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BACKGROUND: A safe and effective vaccine to prevent chronic hepatitis C virus (HCV) infection is a critical component of efforts to eliminate the disease. METHODS: In this phase 1–2 randomized, double-blind, placebo-controlled trial, we evaluated a recombinant chimpanzee adenovirus 3 vector priming vaccination followed by a recombinant modified vaccinia Ankara boost; both vaccines encode HCV nonstructural proteins. Adults who were considered to be at risk for HCV infection on the basis of a history of recent injection drug use were randomly assigned (in a 1:1 ratio) to receive vaccine or placebo on days 0 and 56. Vaccine-related serious adverse events, severe local or systemic adverse events, and laboratory adverse events were the primary safety end points. The primary efficacy end point was chronic HCV infection, defined as persistent viremia for 6 months. RESULTS: A total of 548 participants underwent randomization, with 274 assigned to each group. There was no significant difference in the incidence of chronic HCV infection between the groups. In the per-protocol population, chronic HCV infection developed in 14 participants in each group (hazard ratio [vaccine vs. placebo], 1.53; 95% confidence interval [CI], 0.66 to 3.55; vaccine efficacy, −53%; 95% CI, −255 to 34). In the modified intention-to-treat population, chronic HCV infection developed in 19 participants in the vaccine group and 17 in placebo group (hazard ratio, 1.66; 95% CI, 0.79 to 3.50; vaccine efficacy, −66%; 95% CI, −250 to 21). The geometric mean peak HCV RNA level after infection differed between the vaccine group and the placebo group (152.51×10(3) IU per milliliter and 1804.93×10(3) IU per milliliter, respectively). T-cell responses to HCV were detected in 78% of the participants in the vaccine group. The percentages of participants with serious adverse events were similar in the two groups. CONCLUSIONS: In this trial, the HCV vaccine regimen did not cause serious adverse events, produced HCV-specific T-cell responses, and lowered the peak HCV RNA level, but it did not prevent chronic HCV infection. (Funded by the National Institute of Allergy and Infectious Diseases; ClinicalTrials.gov number, NCT01436357.)

The cell cycle of antigen-specific T cells in vivo has been examined by using a few methods, all of which possess some limitations. Bromodeoxyuridine (BrdU) marks cells that are in or recently completed S-phase, and carboxyfluorescein succinimidyl ester (CFSE) detects daughter cells after division. However, these dyes do not allow identification of the cell cycle phase at the time of analysis. An alternative approach is to exploit Ki67, a marker that is highly expressed by cells in all phases of the cell cycle except the quiescent phase G0. Unfortunately, Ki67 does not allow further differentiation as it does not separate cells in S-phase that are committed to mitosis from those in G1 that can remain in this phase, proceed into cycling, or move into G0. Here, we describe a flow cytometric method for capturing a "snapshot" of T cells in different cell cycle phases in mouse secondary lymphoid organs. The method combines Ki67 and DNA staining with major histocompatibility complex (MHC)-peptide-multimer staining and an innovative gating strategy, allowing us to successfully differentiate between antigen-specific CD8 T cells in G0, in G1 and in S-G2/M phases of the cell cycle in the spleen and draining lymph nodes of mice after vaccination with viral vectors carrying the model antigen gag of human immunodeficiency virus (HIV)-1. Critical steps of the method were the choice of the DNA dye and the gating strategy to increase the assay sensitivity and to include highly activated/proliferating antigenspecific T cells that would have been missed by current criteria of analysis. The DNA dye, Hoechst 33342, enabled us to obtain a high-quality discrimination of the G0/ G1 and G2/M DNA peaks, while preserving membrane and intracellular staining. The method has great potential to increase knowledge about T cell response in vivo and to improve immuno-monitoring analysis.

Since the discovery in 1796 by Edward Jenner of vaccinia virus as a way to prevent and finally eradicate smallpox, the concept of using a virus to fight another virus has evolved into the current approaches of viral vectored genetic vaccines. In recent years, key improvements to the vaccinia virus leading to a safer version (Modified Vaccinia Ankara, MVA) and the discovery that some viruses can be used as carriers of heterologous genes encoding for pathological antigens of other infectious agents (the concept of ‘viral vectors’) has spurred a new wave of clinical research potentially providing for a solution for the long sought after vaccines against major diseases such as HIV, TB, RSV and Malaria, or emerging infectious diseases including those caused by filoviruses and coronaviruses. The unique ability of some of these viral vectors to stimulate the cellular arm of the immune response and, most importantly, T lymphocytes with cell killing activity, has also reawakened the interest toward developing therapeutic vaccines against chronic infectious diseases and cancer. To this end, existing vectors such as those based on Adenoviruses have been improved in immunogenicity and efficacy. Along the same line, new vectors that exploit viruses such as Vesicular Stomatitis Virus (VSV), Measles Virus (MV), Lymphocytic choriomeningitis virus (LCMV), cytomegalovirus (CMV), and Herpes Simplex Virus (HSV), have emerged. Furthermore, technological progress toward modifying their genome to render some of these vectors incompetent for replication has increased confidence toward their use in infant and elderly populations. Lastly, their production process being the same for every product has made viral vectored vaccines the technology of choice for rapid development of vaccines against emerging diseases and for ‘personalised’ cancer vaccines where there is an absolute need to reduce time to the patient from months to weeks or days. Here we review the recent developments in viral vector technologies, focusing on novel vectors based on primate derived Adenoviruses and Poxviruses, Rhabdoviruses, Paramixoviruses, Arenaviruses and Herpesviruses. We describe the rationale for, immunologic mechanisms involved in, and design of viral vectored gene vaccines under development and discuss the potential utility of these novel genetic vaccine approaches in eliciting protection against infectious diseases and cancer.

Rabies, caused by RNA viruses in the Genus Lyssavirus, is the most fatal of all infectious diseases. This neglected zoonosis remains a major public health problem in developing countries, causing the death of an estimated 25,000–159,000 people each year, with more than half of them in children. The high incidence of human rabies in spite of effective vaccines is mainly linked to the lack of compliance with the complicated administration schedule, inadequacies of the community public health system for local administration by the parenteral route and the overall costs of the vaccine. The goal of our work was the development of a simple, affordable and effective vaccine strategy to prevent human rabies virus infection. This next generation vaccine is based on a replication-defective chimpanzee adenovirus vector belonging to group C, ChAd155-RG, which encodes the rabies glycoprotein (G). We demonstrate here that a single dose of this vaccine induces protective efficacy in a murine model of rabies challenge and elicits strong and durable neutralizing antibody responses in vaccinated non-human primates. Importantly, we demonstrate that one dose of a commercial rabies vaccine effectively boosts the neutralizing antibody responses induced by ChAd155-RG in vaccinated monkeys, showing the compatibility of the novel vectored vaccine with the current post-exposure prophylaxis in the event of rabies virus exposure. Finally, we demonstrate that antibodies induced by ChAd155-RG can also neutralize European bat lyssaviruses 1 and 2 (EBLV-1 and EBLV-2) found in bat reservoirs., Author summary Rabies still remains a neglected zoonosis after a long history of vaccination. Considering the severity of the disease and its continued high incidence in low-income countries, the development of a next generation vaccine is warranted. We utilized a group C, replication-defective chimpanzee adenovirus vector to develop a novel vaccine against rabies. Mice vaccinated with ChAd155-RG survived after rabies infection and non-human primates injected with a single dose of this vaccine developed strong and durable neutralizing antibody responses which could be effectively boosted with a licensed vaccine, demonstrating the compatibility of the novel vectored vaccine with the current post-exposure prophylaxis in the event of rabies virus exposure. Importantly, we show that ChAd155-RG induced neutralizing antibodies can neutralize also lyssavirus species (EBLV-1 and EBLV-2) found in bat reservoirs. These studies paved the way to the clinical testing of the ChAd155-RG based rabies vaccine as a single-dose, low cost, preventative rabies vaccine candidate.

Oncolytic virotherapy is emerging as a promising therapeutic option for solid tumours. Several oncolytic vectors in clinical testing are based on attenuated viruses; thus, efforts are being taken to develop a new repertoire of oncolytic viruses, based on virulent viral genomes. This possibility, however, raises concerns dealing with the safety features of the virulent phenotypes. We generated a double regulated Herpes simplex type-1 virus (HSV-1), in which tumour cell restricted replicative potential was combined to selective entry via ERBB2 receptor retargeting. The transcriptional control of the viral alpha4 gene encoding for the infected cell protein-4 (ICP4) by the cellular Survivin/BIRC5 promoter conferred a tumour cell-restricted replicative potential to a virulent HSV-1 genome. The combination of the additional ERBB2 retargeting further improved the selectivity for tumour cells, conferring to the double regulated virus a very limited ability to infect and propagate in non-cancerous cells. Accordingly, a suitable replicative and cytotoxic potential was maintained in tumour cell lines, allowing the double regulated virus to synergize in vivo with immune checkpoint (anti-PD-1) blockade in immunocompetent mice. Thus, restricting the replicative spectrum and tropism of virulent HSV-1 genomes by combination of conditional replication and retargeting provides an improved safety, does not alter the oncolytic strength, and is exploitable for its therapeutic potential with immune checkpoint blockade in cancer.

