactive immune responses instead of specific responses against unique vaccine epitopes. Only approximately one-third of the cohort achieved a ≥4-fold increase in titer., Conclusions: In contrast to analyses based on ferret studies, low H3N2 VE in 2012-2013 in adults does not appear to be due to egg adaptation of the vaccine strain. Instead, low VE might have been caused by low vaccine immunogenicity in a subset of the population.

Cold chain-free vaccine technologies are needed to ensure effective vaccine delivery and coverage, particularly in resource-poor countries. However, the immunogenicity and thermostability of spray dried live viral vector-based vaccines such as recombinant adenoviral-vectored vaccines remain to be investigated. To address this issue, we have spray dried human adenoviral (AdHu5)- and chimpanzee adenoviral (AdCh68)-vectored tuberculosis vaccines in a mannitol and dextran matrix. Spray dried powders containing these two vaccines display the morphologic and chemical properties desired for long-term thermostability and vaccination. Upon reconstitution, they effectively transfected the cells in vitro with relatively small losses in viral infectivity related to the spray drying process. Following in vivo vaccination, AdHu5- and AdCh68-vectored vaccines were as immunogenic as the conventional fresh, cryopreserved liquid vaccine samples. Of importance, even after cold chain-free storage, at ambient temperatures and relatively low humidity for 30 and 90days, the vaccines retained their in vivo immunogenicity, while the liquid vaccine samples stored under the same conditions lost their immune-activating capability almost entirely. Our results support further development of our spray drying technologies for generating thermally stable adenoviral-vectored and other viral-vectored vaccines., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

This chapter reviews the performance of viral vectors based on adenoviruses or adeno-associated virus as vaccine carriers for infectious diseases. Replication-defective adenovirus vectors based on multiple human or non-human serotypes have consistently induced potent transgene product-specific B and T cell responses and are increasingly being explored in human clinical trials. The immunogenicity of most vectors based on adeno-associated virus vectors has been poor with the exception of a recently described hybrid vector from rhesus macaques that due to its ability to induce potent responses in mice warrant further investigation., (Copyright © 2016. Published by Elsevier B.V.)

We analyzed gene expression profiles of young and aged mouse CD8 + T cells specific for the nucleoprotein (NP) of influenza A/PR8/34 virus. CD8 + T cells were stimulated either by the NP antigen expressed in its native form or fused into the herpes virus (HSV)-1 glycoprotein D (gD) protein, which blocks signaling through the immunoinhibitory B and T lymphocyte attenuator (BTLA) and CD160 pathways. We show that NP-specific CD8 + T cells from aged mice exhibit numerous differences in gene expression compared to NP-specific CD8 + T cells from young mice, including a significant reduction of expression in genes involved in T cell receptor (TcR) and CD28 signaling. We also show that these changes can be reversed in a sub-population (~50%) of the aged mice by a BTLA/CD160 checkpoint blockade. These results suggest that BTLA/CD160 checkpoint blockade has potential value as a vaccine additive to induce better CD8 + T cell responses in the aged., Competing Interests: Conflicts of Interest: None declared.

Objectives: Over 2 million individuals are infected with HIV type 1 (HIV-1) each year, yet an effective vaccine remains elusive. The most successful HIV-1 vaccine to date demonstrated 31% efficacy. Immune correlate analyses associated HIV-1 envelope (Env)-specific antibodies with protection, thus providing a path toward a more effective vaccine. We sought to test the antibody response from novel prime-boost vaccination with a chimpanzee-derived adenovirus (AdC) vector expressing a subtype C Env glycoprotein (gp)140 combined with either a serologically distinct AdC vector expressing gp140 of a different subtype C isolate or an alum-adjuvanted, partially trimeric gp145 from yet another subtype C isolate., Design: Three different prime-boost regimens were tested in mice: AdC prime-protein boost, protein prime-AdC boost, and AdC prime-AdC boost. Each regimen was tested at two different doses of AdC vector in a total of six experimental groups., Methods: Sera were collected at various time points and evaluated by ELISA for Env-specific antibody binding, isotype, and avidity. Antibody functionality was assessed by pseudovirus neutralization assay., Results: Priming with AdC followed by a protein boost or sequential immunizations with two AdC vectors induced HIV-1 Env-specific binding antibodies, including those to the variable region 2, whereas priming with protein followed by an AdC boost was relatively ineffective. Antibodies that cross-neutralized tier 1 HIV-1 from different subtypes were elicited with vaccine regimens that included immunizations with protein., Conclusion: Our study warrants further investigation of AdC vector and gp145 protein prime-boost vaccines and their ability to protect against acquisition in animal challenge studies., Competing Interests: Disclaimers/Conflicts of Interest Statement: The Ertl and Polonis Labs declare no conflicts of interest. The views expressed are those of the authors and should not be construed to represent the positions of the U.S. Army or the Department of Defense.