Tumors with microsatellite instability (MSI) are caused by a defective DNA mismatch repair system that leads to the accumulation of mutations within microsatellite regions. Indels in microsatellites of coding genes can result in the synthesis of frameshift peptides (FSP). FSPs are tumor-specific neoantigens shared across patients with MSI. In this study, we developed a neoantigen-based vaccine for the treatment of MSI tumors. Genetic sequences from 320 MSI tumor biopsies and matched healthy tissues in The Cancer Genome Atlas database were analyzed to select shared FSPs. Two hundred nine FSPs were selected and cloned into nonhuman Great Ape Adenoviral and Modified Vaccinia Ankara vectors to generate a viral-vectored vaccine, referred to as Nous-209. Sequencing tumor biopsies of 20 independent patients with MSI colorectal cancer revealed that a median number of 31 FSPs out of the 209 encoded by the vaccine was detected both in DNA and mRNA extracted from each tumor biopsy. A relevant number of peptides encoded by the vaccine were predicted to bind patient HLA haplotypes. Vaccine immunogenicity was demonstrated in mice with potent and broad induction of FSP-specific CD8 and CD4 T-cell responses. Moreover, a vaccine-encoded FSP was processed in vitro by human antigen-presenting cells and was subsequently able to activate human CD8 T cells. Nous-209 is an “off-the-shelf” cancer vaccine encoding many neoantigens shared across sporadic and hereditary MSI tumors. These results indicate that Nous-209 can induce the optimal breadth of immune responses that might achieve clinical benefit to treat and prevent MSI tumors. Significance: These findings demonstrate the feasibility of an “off-the-shelf” vaccine for treatment and prevention of tumors harboring frameshift mutations and neoantigenic peptides as a result of microsatellite instability.

BackgroundA number of different immune pathways are involved in the effective killing of cancer cells, collectively named as the ‘Cancer Immunity Cycle’. Anti-PD-1 checkpoint blockade (CPB) therapy is active on one of these pathways and reinvigorates anticancer T cell immunity, leading to long-term responses in a limited fraction of patients with cancer. We have previously shown that neoantigens-based adenovirus vectored vaccine in combination with anti-PD-1 further expands pre-existing anticancer immunity and elicits novel neoantigen-specific T cells thereby increasing efficacy to 50% of tumor clearance in mice. Here we added a third component to the CPB plus vaccine combination, which is able to modify the suppressive tumor microenvironment by reducing the number of tumor-infiltrating regulatory T cells (Tregs), as strategy for improving the therapeutic efficacy and overcoming resistance.MethodsThe antitumor efficacy of anti-PD-1, neoantigen vaccine and Treg modulating agents, either Bempegaldesleukin (BEMPEG: NKTR-214) or an anti-CTLA-4 mAb with Treg-depleting activity, was investigated in murine tumor models. We evaluated tumor growth in treated animals, neoantigen-specific T cells in tumors, tumor-infiltrating lymphocytes (TILs) and intratumoral Tregs.ResultsThe addition of BEMPEG or anti-CTLA-4 to the combination of vaccine and anti-PD-1 led to complete eradication of large tumors in nearby 100% of treated animals, in association with expansion and activation of cancer neoantigen-specific T cells and reduction of tumor-infiltrating Tregs.ConclusionThese data support the notion that the integrated regulation of three steps of the cancer immunity cycle, including expansion of neoantigen-specific T cells, reversal of the exhausted T cell phenotype together with the reduction of intratumoral Tregs may represent a novel rationally designed drug combination approach to achieve higher cure rates.

BackgroundThe unprecedented 2014 epidemic of Ebola virus disease (EVD) prompted an international response to accelerate the availability of a preventive vaccine. A replication-defective recombinant chimpanzee adenovirus type 3–vectored ebolavirus vaccine (cAd3-EBO), encoding the glycoprotein from Zaire and Sudan species, that offers protection in the nonhuman primate model, was rapidly advanced into phase 1 clinical evaluation. MethodsWe conducted a phase 1, dose-escalation, open-label trial of cAd3-EBO. Twenty healthy adults, in sequentially enrolled groups of 10 each, received vaccination intramuscularly in doses of 2×1010 particle units or 2×1011 particle units. Primary and secondary end points related to safety and immunogenicity were assessed throughout the first 8 weeks after vaccination; in addition, longer-term vaccine durability was assessed at 48 weeks after vaccination. ResultsIn this small study, no safety concerns were identified; however, transient fever developed within 1 day after vaccinat...

The novel antibody-based immunotherapy in oncology exploits the activation of immune system mediated by immunomodulatory antibodies specific for immune checkpoints. Among them, the programmed death ligand-1 (PD-L1) is of particular interest as it is expressed not only on T-cells, but also on other immune cells and on a large variety of cancer cells, such as breast cancer cells, considering its high expression in both ErbB2-positive and Triple Negative Breast Cancers. We demonstrate here that PD-L1_1, a novel anti-PD-L1 T -cell stimulating antibody, inhibits PD-L1-tumor cell growth also by affecting the intracellular MAPK pathway and by activating caspase 3. Similar in vitro results were obtained for the first time here also with the clinically validated anti-PD-L1 mAb Atezolizumab and in vivo with another validated anti-mouse anti-PD-L1 mAb. Moreover, we found that two high affinity variants of PD-L1_1 inhibited tumor cell viability more efficiently than the parental PD-L1_1 by affecting the same MAPK pathways with a more potent effect. Altogether, these results shed light on the role of PD-L1 in cancer cells and suggest that PD-L1_1 and its high affinity variants could become powerful antitumor weapons to be used alone or in combination with other drugs such as the anti-ErbB2 cAb already successfully tested in in vitro combinatorial treatments.

Although clonal expansion is a hallmark of adaptive immunity, the location(s) where antigen‐responding T cells enter cell cycle and complete it have been poorly explored. This lack of knowledge stems partially from the limited experimental approaches available. By using Ki67 plus DNA staining and a novel strategy for flow cytometry analysis, we distinguished antigen‐specific CD8 T cells in G0, in G1 and in S‐G2/M phases of cell cycle after intramuscular vaccination of BALB/c mice with antigen‐expressing viral vectors. Antigen‐specific cells in S‐G2/M were present at early times after vaccination in lymph nodes (LNs), spleen and, surprisingly, also in the blood, which is an unexpected site for cycling of normal non‐leukaemic cells. Most proliferating cells had high scatter profile and were undetected by current criteria of analysis, which under‐estimated up to 6 times antigen‐specific cell frequency in LNs. Our discovery of cycling antigen‐specific CD8 T cells in the blood opens promising translational perspectives.

Canine malignant melanoma is a highly aggressive tumor with a low survival rate and represents an ideal spontaneous model for the human counterpart. Considerable progress has been recently obtained, but the therapeutic success for canine melanoma is still challenging. Little is known about the mechanisms beyond pathogenesis and melanoma development, and the molecular response to radiotherapy has never been explored before. A faster and deeper understanding of cancer mutational processes and developing mechanisms are now possible through next generation sequencing technologies. In this study, we matched whole exome and transcriptome sequencing in four dogs affected by malignant melanoma at diagnosis and at disease progression to identify possible genetic mechanisms associated with therapy failure. According to previous studies, a genetic similarity between canine malignant melanoma and its human counterpart was observed. Several somatic mutations were functionally related to MAPK, PI3K/AKT and p53 signaling pathways, but located in genes other than BRAF, RAS and KIT. At disease progression, several mutations were related to therapy effects. Natural killer cell-mediated cytotoxicity and several immune-system-related pathways resulted activated opening a new scenario on the microenvironment in this tumor. In conclusion, this study suggests a potential role of the immune system associated to radiotherapy in canine melanoma, but a larger sample size associated with functional studies are needed. This article is protected by copyright. All rights reserved.

Exhaustion of antiviral CD8(+) T cells contributes to persistence of hepatitis C viral (HCV) infection. This immune response has proved difficult to restore by therapeutic vaccination, even when HCV replication is suppressed using antiviral regimens containing type I interferon. Because immunomodulatory effects of type I interferon may be a factor in poor T-cell priming, we undertook therapeutic vaccination in two chronically infected chimpanzees during treatment with a direct-acting antiviral (DAA) targeting the HCV NS5b polymerase protein. Immunization with genetic vaccines encoding the HCV NS3-NS5b nonstructural proteins during DAA treatment resulted in a multifunctional CD8(+) T-cell response. However, these antiviral CD8(+) T cells did not prevent persistent replication of DAA-resistant HCV variants that emerged during treatment. Most vaccine-induced CD8(+) T cells targeted class I epitopes that were not conserved in the circulating virus. Exhausted intrahepatic CD8(+) T-cell targeting-conserved epitopes did not expand after vaccination, with a notable exception. A sustained, multifunctional CD8(+) T-cell response against at least one intact class I epitope was detected in blood after vaccination. Persistence of HCV was not due to mutational escape of this epitope. Instead, failure to control HCV replication was likely caused by localized exhaustion in the liver, where CD8(+) T-cell expression of the inhibitory receptor programmed cell death 1 increased 25-fold compared with those in circulation. CONCLUSION: Treatment with a DAA during therapeutic vaccination provided transient control of HCV replication and a multifunctional T-cell response, primarily against nonconserved class I epitopes; exhaustion of liver-infiltrating CD8(+) T cells that target conserved epitopes may not be averted when DAA therapy fails prematurely due to emergence of resistant HCV variants.

Neoantigens (nAgs) are promising tumor antigens for cancer vaccination with the potential of inducing robust and selective T cell responses. Genetic vaccines based on Adenoviruses derived from non-human Great Apes (GAd) elicit strong and effective T cell-mediated immunity in humans. Here, we investigate for the first time the potency and efficacy of a novel GAd encoding multiple neoantigens. Prophylactic or early therapeutic vaccination with GAd efficiently control tumor growth in mice. In contrast, combination of the vaccine with checkpoint inhibitors is required to eradicate large tumors. Gene expression profile of tumors in regression shows abundance of activated tumor infiltrating T cells with a more diversified TCR repertoire in animals treated with GAd and anti-PD1 compared to anti-PD1. Data suggest that effectiveness of vaccination in the presence of high tumor burden correlates with the breadth of nAgs-specific T cells and requires concomitant reversal of tumor suppression by checkpoint blockade., Vaccination against neo-antigens has resulted in an effective antitumor response in several models. Here, the authors show that delivery of larger sets of neo-antigens using an adenovirus-based vaccination platform, results in much better tumor protection when combined with checkpoint blockade in a mouse model of advanced disease.

Recombinant adenoviral vectors (rAds) are lead vaccine candidates for protection against a variety of pathogens, including Ebola, HIV, tuberculosis, and malaria, due to their ability to potently induce T cell immunity in humans. However, the ability to induce protective cellular immunity varies among rAds. Here, we assessed the mechanisms that control the potency of CD8 T cell responses in murine models following vaccination with human-, chimpanzee-, and simian-derived rAds encoding SIV-Gag antigen (Ag). After rAd vaccination, we quantified Ag expression and performed expression profiling of innate immune response genes in the draining lymph node. Human-derived rAd5 and chimpanzee-derived chAd3 were the most potent rAds and induced high and persistent Ag expression with low innate gene activation, while less potent rAds induced less Ag expression and robustly induced innate immunity genes that were primarily associated with IFN signaling. Abrogation of type I IFN or stimulator of IFN genes (STING) signaling increased Ag expression and accelerated CD8 T cell response kinetics but did not alter memory responses or protection. These findings reveal that the magnitude of rAd-induced memory CD8 T cell immune responses correlates with Ag expression but is independent of IFN and STING and provide criteria for optimizing protective CD8 T cell immunity with rAd vaccines.

Currently available prophylactic vaccines have no therapeutic efficacy for preexisting human papillomavirus (HPVs) infections, do not target all oncogenic HPVs and are insufficient to eliminate the burden of HPV induced cancer. We aim to develop an alternative HPV vaccine which is broadly effective and capable of clearing preexisting infection. In an initial attempt to develop a broadly reactive therapeutic vaccine, we designed a putative papillomavirus (PV) ancestor antigen (circulating sequence derived antigenic sequences E1E2-CDSE1E2) based on the conserved E1 and E2 protein sequences from existing oncogenic HPV strains. This antigen was found to be as related to circulating oncogenic Macaca fascicularis papillomaviruses (MfPVs) as to oncogenic HPVs. The CDSE1E2 antigen was fused to a T-cell adjuvant and encoded in chimpanzee 3 and 63 adenoviral vectors. We first showed that the combination of these 2 vaccines induced long-lasting potent CDSE1E2 specific T cell responses in outbred mice. This prime-boost regimen was then tested in female macaques naturally infected with MfPVs. All immunized animals (16/16) responded to the vaccine antigen but with reduced cross-reactivity against existing PVs. Preexisting MfPV infections did not prime vaccine inducible immune responses. Importantly, immunized oncogenic MfPV type 3 (MfPV3) infected animals that responded toward MfPV3 were able to diminish cervical MfPV3 DNA content. Although insufficient breadth was achieved, our results suggest that a relevant level of E1E2 specific T cell immunity is achievable and might be sufficient for the elimination of PV infection. Importantly, naturally infected macaques, offer a relevant model for testing vaccines aimed at eliminating mucosal PV infections.

The oncolytic herpes simplex virus (HSV) that has been approved for clinical practice and those HSVs in clinical trials are attenuated viruses, often with the neurovirulence gene γ 1 34.5 and additional genes deleted. One strategy to engineer nonattenuated oncolytic HSVs consists of retargeting the viral tropism to a cancer-specific receptor of choice, exemplified by HER2 (human epidermal growth factor receptor 2), which is present in breast, ovary, and other cancers, and in detargeting from the natural receptors. Because the HER2-retargeted HSVs strictly depend on this receptor for infection, the viruses employed in preclinical studies were cultivated in HER2-positive cancer cells. The production of clinical-grade viruses destined for humans should avoid the use of cancer cells. Here, we engineered the R-213 recombinant, by insertion of a 20-amino-acid (aa) short peptide (named GCN4) in the gH of R-LM113; this recombinant was retargeted to HER2 through insertion in gD of a single-chain antibody (scFv) to HER2. Next, we generated a Vero cell line expressing an artificial receptor (GCN4R) whose N terminus consists of an scFv to GCN4 and therefore is capable of interacting with GCN4 present in gH of R-213. R-213 replicated as well as R-LM113 in SK-OV-3 cells, implying that addition of the GCN4 peptide was not detrimental to gH. R-213 grew to relatively high titers in Vero-GCN4R cells, efficiently spread from cell to cell, and killed both Vero-GCN4R and SK-OV-3 cells, as expected for an oncolytic virus. Altogether, Vero-GCN4R cells represent an efficient system for cultivation of retargeted oncolytic HSVs in non-cancer cells. IMPORTANCE There is growing interest in viruses as oncolytic agents, which can be administered in combination with immunotherapeutic compounds, including immune checkpoint inhibitors. The oncolytic HSV approved for clinical practice and those in clinical trials are attenuated viruses. An alternative to attenuation is a cancer specificity achieved by tropism retargeting to selected cancer receptors. However, the retargeted oncolytic HSVs strictly depend on cancer receptors for infection. Here, we devised a strategy for in vitro cultivation of retargeted HSVs in non-cancer cells. The strategy envisions a double-retargeting approach: one retargeting is via gD to the cancer receptor, and the second retargeting is via gH to an artificial receptor expressed in Vero cells. The double-retargeted HSV uses alternatively the two receptors to infect cancer cells or producer cells. A universal non-cancer cell line for growth of clinical-grade retargeted HSVs represents a step forward in the translational phase.

In recent years, replication-defective chimpanzee-derived adenoviruses have been extensively evaluated as genetic vaccines. These vectors share desirable properties with human adenoviruses like the broad tissue tropism and the ease of large-scale manufacturing. Additionally, chimpanzee adenoviruses have the advantage to overcome the negative impact of pre-existing anti-human adenovirus immunity. Areas covered: Here the authors review current pre-clinical research and clinical trials that utilize chimpanzee-derived adenoviral vectors as vaccines. A wealth of studies are ongoing to evaluate different vector backbones and administration routes with the aim of improving immune responses. The challenges associated with the identification of an optimal chimpanzee vector and immunization strategies for different immunological outcomes will be discussed. Expert commentary: The demonstration that chimpanzee adenoviruses can be safely used in humans has paved the way to the use of a whole new array of vectors of different serotypes. However, so far no predictive signature of vector immunity in humans has been identified. The high magnitude of T cell responses elicited by chimpanzee adenoviruses has allowed dissecting the qualitative aspects that may be important for protective immunity. Ultimately, only the results from the most clinically advanced products will help establish the efficacy of the vaccine vector platform in the field of disease prevention.