Adenovirus (Ad) is used extensively for construction of viral vectors, most commonly with deletion in its E1 and/or E3 genomic regions. Previously, our attempts to insert envelope proteins (Env) of HIV-1 into such vectors based on chimpanzee-derived Ad (AdC) viruses were thwarted. Here, we describe that genetic instability of an E1- and E3-deleted AdC vector of serotype C6 expressing Env of HIV-1 can be overcome by reinsertion of E3 sequences with anti-apoptotic activities. This partial E3 deletion presumably delays premature death of HEK-293 packaging cell lines due to Env-induced cell apoptosis. The same partial E3 deletion also allows for the generation of stable glycoprotein 140 (gp140)- and gp160-expressing Ad vectors based on AdC7, a distinct AdC serotype. Env-expressing AdC vectors containing the partial E3 deletion are genetically stable upon serial cell culture passaging, produce yields comparable to those of other AdC vectors, and induce transgene product-specific antibody responses in mice. A partial E3 deletion thereby allows expansion of the repertoire of transgenes that can be expressed by Ad vectors., Competing Interests: Author Disclosure No competing financial interests exist.

Adeno-associated viral (AAV) vectors are currently being tested in multiple clinical trials for liver-directed gene transfer to treat the bleeding disorders hemophilia A and B and metabolic disorders. The optimal viral capsid for transduction of human hepatocytes has been under active investigation, but results across various models are inconsistent. We tested in vivo transduction in "humanized" mice. Methods to quantitate percent AAV transduced human and murine hepatocytes in chimeric livers were optimized using flow cytometry and confocal microscopy with image analysis. Distinct transduction efficiencies were noted following peripheral vein administration of a self-complementary vector expressing a gfp reporter gene. An engineered AAV3 capsid with two amino acid changes, S663V+T492V (AAV3-ST), showed best efficiency for human hepatocytes (~3-times, ~8-times, and ~80-times higher than for AAV9, AAV8, and AAV5, respectively). AAV5, 8, and 9 were more efficient in transducing murine than human hepatocytes. AAV8 yielded the highest transduction rate of murine hepatocytes, which was 19-times higher than that for human hepatocytes. In summary, our data show substantial differences among AAV serotypes in transduction of human and mouse hepatocytes, are the first to report on AAV5 in humanized mice, and support the use of AAV3-based vectors for human liver gene transfer.

The tumor stroma, which is essential to support growth and metastasis of malignant cells, provides targets for active immunotherapy of cancer. Previous studies have shown that depleting fibroblast activation protein (FAP)-expressing stromal cells reduces tumor progression and concomitantly increases tumor antigen (TA)-specific T cell responses. However the underlying pathways remain ill defined. Here we identify that immunosuppressive cells (ISCs) from tumor-bearing mice impose metabolic stress on CD8+T cells, which is associated with increased expression of the co-inhibitor PD-1. In two mouse melanoma models, depleting FAP+ stroma cells from the tumor microenvironment (TME) upon vaccination with an adenoviral-vector reduces frequencies and functions of ISCs. This is associated with changes in the cytokine/chemokine milieu in the TME and decreased activity of STAT6 signaling within ISCs. Decreases in ISCs upon FAP+stromal cell depletion is associated with reduced metabolic stress of vaccine-induced tumor infiltrating CD8+T cells and their delayed progression towards functional exhaustion, resulting in prolonged survival of tumor-bearing mice., Competing Interests: The authors declare that they have no conflict of interest.

We analyzed age-related defects in B cell populations from young and aged mice. Microarray analysis of bone marrow resident antibody secreting cells (ASCs) showed significant changes upon aging, affecting multiple genes, pathways and functions including those that play a role in immune regulation, humoral immune responses, chromatin structure and assembly, cell metabolism and the endoplasmic reticulum (ER) stress response. Further analysis showed upon aging defects in energy production through glucose catabolism with reduced oxidative phosphorylation. In addition aged B cells had increased levels of reactive oxygen-species (ROS), which was linked to enhanced expression of the co-inhibitor programmed cell death (PD)-1.