Hepatitis C virus (HCV)-induced endstage liver disease is currently a major indication for liver transplantation. After transplantation the donor liver inevitably becomes infected with the circulating virus. Monoclonal antibodies (mAbs) against the HCV coreceptor scavenger receptor class B type I (SR-BI) inhibit HCV infection of different genotypes, both in cell culture and in humanized mice. Anti-SR-BI mAb therapy is successful even when initiated several days after HCV exposure, supporting its potential applicability to prevent HCV reinfection of liver allografts. However, HCV variants with reduced SR-BI dependency have been described in the literature, which could potentially limit the use of SR-BI targeting therapy. In this study we show, both in a preventative and postexposure setting, that humanized mice infected with HCV variants exhibiting increased in vitro resistance to SR-BI-targeting molecules remain responsive to anti-SR-BI mAb therapy in vivo. A 2-week antibody therapy readily cleared HCV RNA from the circulation of infected humanized mice. We found no evidence supporting increased SR-BI-receptor dependency of viral particles isolated from humanized mice compared to cell culture-produced virus. However, we observed that, unlike wild-type virus, the in vitro infectivity of the resistant variants was inhibited by both human high density lipoprotein (HDL) and very low density lipoprotein (VLDL). The combination of mAb1671 with these lipoproteins further increased the antiviral effect. Conclusion: HCV variants that are less dependent on SR-BI in vitro can still be efficiently blocked by an anti-SR-BI mAb in humanized mice. Since these variants are also more susceptible to neutralization by anti-HCV envelope antibodies, their chance of emerging during anti-SR-BI therapy is severely reduced. Our data indicate that anti-SR-BI receptor therapy could be an effective way to prevent HCV infection in a liver transplant setting. (Hepatology 2014;60:1508–1518)

Recombinant adenoviral vectors (rAds) are the most potent recombinant vaccines for eliciting CD8+ T cell–mediated immunity in humans; however, prior exposure from natural adenoviral infection can decrease such responses. In this study we show low seroreactivity in humans against simian- (sAd11, sAd16) or chimpanzee-derived (chAd3, chAd63) compared with human-derived (rAd5, rAd28, rAd35) vectors across multiple geographic regions. We then compared the magnitude, quality, phenotype, and protective capacity of CD8+ T cell responses in mice vaccinated with rAds encoding SIV Gag. Using a dose range (1 × 107–109 particle units), we defined a hierarchy among rAd vectors based on the magnitude and protective capacity of CD8+ T cell responses, from most to least, as: rAd5 and chAd3, rAd28 and sAd11, chAd63, sAd16, and rAd35. Selection of rAd vector or dose could modulate the proportion and/or frequency of IFN-γ+TNF-α+IL-2+ and KLRG1+CD127−CD8+ T cells, but strikingly ∼30–80% of memory CD8+ T cells coexpressed CD127 and KLRG1. To further optimize CD8+ T cell responses, we assessed rAds as part of prime-boost regimens. Mice primed with rAds and boosted with NYVAC generated Gag-specific responses that approached ∼60% of total CD8+ T cells at peak. Alternatively, priming with DNA or rAd28 and boosting with rAd5 or chAd3 induced robust and equivalent CD8+ T cell responses compared with prime or boost alone. Collectively, these data provide the immunologic basis for using specific rAd vectors alone or as part of prime-boost regimens to induce CD8+ T cells for rapid effector function or robust long-term memory, respectively.

Long-acting injectable antiretroviral (LA-ARV) drugs with low toxicity profiles and propensity for drug-drug interactions are a goal for future ARV regimens. C34-PEG4-Chol is a novel cholesterol tagged LA HIV-fusion-inhibitor (FI). We assessed pre-clinical toxicology and first-in-human administration of C34-PEG4-Chol. Pre-clinical toxicology was conducted in 2 species. HIV-positive men were randomised to a single subcutaneous dose of C34-PEG4-Chol at incrementing doses or placebo. Detailed clinical (including injection site reaction (ISR) grading), plasma pharmacokinetic (time-to-minimum-effective-concentration (MEC, 25 ng/mL) and pharmacodynamic (plasma HIV RNA) parameters were assessed. In both mice and dogs, no-observed-adverse effect level (NOAEL) was observed at a 12 mg/kg/dose after two weeks. Of 5 men enrolled, 3 received active drug (10 mg, 10 mg and 20 mg). In 2 individuals grade 3 ISR occurred and the study was halted. Both ISR emerged within 12 hours of active drug dosing. No systemic toxicities were observed. The time-to-MEC was >72 and >96 hours after 10 and 20 mg dose, respectively, and mean change in HIV RNA was −0.9 log10 copies/mL. These human pharmacodynamic and pharmacokinetic data, although limited to 3 subjects, of C34-PEG-4-Chol suggest continuing evaluation of this agent as a LA-ARV. However, alternative administration routes must be explored.

Malaria remains a significant global health burden and a vaccine would make a substantial contribution to malaria control. Chimpanzee Adenovirus 63 Modified Vaccinia Ankara Multiple epitope thrombospondin adhesion protein (ME-TRAP) and vaccination has shown significant efficacy against malaria sporozoite challenge in malaria-naive European volunteers and against malaria infection in Kenyan adults. Infants are the target age group for malaria vaccination; however, no studies have yet assessed T-cell responses in children and infants. We enrolled 138 Gambian and Burkinabe children in four different age-groups: 2-6 years old in The Gambia; 5-17 months old in Burkina Faso; 5-12 months old, and also 10 weeks old, in The Gambia; and evaluated the safety and immunogenicity of Chimpanzee Adenovirus 63 Modified Vaccinia Ankara ME-TRAP heterologous prime-boost immunization. The vaccines were well tolerated in all age groups with no vaccine-related serious adverse events. T-cell responses to vaccination peaked 7 days after boosting with Modified Vaccinia Ankara, with T-cell responses highest in 10 week-old infants. Heterologous prime-boost immunization with Chimpanzee Adenovirus 63 and Modified Vaccinia Ankara ME-TRAP was well tolerated in infants and children, inducing strong T-cell responses. We identify an approach that induces potent T-cell responses in infants, which may be useful for preventing other infectious diseases requiring cellular immunity.

Malaria is a major health problem as nearly half of the human population is exposed to this parasite causing around 600 million clinical cases annually. Prime-boost regimes using simian adenoviral vectors and MVA expressing the clinically relevant Plasmodium falciparum ME.TRAP antigen have shown outstanding protective efficacy in mouse models. We now extend those observations to macaque monkeys. Immunisation with AdCh63 elicited a median response of 869 IFN-γ SFC/million PBMCs to ME.TRAP and responses were boosted by MVA to reach 5256 SFC/million PBMCs, increasing at the same time the breadth of the T cell responses to cover the complete ME.TRAP antigen. Intramuscular vaccination was more immunogenic than the intradermal route, and MVA could be used repeatedly for up to 3 times to boost adenovirus-primed responses. An interval of 16 weeks between repeated MVA injections was optimal to enhance cytokine production by T cells and improve the CD8 multifunctional responses. Antibodies to TRAP were exceptionally high and maintained for a long period of time after the prime-boost regime. These results in non-human primates highlight the potential of this vaccination regime and encourage its future use in clinical trials. © 2010 Elsevier Ltd.

Replication defective Adenovirus vectors based on the human serotype 5 (Ad5) have been shown to induce protective immune responses against diverse pathogens and cancer in animal models and to elicit robust and sustained cellular immunity in humans. However, most humans have anti-Ad5 neutralising antibodies that can impair the immunological potency of such vaccines. Here we show that most other human Adenoviruses from rare serotypes are far less potent as vaccine vectors than Ad5 in mice and non-human primates, casting doubt on their potential efficacy in humans. To identify novel vaccine carriers suitable for vaccine delivery in humans we isolated and sequenced over a thousand Adenovirus strains from chimpanzees (ChAd). Replication-defective vectors were generated from different ChAd serotypes and were screened for neutralization by human sera and for ability to grow in human cell lines already approved for clinical studies. Most importantly, we devised a screening strategy to rank the ChAd vectors by immunological potency in mice which predicts their immunogenicity in non-human primates and humans. The vectors studied varied by up to a thousand-fold in potency for CD8 T cell induction in mice. Two of the most potent ChAd vectors were selected for clinical studies as carriers for Malaria and Hepatitis C virus (HCV) genetic vaccines. These ChAd vectors were found to be safe and immunologically potent in Phase I clinical trials thereby validating our screening approach. The ChAd vectors that we have developed represent a large collection of non cross-reactive, potent vectors that can be exploited for diverse vaccine strategies.

Respiratory syncytial virus (RSV) causes respiratory infection in annual epidemics, with infants and the elderly at particular risk of developing severe disease and death. However, despite its importance, no vaccine exists. The chimpanzee adenovirus, PanAd3-RSV, and modified vaccinia virus Ankara, MVA-RSV, are replication-defective viral vectors encoding the RSV fusion (F), nucleocapsid (N), and matrix (M2-1) proteins for the induction of humoral and cellular responses. We performed an open-label, dose escalation, phase 1 clinical trial in 42 healthy adults in which four different combinations of prime/boost vaccinations were investigated for safety and immunogenicity, including both intramuscular (IM) and intranasal (IN) administration of the adenovirus-vectored vaccine. The vaccines were safe and well tolerated, with the most common reported adverse events being mild injection site reactions. No vaccine-related serious adverse events occurred. RSV neutralizing antibody titers rose in response to IM prime with PanAd3-RSV and after IM boost for individuals primed by the IN route. Circulating anti-F immunoglobulin G (IgG) and IgA antibody-secreting cells (ASCs) were observed after the IM prime and IM boost. RSV-specific T cell responses were increased after the IM PanAd3-RSV prime and were most efficiently boosted by IM MVA-RSV. Interferon-γ (IFN-γ) secretion after boost was from both CD4(+) and CD8(+) T cells, without detectable T helper cell 2 (TH2) cytokines that have been previously associated with immune pathogenesis following exposure to RSV after the formalin-inactivated RSV vaccine. In conclusion, PanAd3-RSV and MVA-RSV are safe and immunogenic in healthy adults. These vaccine candidates warrant further clinical evaluation of efficacy to assess their potential to reduce the burden of RSV disease.

Efficacy trials of antibody-inducing protein-in-adjuvant vaccines targeting the blood-stage Plasmodium falciparum malaria parasite have so far shown disappointing results. The induction of cell-mediated responses in conjunction with antibody responses is thought to be one alternative strategy that could achieve protective efficacy in humans. Here, we prepared chimpanzee adenovirus 63 (ChAd63) and modified vaccinia virus Ankara (MVA) replication-deficient vectors encoding the well-studied P. falciparum blood-stage malaria antigen merozoite surface protein 1 (MSP1). A phase Ia clinical trial was conducted in healthy adults of a ChAd63-MVA MSP1 heterologous prime-boost immunization regime. The vaccine was safe and generally well tolerated. Fewer systemic adverse events (AEs) were observed following ChAd63 MSP1 than MVA MSP1 administration. Exceptionally strong T-cell responses were induced, and these displayed a mixed of CD4 and CD8 phenotype. Substantial MSP1-specific serum immunoglobulin G (IgG) antibody responses were also induced, which were capable of recognizing native parasite antigen, but these did not reach titers sufficient to neutralize P. falciparum parasites in vitro. This viral vectored vaccine regime is thus a leading approach for the induction of strong cellular and humoral immunogenicity against difficult disease targets in humans. Further studies are required to assess whether this strategy can achieve protective efficacy against blood-stage malaria infection. © The American Society of Gene and Cell Therapy.

Objective: Direct-acting antivirals (DAAs) inhibit hepatitis C virus (HCV) infection by targeting viral proteins that play essential roles in the replication process. However, selection of resistance-associated variants (RAVs) during DAA therapy has been a cause of therapeutic failure. In this study, we wished to address whether such RAVs could be controlled by the co-administration of host-targeting entry inhibitors that prevent intrahepatic viral spread. Design: We investigated the effect of adding an entry inhibitor (the anti-scavenger receptor class B type I mAb1671) to a DAA monotherapy (the protease inhibitor ciluprevir) in human-liver mice chronically infected with HCV of genotype 1b. Clinically relevant non-laboratory strains were used to achieve viraemia consisting of a cloud of related viral variants (quasispecies) and the emergence of RAVs was monitored at high resolution using next-generation sequencing. Results: HCV-infected human-liver mice receiving DAA monotherapy rapidly experienced on-therapy viral breakthrough. Deep sequencing of the HCV protease domain confirmed the manifestation of drug-resistant mutants upon viral rebound. In contrast, none of the mice treated with a combination of the DAA and the entry inhibitor experienced on-therapy viral breakthrough, despite detection of RAV emergence in some animals. Conclusions: This study provides preclinical in vivo evidence that addition of an entry inhibitor to an anti-HCV DAA regimen restricts the breakthrough of DAA-resistant viruses. Our approach is an excellent strategy to prevent therapeutic failure caused by on-therapy rebound of DAA-RAVs. Inclusion of an entry inhibitor to the newest DAA combination therapies may further increase response rates, especially in difficult-to-treat patient populations.

BACKGROUND\ud The West African outbreak of Ebola virus disease that peaked in 2014 has caused more than 11,000 deaths. The development of an effective Ebola vaccine is a priority for control of a future outbreak.\ud \ud METHODS\ud In this phase 1 study, we administered a single dose of the chimpanzee adenovirus 3 (ChAd3) vaccine encoding the surface glycoprotein of Zaire ebolavirus (ZEBOV) to 60 healthy adult volunteers in Oxford, United Kingdom. The vaccine was administered in three dose levels — 1×1010 viral particles, 2.5×1010 viral particles, and 5×1010 viral particles — with 20 participants in each group. We then assessed the effect of adding a booster dose of a modified vaccinia Ankara (MVA) strain, encoding the same Ebola virus glycoprotein, in 30 of the 60 participants and evaluated a reduced prime–boost interval in another 16 participants. We also compared antibody responses to inactivated whole Ebola virus virions and neutralizing antibody activity with those observed in phase 1 studies of a recombinant vesicular stomatitis virus–based vaccine expressing a ZEBOV glycoprotein (rVSV-ZEBOV) to determine relative potency and assess durability.\ud \ud RESULTS\ud No safety concerns were identified at any of the dose levels studied. Four weeks after immunization with the ChAd3 vaccine, ZEBOV-specific antibody responses were similar to those induced by rVSV-ZEBOV vaccination, with a geometric mean titer of 752 and 921, respectively. ZEBOV neutralization activity was also similar with the two vaccines (geometric mean titer, 14.9 and 22.2, respectively). Boosting with the MVA vector increased virus-specific antibodies by a factor of 12 (geometric mean titer, 9007) and increased glycoprotein-specific CD8+ T cells by a factor of 5. Significant increases in neutralizing antibodies were seen after boosting in all 30 participants (geometric mean titer, 139; P

The use of adenovirus (Ad) as vaccine vectors is hindered by pre-existing immunity to human Ads in most of the human population. In order to overcome this limitation, uncommon alternative Ad serotypes need to be utilized, In this study, an E1-E3 deleted recombinant Ad based on the chimpanzee serotype 3 (ChAd3) was engineered to express human carcinoembryonic antigen (CEA) protein or rat neu extracellular/transmembrane domains (ECD.TM). ChAd3 vectors were tested in CEA transgenic (CEA.Tg) and BALB/NeuT mice, which show immunologic tolerance to these antigens. ChAd3 is capable of inducing an immune response comparable to that of hAd5 serotype-based vectors, thus breaking tolerance to tumor associated antigens (TAAs) and achieving anti-tumor effects. Of importance is that ChAd3 can overcome hAd5 pre-existing immunity and work in conjunction with DNA electroporation (DNA-EP) and other Ad vaccines based on common human serotypes. (C) 2009 Elsevier Ltd. All rights reserved.