Although cancer immunotherapy has achieved significant breakthroughs in recent years, its overall efficacy remains limited in the majority of patients. One major barrier is exhaustion of tumor antigen-specific CD8(+) tumor-infiltrating lymphocytes (TILs), which conventionally has been attributed to persistent stimulation with antigen within the tumor microenvironment (TME). A series of recent studies have highlighted that the TME poses significant metabolic challenges to TILs, which may contribute to their functional exhaustion. Hypoxia increases the expression of coinhibitors on activated CD8(+) T cells, which in general reduces the T cells' effector functions. It also impairs the cells' ability to gain energy through oxidative phosphorylation. Glucose limitation increases the expression of programed cell death protein-1 and reduces functions of activated CD8(+) T cells. A combination of hypoxia and hypoglycemia, as is common in solid tumors, places CD8(+) TILs at dual metabolic jeopardy by affecting both major pathways of energy production. Recently, a number of studies addressed the effects of metabolic stress on modulating CD8(+) T cell metabolism, differentiation, and functions. Here, we discuss recent findings on how different types of metabolic stress within the TME shape the tumor-killing capacity of CD8(+) T cells. We propose that manipulating the metabolism of TILs to more efficiently utilize nutrients, especially during intermittent periods of hypoxia could maximize their performance, prolong their survival and improve the efficacy of active cancer immunotherapy.

We tested antibody responses to the trivalent inactivated influenza vaccine (TIV) in 34 aged individuals (>65 yrs) during the 2012/13 vaccination seasons. Nearly all had been vaccinated the previous year although the time interval between the two vaccine doses differed. One subgroup was re-vaccinated in 2012/13 within 6-9 months of their 2011/12 vaccination, the other received the two doses of vaccine in the typical ~12 month interval. Unexpectedly the sub-cohort with early revaccination exhibited significantly increased response rates and antibody titers to TIV compared to their normally re-vaccinated aged counter parts. Microarray analyses of gene expression in whole blood RNA taken at the day of the 2012/13 re-vaccination revealed statistically significant differences in expression of 754 genes between the individuals with early re-vaccination compared to subjects vaccinated in a normal 12 month interval. These observations suggest that TIV has long-lasting effects on the immune system affecting B cell responses as well as the transcriptome of peripheral blood mononuclear cells and this residual effect may augment vaccination response in patients where the effect of the previous vaccination has not yet diminished.

Pulmonary tuberculosis (TB) remains to be a major global health problem despite many decades of parenteral use of Bacillus Calmette-Guérin (BCG) vaccine. Developing safe and effective respiratory mucosal TB vaccines represents a unique challenge. Over the past decade or so, the human serotype 5 adenovirus (AdHu5)-based TB vaccine has emerged as one of the most promising candidates based on a plethora of preclinical and early clinical studies. However, anti-AdHu5 immunity widely present in the lung of humans poses a serious gap and limitation to its real-world applications. In this study we have developed a novel chimpanzee adenovirus 68 (AdCh68)-vectored TB vaccine amenable to the respiratory route of vaccination. We have evaluated AdCh68-based TB vaccine for its safety, T-cell immunogenicity, and protective efficacy in relevant animal models of human pulmonary TB with or without parenteral BCG priming. We have also compared AdCh68-based TB vaccine with its AdHu5 counterpart in both naive animals and those with preexisting anti-AdHu5 immunity in the lung. We provide compelling evidence that AdCh68-based TB vaccine is not only safe when delivered to the respiratory tract but, importantly, is also superior to its AdHu5 counterpart in induction of T-cell responses and immune protection, and limiting lung immunopathology in the presence of preexisting anti-AdHu5 immunity in the lung. Our findings thus suggest AdCh68-based TB vaccine to be an ideal candidate for respiratory mucosal immunization, endorsing its further clinical development in humans.