Background The unprecedented 2014 epidemic of Ebola virus disease (EVD) has prompted an international response to accelerate the availability of a preventive vaccine. A replicationdefective recombinant chimpanzee adenovirus type 3–vectored ebolavirus vaccine (cAd3-EBO), encoding the glycoprotein from Zaire and Sudan species that offers protection in the nonhuman primate model, was rapidly advanced into phase 1 clinical evaluation. Methods We conducted a phase 1, dose-escalation, open-label trial of cAd3-EBO. Twenty healthy adults, in sequentially enrolled groups of 10 each, received vaccination intramuscularly in doses of 2×10 10 particle units or 2×10 11 particle units. Primary and secondary end points related to safety and immunogenicity were assessed throughout the first 4 weeks after vaccination. Results In this small study, no safety concerns were identified; however, transient fever developed within 1 day after vaccination in two participants who had received the 2×10 11 particle-unit dose. Glycoprotein-specific antibodies were induced in all 20 participants; the titers were of greater magnitude in the group that received the 2×10 11 particle-unit dose than in the group that received the 2×10 10 particle-unit dose (geometric mean titer against the Zaire antigen, 2037 vs. 331; P = 0.001). Glycoproteinspecific T-cell responses were more frequent among those who received the 2x10 11 particle-unit dose than among those who received the 2×10 10 particle-unit dose, with a CD4 response in 10 of 10 participants versus 3 of 10 participants (P = 0.004) and a CD8 response in 7 of 10 participants versus 2 of 10 participants (P = 0.07). Conclusions Reactogenicity and immune responses to cAd3-EBO vaccine were dose-dependent. At the 2×10 11 particle-unit dose, glycoprotein Zaire–specific antibody responses were in the range reported to be associated with vaccine-induced protective immunity in challenge studies involving nonhuman primates. Clinical trials assessing cAd3-EBO are ongoing. (Funded by the Intramural Research Program of the National Institutes of Health; VRC 207 ClinicalTrials.gov number, NCT02231866.)

Human respiratory syncytial virus (HRSV) is a major cause of lower respiratory tract disease in children and the elderly for which there is still no effective vaccine. We have previously shown that PanAd3-RSV, which is a chimpanzee adenovirus-vectored vaccine candidate that expresses a secreted form of the HRSV F protein together with the N and M2-1 proteins of HRSV, is immunogenic in rodents and nonhuman primates, and protects mice and cotton rats from HRSV challenge. Because the extent to which protection demonstrated in rodent models will translate to humans is unclear, we have exploited the calf model of bovine RSV (BRSV) infection, which mimics HRSV disease in children more closely than do experimental models of unnatural laboratory hosts, to evaluate the safety and efficacy of the PanAd3-RSV vaccine. We show that PanAd3-RSV alone and in combination with a modified vaccinia Ankara expressing the same HRSV antigens (MVA-RSV) induced neutralizing antibodies and cellular immunity in young seronegative calves and protected against upper and lower respiratory tract infection and pulmonary disease induced by heterologous BRSV challenge. There was no evidence either of enhanced pulmonary pathology or of enhanced respiratory disease in vaccinated calves after BRSV challenge. These findings support the continued evaluation of the vectored RSV vaccines in man.

Malaria transmission-blocking vaccines (TBVs) target the development of Plasmodium parasites within the mosquito, with the aim of preventing malaria transmission from one infected individual to another. Different vaccine platforms, mainly protein-in-adjuvant formulations delivering the leading candidate antigens, have been developed independently and have reported varied transmission-blocking activities (TBA). Here, recombinant chimpanzee adenovirus 63, ChAd63 and modified vaccinia virus Ankara, MVA, expressing AgAPN1, Pfs230-C, Pfs25 and Pfs48/45 were generated. Antibody responses primed individually against all antigens by ChAd63 immunization in BALB/c mice were boosted by the administration of MVA expressing the same antigen. These antibodies exhibited a hierarchy of inhibitory activity against the NF54 laboratory strain of P. falciparum in Anopheles stephensi mosquitoes using the standard membrane feeding assay (SMFA), with anti-Pfs230-C and anti-Pfs25 antibodies giving complete blockade. The observed rank order of inhibition was replicated against P. falciparum African field isolates in A. gambiae in direct membrane feeding assays (DMFA). TBA achieved was IgG concentration dependent. This study provides the first head-to-head comparative analysis of leading antigens using two different parasite sources in two different vector species and can be used to guide selection of TBVs for future clinical development using the viral-vectored delivery platform.

Objectives Plasmodium falciparum sporozoites, deposited in the skin by infected Anopheles mosquitoes taking a blood meal, cross the endothelium of skin capillaries and travel to the liver where they traverse Kupffer cells and hepatocytes to finally invade a small number of the latter. In hepatocytes, sporozoites replicate, differentiate and give rise to large numbers of merozoites that are released into the bloodstream where they invade red blood cells, thus initiating the symptomatic blood stage. Using in vitro systems and rodent models, it has been shown that the hepatocyte receptors CD81 and scavenger receptor type B class I (SR-BI) play a pivotal role during sporozoite invasion. We wanted to evaluate whether these two entry factors are genuine drug targets for the prevention of P. falciparum infection in humans. Methods Immunodeficient mice of which the liver is largely repopulated by human hepatocytes were treated with monoclonal antibodies blocking either CD81 or SR-BI 1 day prior to challenge with infected mosquitoes. P. falciparum infection of the liver was demonstrated using a qPCR assay. Results In human liver chimeric mice, an antibody directed against CD81 completely blocked P. falciparum sporozoite invasion while SR-BI-specific monoclonal antibodies did not influence in vivo infection. Conclusions These observations confirm the role of CD81 in liver-stage malaria and question that of SR-BI. CD81 might be a valuable drug target for the prevention of malaria.

Efficient vaccination against viral agents requires a strong T-cell-mediated immune response to clear viral-infected cells. Optimal vaccination can be achieved by administration of recombinant viral vectors encoding phatogen antigens. Adenoviral vectors have attracted considerable attention as potential viral vectors for genetic vaccination owing to their favorable safety profile and potent transduction efficiency following intramuscular injection. However, the neutralizing antibody response against adenoviral capsid proteins following adenoviral vectors injection limits the success of vaccination protocols based on multiple administrations of the same adenoviral serotype. In this work, we describe efficient immunization of rhesus macaques, the preferred model for preclinical assessment, with an HCV candidate vaccine by heterologous priming-boosting with adenoviral vectors based on different serotypes. The induced responses are broad and show significant cross-strain reactivity. Boosting can be delayed for over 2 years after priming, indicating that there is long-term maintenance of resting memory cells.

Background Intramuscular plasmid injection followed by electroporation is an efficient method for gene therapy or vaccination. Several protocols have been described that give good transduction. levels with several reporter genes. Methods In this work we have explored the efficiency of gene delivery upon variation of the different electrical parameters such as pulse length frequency and voltage monitoring both on short- and long-term protein production. Results Having defined the best performing parameters, we have designed a short electric treatment that gives good levels of plasmid-encoded protein in different species such as mice, rabbits and monkeys. Copyright (C) 2005 John Wiley & Sons, Ltd.

We discovered that the hepatitis C virus (HCV) envelope glycoprotein E2 binds to human hepatoma cell lines independently of the previously proposed HCV receptor CD81. Comparative binding studies using recombinant E2 from the most prevalent 1a and 1b genotypes revealed that E2 recognition by hepatoma cells is independent from the viral isolate, while E2–CD81 interaction is isolate specific. Binding of soluble E2 to human hepatoma cells was impaired by deletion of the hypervariable region 1 (HVR1), but the wild-type phenotype was recovered by introducing a compensatory mutation reported previously to rescue infectivity of an HVR1-deleted HCV infectious clone. We have identified the receptor responsible for E2 binding to human hepatic cells as the human scavenger receptor class B type I (SR-BI). E2–SR-BI interaction is very selective since neither mouse SR-BI nor the closely related human scavenger receptor CD36, were able to bind E2. Finally, E2 recognition by SR-BI was competed out in an isolate-specific manner both on the hepatoma cell line and on the human SR-BI-transfected cell line by an anti-HVR1 monoclonal antibody.

Despite the recent progress in the development of new antiviral agents, hepatitis C virus (HCV) infection remains a major global health problem, and there is a need for a preventive vaccine. We previously reported that adenoviral vectors expressing HCV nonstructural proteins elicit protective T cell responses in chimpanzees and were immunogenic in healthy volunteers. Furthermore, recombinant HCV E1E2 protein formulated with adjuvant MF59 induced protective antibody responses in chimpanzees and was immunogenic in humans. To develop an HCV vaccine capable of inducing both T cell and antibody responses, we constructed adenoviral vectors expressing full-length and truncated E1E2 envelope glycoproteins from HCV genotype 1b. Heterologous prime-boost immunization regimens with adenovirus and recombinant E1E2 glycoprotein (genotype 1a) plus MF59 were evaluated in mice and guinea pigs. Adenovirus prime and protein boost induced broad HCV-specific CD8 + and CD4 + T cell responses and functional Th1-type IgG responses. Immune sera neutralized luciferase reporter pseudoparticles expressing HCV envelope glycoproteins (HCVpp) and a diverse panel of recombinant cell culture-derived HCV (HCVcc) strains and limited cell-to-cell HCV transmission. This study demonstrated that combining adenovirus vector with protein antigen can induce strong antibody and T cell responses that surpass immune responses achieved by either vaccine alone. IMPORTANCE HCV infection is a major health problem. Despite the availability of new directly acting antiviral agents for treating chronic infection, an affordable preventive vaccine provides the best long-term goal for controlling the global epidemic. This report describes a new anti-HCV vaccine targeting the envelope viral proteins based on adenovirus vector and protein in adjuvant. Rodents primed with the adenovirus vaccine and boosted with the adjuvanted protein developed cross-neutralizing antibodies and potent T cell responses that surpassed immune responses achieved with either vaccine component alone. If combined with the adenovirus vaccine targeting the HCV NS antigens now under clinical testing, this new vaccine might lead to a stronger and broader immune response and to a more effective vaccine to prevent HCV infection. Importantly, the described approach represents a valuable strategy for other infectious diseases in which both T and B cell responses are essential for protection.

Memory CD8+ T cells generated by spontaneous resolution of hepatitis C virus (HCV) infection rapidly control secondary infections and reduce the risk of virus persistence. Here, CD8+ T-cell immunity and response to reinfection were assessed in a chimpanzee cured of an earlier chronic infection with an interferon (IFN)-free antiviral regimen. CD8+ T cells expanded from liver immediately before and 2 years after cure of chronic infection with two direct-acting antivirals (DAAs) targeted epitopes in the E2, nonstructural (NS)5a, and NS5b proteins. A second infection to assess CD8+ T-cell responsiveness resulted in rapid suppression of HCV replication by week 2, but viremia rebounded 3 weeks later and the infection persisted. The E2, NS5a, and NS5b proteins remained dominant CD8+ T-cell targets after reinfection. Resurgent HCV replication was temporally associated with mutational escape of NS5a and NS5b class I epitopes that had also mutated during the first chronic infection. Two epitopes in E2 remained intact throughout both persistent infections. Intrahepatic CD8+ T cells targeting intact and escape-prone epitopes differed in expression of phenotypic markers of functional exhaustion 2 years after successful DAA therapy and in the capacity to expand in liver upon reinfection.The intrahepatic HCV-specific CD8+ T-cell repertoire established during chronic infection was narrowly focused, but very stable, after cure with DAA. Existing intrahepatic CD8+ T cells targeting dominant epitopes of the challenge virus failed to prevent persistence. Vaccination after DAA cure may be necessary to broaden T-cell responses and reduce the risk of a second persistent infection.

Antivector immunity may limit the immunogenicity of adenovirus vector vaccines. We tested sera from individuals immunized with adenovirus type 4 and 7 (Ad4 and Ad7, respectively) vaccine or naturally infected with Ad4 for their ability to neutralize a panel of E1-deleted human and chimpanzee adenoviruses (ChAd). Small statistically significant increases in titers to ChAd63, ChAd3, human Ad35, and human Ad5 were observed. Neutralizing antibodies elicited by Ad4 infection or immunization results in a small amount of adenovirus cross-reactivity.

A protective vaccine against hepatitis C virus (HCV) remains an unmet clinical need. HCV infects millions of people worldwide and is a leading cause of liver cirrhosis and hepatocellular cancer. Animal challenge experiments, immunogenetics studies, and assessment of host immunity during acute infection highlight the critical role that effective T cell immunity plays in viral control. In this first-in-man study, we have induced antiviral immunity with functional characteristics analogous to those associated with viral control in natural infection, and improved upon a vaccine based on adenoviral vectors alone. We assessed a heterologous prime-boost vaccination strategy based on a replicative defective simian adenoviral vector (ChAd3) and modified vaccinia Ankara (MVA) vector encoding the NS3, NS4, NS5A, and NS5B proteins of HCV genotype 1b. Analysis used single-cell mass cytometry and human leukocyte antigen class I peptide tetramer technology in healthy human volunteers. We show that HCV-specific T cells induced by ChAd3 are optimally boosted with MVA, and generate very high levels of both CD8(+) and CD4(+) HCV-specific T cells targeting multiple HCV antigens. Sustained memory and effector T cell populations are generated, and T cell memory evolved over time with improvement of quality (proliferation and polyfunctionality) after heterologous MVA boost. We have developed an HCV vaccine strategy, with durable, broad, sustained, and balanced T cell responses, characteristic of those associated with viral control, paving the way for the first efficacy studies of a prophylactic HCV vaccine.

Despite viral vectors being potent inducers of antigen-specific T cells, strategies to further improve their immunogenicity are actively pursued. Of the numerous approaches investigated, fusion of the encoded antigen to major histocompatibility complex class II-associated invariant chain (Ii) has been reported to enhance CD8(+) T-cell responses. We have previously shown that adenovirus vaccine encoding nonstructural (NS) hepatitis C virus (HCV) proteins induces potent T-cell responses in humans. However, even higher T-cell responses might be required to achieve efficacy against different HCV genotypes or therapeutic effect in chronically infected HCV patients. In this study, we assessed fusion of the HCV NS antigen to murine and human Ii expressed by the chimpanzee adenovirus vector ChAd3 or recombinant modified vaccinia Ankara in mice and nonhuman primates (NHPs). A dramatic increase was observed in outbred mice in which vaccination with ChAd3 expressing the fusion antigen resulted in a 10-fold increase in interferon-γ(+) CD8(+) T cells. In NHPs, CD8(+) T-cell responses were enhanced and accelerated with vectors encoding the Ii-fused antigen. These data show for the first time that the enhancement induced by vector vaccines encoding li-fused antigen was not species specific and can be translated from mice to NHPs, opening the way for testing in humans.

Ebolavirus disease causes high mortality, and the current outbreak has spread unabated through West Africa. Human adenovirus type 5 vectors (rAd5) encoding ebolavirus glycoprotein (GP) generate protective immunity against acute lethal Zaire ebolavirus (EBOV) challenge in macaques, but fail to protect animals immune to Ad5, suggesting natural Ad5 exposure may limit vaccine efficacy in humans. Here we show that a chimpanzee-derived replication-defective adenovirus (ChAd) vaccine also rapidly induced uniform protection against acute lethal EBOV challenge in macaques. Because protection waned over several months, we boosted ChAd3 with modified vaccinia Ankara (MVA) and generated, for the first time, durable protection against lethal EBOV challenge.

We describe an improved genetic immunization strategy for eliciting a full spectrum of anti-hepatitis C virus (HCV) envelope 2 (E2) glycoprotein responses in mammals through electrical gene transfer (EGT) of plasmid DNA into muscle fibers. Intramuscular injection of a plasmid encoding a cross-reactive hypervariable region 1 (HVR1) peptide mimic fused at the N terminus of the E2 ectodomain, followed by electrical stimulation treatment in the form of high-frequency, low-voltage electric pulses, induced more than 10-fold-higher expression levels in the transfected mouse tissue. As a result of this substantial increment of in vivo antigen production, the humoral response induced in mice, rats, and rabbits ranged from 10- to 30-fold higher than that induced by conventional naked DNA immunization. Consequently, immune sera from EGT-treated mice displayed a broader cross-reactivity against HVR1 variants from natural isolates than sera from injected animals that were not subjected to electrical stimulation. Cellular response against E2 epitopes specific for helper and cytotoxic T cells was significantly improved by EGT. The EGT-mediated enhancement of humoral and cellular immunity is antigen independent, since comparable increases in antibody response against ciliary neurotrophic factor or in specific anti-human immunodeficiency virus type 1 gag CD8+T cells were obtained in rats and mice. Thus, the method described potentially provides a safe, low-cost treatment that may be scaled up to humans and may hold the key for future development of prophylactic or therapeutic vaccines against HCV and other infectious diseases.