Virus-neutralizing antibody and B cell responses to influenza A viruses were measured in 35 aged and 28 middle-aged individuals following vaccination with the 2012 and 2013 trivalent inactivated influenza vaccines. Antibody responses to the vaccine strains were lower in the aged. An analysis of B cell subsets by flow cytometry with stains for immunoregulators showed that B cells of multiple subsets from the aged as compared to younger human subjects showed differences in the expression of the co-inhibitor B and T lymphocyte attenuator (BTLA). Expression of BTLA inversely correlated with age and appears to be linked to shifting the nature of the response from IgM to IgG. High BTLA expression on mature B cells was linked to higher IgG responses to the H1N1 virus. Finally, high BTLA expression on isotype switched memory B cells was linked to better preservation of virus neutralizing antibody titers and improved recall responses to vaccination given the following year.

Unlabelled: Influenza infection causes severe disease and death in humans. In traditional vaccine research and development, a single high-dose virus challenge of animals is used to evaluate vaccine efficacy. This type of challenge model may have limitations. In the present study, we developed a novel challenge model by infecting mice repeatedly in short intervals with low doses of influenza A virus. Our results show that compared to a single high-dose infection, mice that received repeated low-dose challenges showed earlier morbidity and mortality and more severe disease. They developed higher vial loads, more severe lung pathology, and greater inflammatory responses and generated only limited influenza A virus-specific B and T cell responses. A commercial trivalent influenza vaccine protected mice against a single high and lethal dose of influenza A virus but was ineffective against repeated low-dose virus challenges. Overall, our data show that the repeated low-dose influenza A virus infection mouse model is more stringent and may thus be more suitable to select for highly efficacious influenza vaccines., Importance: Influenza epidemics and pandemics pose serious threats to public health. Animal models are crucial for evaluating the efficacy of influenza vaccines. Traditional models based on a single high-dose virus challenge may have limitations. Here, we describe a new mouse model based on repeated low-dose influenza A virus challenges given within a short period. Repeated low-dose challenges caused more severe disease in mice, associated with higher viral loads and increased lung inflammation and reduced influenza A virus-specific B and T cell responses. A commercial influenza vaccine that was shown to protect mice from high-dose challenge was ineffective against repeated low-dose challenges. Overall, our results show that the low-dose repeated-challenge model is more stringent and may therefore be better suited for preclinical vaccine efficacy studies., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Influenza virus (IV) infection is a major public health problem, causing millions of cases of severe illness and as many as 500 000 deaths each year worldwide. Given the limitations of current prevention or treatment of acute influenza, novel therapies are needed. RNA interference (RNAi) through microRNAs (miRNA) is an emerging technology that can suppress virus replication in vitro and in vivo. Here, we describe a novel strategy for the treatment of infuenza based on RNAi delivered by a replication-defective adenovirus (Ad) vector, derived from chimpanzee serotype 68 (AdC68). Our results showed that artificial miRNAs (amiRNAs) specifically targeting conserved regions of the IV genome could effectively inhibit virus replication in human embryonic kidney 293 cells. Moreover, our results demonstrated that prophylactic treatment with AdC68 expressing amiRNAs directed against M1, M2 or nucleoprotein genes of IV completely protected mice from homologous A/PR8 virus challenge and partially protected the mice from heterologous influenza A virus strains such as H9N2 and H5N1. Collectively, our data demonstrate that amiRNAs targeting the conserved regions of influenza A virus delivered by Ad vectors should be pursued as a novel strategy for prophylaxis of IV infection in humans and animals.

An effective T-cell-based AIDS vaccine should induce strong HIV-specific CD8(+) T cells in mucosal tissues without increasing the availability of target cells for the virus. Here, we evaluated five immunization strategies that include Human adenovirus-5 (AdHu5), Chimpanzee adenovirus-6 (AdC6) or -7 (AdC7), Vaccinia virus (VV), and DNA given by electroporation (DNA/EP), all expressing Simian immunodeficiency virus group specific antigen/transactivator of transcription (SIV(mac239Gag/Tat)). Five groups of six rhesus macaques (RMs) each were vaccinated with DNA/EP-AdC6-AdC7, VV-AdC6-AdC7, DNA/-EP-VV-AdC6, DNA/EP-VV-AdC7, or AdHu5-AdHu5-AdHu5 and were challenged repeatedly with low-dose intrarectal SIVmac239. Upon challenge, there were no significant differences among study groups in terms of virus acquisition or viral load after infection. When taken together, the immunization regimens did not protect against SIV acquisition compared with controls but did result in an ∼ 1.6-log decline in set-point viremia. Although all immunized RMs had detectable SIV-specific CD8(+) T cells in blood and rectal mucosa, we found no correlation between the number or function of these SIV-specific CD8(+) T cells and protection against SIV acquisition. Interestingly, RMs experiencing breakthrough infection showed significantly higher prechallenge levels of CD4(+)C-C chemokine receptor type 5 (CCR5)(+)HLA-DR(+) T cells in the rectal biopsies (RB) than animals that remained uninfected. In addition, among the infected RMs, the percentage of CD4(+)CCR5(+)Ki-67(+) T cells in RBs prechallenge correlated with higher early viremia. Overall, these data suggest that the levels of activated CD4(+)CCR5(+) target T cells in the rectal mucosa may predict the risk of SIV acquisition in RMs vaccinated with vectors that express SIVGag/Tat.