Hepatitis C virus (HCV) glycoprotein E2 binds to human cells by interacting with the CD81 molecule, which has been proposed to be the viral receptor. A correlation between binding to CD81 and species permissiveness to HCV infection has also been reported. We have determined the sequence of CD81 from the tamarin, a primate species known to be refractory to HCV infection. Tamarin CD81 (t-CD81) differs from the human molecule at 5 amino acid positions (155, 163, 169, 180, and 196) within the large extracellular loop (LEL), where the binding site for E2 has been located. Soluble recombinant forms of human CD81 (h-CD81), t-CD81, and African green monkey CD81 (agm-CD81) LEL molecules were analyzed by enzyme-linked immunosorbent assay for binding to E2 glycoprotein. Both h-CD81 and t-CD81 molecules were able to bind E2. Competition experiments showed that the two receptors cross-compete and that the t-CD81 binds with stronger affinity than the human molecule. Recently, h-CD81 residue 186 has been characterized as the critical residue involved in the interaction with E2. Recombinant CD81 mutant proteins were expressed to test whether human and tamarin receptors interacted with E2 in a comparable manner. Mutation of residue 186 (F186L) dramatically reduced the binding capability of t-CD81, a result that has already been demonstrated for the human receptor, whereas the opposite mutation (L186F) in agm-CD81 resulted in a neat gain of binding activity. Finally, the in vitro data were confirmed by detection of E2 binding to cotton-top tamarin (Saguinus oedipus) cell line B95-8 expressing endogenous CD81. These results indicate that the binding of E2 to CD81 is not predictive of an infection-producing interaction between HCV and host cells.

Induction of antigen-specific CD8+ T cells offers the prospect of immunization against many infectious diseases, but no subunit vaccine has induced CD8+ T cells that correlate with efficacy in humans. Here we demonstrate that a replication-deficient chimpanzee adenovirus vector followed by a modified vaccinia virus Ankara booster induces exceptionally high frequency T-cell responses (median >2400 SFC/106 peripheral blood mononuclear cells) to the liver-stage Plasmodium falciparum malaria antigen ME-TRAP. It induces sterile protective efficacy against heterologous strain sporozoites in three vaccinees (3/14, 21%), and delays time to patency through substantial reduction of liver-stage parasite burden in five more (5/14, 36%), P=0.008 compared with controls. The frequency of monofunctional interferon-γ-producing CD8+ T cells, but not antibodies, correlates with sterile protection and delay in time to patency (Pcorrected=0.005). Vaccine-induced CD8+ T cells provide protection against human malaria, suggesting that a major limitation of previous vaccination approaches has been the insufficient magnitude of induced T cells., Induction of protective immunity mediated by CD8+ T cells has been a long sought goal in vaccinology. Here, Ewer et al. report induction of protective efficacy against Plasmodium falciparum malaria in a phase IIa prime-boost vaccine trial where efficacy correlates strongly with induced CD8 T-cell responses.

Highlights • Three novel vaccines were tested in 2 GLP toxicity studies in BALB/c mice. • Vaccines were vectored by DNA, simian adenovirus and modified vaccinia virus Ankara. • All 3 vaccines expressed the same conserved regions of HIV-1. • Intramuscular administration had no systemic toxicity. • All changes were consistent with i.m. delivery and immune response induction., Background The systemic toxicity of three candidate HIV-1 vaccines plasmid pSG2.HIVconsv DNA (D), ChAdV63.HIVconsv (C) and MVA.HIVconsv (M) expressing chimeric immunogen derived from the most conserved regions of the HIV-1 proteome was evaluated in two repeat-dose studies in the male and female BALB/c mice. Methods In study UNO011, mice received three doses of 2 × 107 plaque-forming units of MVA.HIVconsv vaccine (MMM). In study UNO012, mice received 3 doses of 50 μg of pSG2.HIVconsv DNA followed by a single dose of 5.95 × 109 virus particles of ChAdV63.HIVconsv vaccine (DDDC). Similarly constituted control groups received the vehicle alone (phosphate buffered saline) at the same volume-dose. All vaccines were administered by intramuscular needle injection into the right hind limb at 14-day intervals and animals were sacrificed 7 days after the last dose. Assessment of local and systemic toxicity was made. Induction of HIV-1-specific responses was confirmed. Parameters assessed included clinical condition, body weight, food consumption, ophthalmoscopy, haematology, blood chemistry, organ weight and macroscopic and microscopic pathology. Results In both studies, treatment with the candidate vaccines elicited strong HIV-1-specific T-cell responses. The vaccine treatment was well-tolerated without any adverse systemic toxicological changes. The local toxicity findings observed in these studies were consistent with the predicted response to a vaccine/substance administration by intramuscular injection. Conclusions The three novel anti-HIV-1 vaccines were well tolerated when administered by intramuscular injection to BALB/c mice. These results supported an application for authorisation by the Medicines and Healthcare Products Regulatory Agency of the UK to test these vaccines for the first time in phase I clinical trials in healthy both uninfected subjects and HIV-1-infected patients stable on antiretroviral treatment.

Replication-defective chimpanzee adenovirus vectors are emerging as a promising new class of genetic vaccine carriers. Chimpanzee adenovirus vectors have now reached the clinical stage and appear to be endowed with all the properties needed for human vaccine development, including high quality and magnitude of the immune response induced against the encoded antigens, good safety and ease of manufacturing on a large-scale basis. Here the authors review the recent findings of this novel class of adenovirus vectors and compare their properties to other clinical stage vaccine vectors derived from poxvirus, alphavirus and human adenovirus.

A full-length cDNA of the rat liver Nuclear Factor 1 (NF1L21) has been cloned and expressed in S. cerevisiae to analyse the architecture of its activation domain. NF1L21 displays a specific DNA-binding activity, as well as the ability to activate transcription from an artificial NF 1-responsive promoter in yeast. Interaction of two or more NF1L21 molecules with multiple sites on the same promoter activated transcription in a synergistic fashion. Functional analysis of the activation domain of NF1L21 reveals a tripartite structure. Two distinct positive elements are required for NF1L21 -mediated transcription activation. A proline-rich element sandwiched between these two positive domains attenuates their transactivation potential. A shorter NF1L variant (NFlL4) in which the distal positive element is replaced by a different sequence was also isolated. NF1L4 displays the same DNA-binding activity and dimerisation properties as NF1L21, but is unable to activate transcription in yeast.

The construction of new and increasingly diverse libraries, as well as the implementation of more powerful selection schemes, has led to the identification of linear peptides that mimic complex epitopes. Phage display techniques are allowing the selection of disease-related peptides, which reproduce the antigenic and immunogenic properties of natural antigens, using whole sera from patients. The range of applications of phage technology has been extended to include the search for peptides binding to molecules other than antibodies, such as cell receptors and enzymes.

Among approximately 1000 adenoviruses from chimpanzees and bonobos studied recently, the Pan Adenovirus type 3 (PanAd3, isolated from a bonobo, Pan paniscus) has one of the best profiles for a vaccine vector, combining potent transgene immunogenicity with minimal pre-existing immunity in the human population. In this study, we inserted into a replication defective PanAd3 a transgene expressing a fusion protein of conserved influenza antigens nucleoprotein (NP) and matrix 1 (M1). We then studied antibody and T cell responses as well as protection from challenge infection in a mouse model. A single intranasal administration of PanAd3-NPM1 vaccine induced strong antibody and T cell responses, and protected against high dose lethal influenza virus challenge. Thus PanAd3 is a promising candidate vector for vaccines, including universal influenza vaccines.

BACKGROUND & AIMS: T cells are an important component for development of a vaccine against hepatitis C virus (HCV), but little is known about the features of successful vaccine-induced T cells. METHODS: We compared the phenotype, function, and kinetics of vaccine-induced and infection-induced T cells in chimpanzees with HCV infection using multicolor flow cytometry and real-time polymerase chain reaction. RESULTS: In chimpanzees successfully vaccinated with recombinant adenovirus and DNA against HCV NS3-5, HCV-specific T cells appeared earlier, maintained better functionality, and persisted at higher frequencies for a longer time after HCV challenge, than those of mock-vaccinated chimpanzees. Vaccine-induced T cells displayed higher levels of CD127, a marker of memory precursors, and lower levels of programmed death-1 (PD-1) than infection-induced T cells. Vaccine-induced, but not infection-induced, T cells were multifunctional; their ability to secrete interferon gamma and tumor necrosis factor ?? correlated with early expression of CD127 but not PD-1. Based on a comparison of vaccine-induced and infection-induced T cells from the same chimpanzee, the CD127(+) memory precursor phenotype was induced by the vaccine itself rather than by low viremia. In contrast, induction of PD-1 correlated with viremia, and levels of intrahepatic PD-1, PD-L1, and 2,5-OAS-1 messenger RNAs correlated with peak titers of HCV. CONCLUSIONS: Compared with infection, vaccination-induced HCV-specific CD127(+) T cells with high functionality that persisted at higher levels for a longer time. Control of viremia prevented up-regulation of PD-1 on T cells and induction of PD-1, PD-L1, and 2,5-OAS-1 in the liver. Early development of a memory T-cell phenotype and, via control of viremia, attenuation of the inhibitory PD1-PD-L1 pathway might be necessary components of successful vaccine-induced protection against HCV.