The immune system represents a significant barrier to successful gene therapy with adeno-associated viral (AAV) vectors. In particular, adaptive immune responses to the viral capsid or the transgene product are of concern. The sensing of AAV by toll-like receptors (TLRs) TLR2 and TLR9 has been suggested to play a role in innate immunity to the virus and may also shape subsequent adaptive immune responses. Here, we investigated the functions of TLR2, TLR9 and the downstream signaling adaptor MyD88 in antibody and CD8+ T-cell responses. Antibody formation against the transgene product occurred largely independently of TLR signaling following gene transfer with AAV1 or AAV2 vectors, whereas loss of signaling through the TLR9-MyD88 pathway substantially reduced CD8+ T-cell responses. In contrast, MyD88 (but neither of the TLRs) regulated antibody responses to capsid. B cell-intrinsic MyD88 was required for the formation of anti-capsid IgG2c independently of vector serotype or route of administration. However, MyD88(-/-) mice instead produced anti-capsid IgG1 that emerged with delayed kinetics but nonetheless completely prevented in vivo readministration. We conclude that there are distinct roles for TLR9 and MyD88 in promoting adaptive immune responses to AAV-mediated gene transfer and that there are redundant MyD88-dependent and MyD88-independent mechanisms that stimulate neutralizing antibody formation against AAV., (© 2015 S. Karger AG, Basel.)

Survivin is highly expressed in most human tumors and fetal tissue, and absent in terminally differentiated cells. It promotes tumor cell proliferation by negatively regulating cell apoptosis and facilitating cell division. Survivin's selective expression pattern suggests that it might be a suitable target for cancer therapy, which would promote death of transformed but not normal cells. This was tested using artificial microRNAs (amiRNAs) targeting survivin. After screening, two effective amiRNAs, which knocked down survivin expression, were identified and cloned into a replication-defective adenoviral vector. Tumor cells infected with the recombinant vector downregulated expression of survivin and underwent apoptotic cell death. Further studies showed that apoptosis was associated with increases in caspase 3 and cleaved Poly (ADP-ribose) polymerase, and activation of the p53 signaling pathway. Furthermore, amiRNA treatment caused blockade of mitosis and cell cycle arrest at the G2/M phase. In vivo, survivin-targeting amiRNAs expressed by adenoviral vectors effectively delayed growth of hepatocellular and cervical carcinomas in mouse xenograft models. These results indicate that silencing of survivin by amiRNA has potential for treatment of cancer.

In this study, we tested the effect of neutralizing Abs to different serotypes of E1-deleted Ad vectors on the immunogenicity of the homologous Ad vector or a vector derived from a heterologous serotype. Our results showed that, as expected, even low titers of passively transferred neutralizing Abs significantly reduced the homologous vectors' ability to elicit transgene-specific CD8(+) T cell responses. In addition, Abs changed the fate of transgene product-specific CD8(+) T cells by promoting their transition into the central memory cell pool, which resulted in markedly enhanced expansion of transgene product-specific CD8(+) T cells after a boost with a heterologous Ad vector. Non-neutralizing Abs specific to a distinct Ad serotype had no effect on the magnitude of transgene product-specific CD8(+) T cells induced by a heterologous Ad vector, nor did such Abs promote induction of more resting memory CD8(+) T cells. These results show that Abs to an Ad vaccine carrier affect not only the magnitude but also the profile of a vector-induced CD8(+) T cell response., (© 2014 Society for Leukocyte Biology.)