Recombinant Adenovirus Serotype Research Articles

Airway basal cells from human-induced pluripotent stem cells: a new frontier in cystic fibrosis research.

Human-induced airway basal cells (hiBCs) derived from human-induced pluripotent stem cells (hiPSCs) offer a promising cell model for studying lung diseases, regenerative medicine, and developing new gene therapy methods. We analyzed existing differentiation protocols and proposed our own protocol for obtaining hiBCs, which involves step-by-step differentiation of hiPSCs into definitive endoderm, anterior foregut endoderm, NKX2.1+ lung progenitors, and cultivation on basal cell medium with subsequent cell sorting using the surface marker CD271 (NGFR). We derived hiBCs from two healthy cell lines and three cell lines with cystic fibrosis (CF). The obtained hiBCs, expressing basal cell markers (NGFR, KRT5, and TP63), could differentiate into lung organoids (LOs). We demonstrated that LOs derived from hiBCs can assess cystic fibrosis transmembrane conductance regulator (CFTR) channel function using the forskolin-induced swelling (FIS) assay. We also carried out non-viral (electroporation) and viral (recombinant adeno-associated virus (rAAV)) serotypes 6 and 9 and recombinant adenovirus (rAdV) serotype 5 transgene delivery to hiBCs and showed that rAAV serotype 6 is most effective against hiBCs, potentially applicable for gene therapy research.

High-Titer Recombinant Adenovirus 26 Vector GMP Manufacturing in HEK 293 Cells with a Stirred Single-Use Bioreactor for COVID-19 Vaccination Purposes.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2 virus) pandemic has shown the importance of pursuing various vaccine manufacturing strategies. In the present study, the HEK 293 cells were infected with recombinant adenovirus serotype 26 (rAd26), and the effects of critical process parameters (CPPs) including viable cell density (VCD) at infection time (0.5 × 106, 0.8 × 106, 1.4 × 106, 1.8 × 106, and 2.5 × 106 cells/mL), the multiplicity of infection (MOI) = 3, 6, 9, 12, and 15, and two aeration strategies (high-speed agitation with a sparging system and low-speed agitation with an overlay system) were investigated experimentally. The results of small-scale experiments in 2 L shake flasks (SF 2L) demonstrated that the initial VCD and MOI could affect the cell proliferation and viability. The results at these experiments showed that VCD = 1.4 × 106 cells/mL and MOI = 9 yielded TCID50 /mL = 108.9, at 72 h post-infection (hpi), while the virus titer at VCD = 0.5 × 106 and 0.8 × 106 cells/mL was lower compared to that of VCD = 1.4 × 106 cells/mL. Moreover, our findings showed that VCDs > 1.8 × 106 cells/m with MOI = 9 did not have a positive effect on TCID50 /mL and MOI = 3 and 6 were less efficient, whereas MOI > 12 decreased the viability drastically. In the next step, the optimized CPPs in a small scale were exploited in a 200 L single-use bioreactor (SUB), with good manufacturing practice (GMP) conditions, at RPM = 25 with an overlay system, yielding high-titer rAd26 manufacturing, i.e., TCID50/mL = 108.9, at 72 hpi.

Method for obtaining recombinant antibodies produced by a cell line transduced with recombinant adenoviruses

Objectives. To develop a technology for obtaining recombinant antibodies in a suspension culture of human HEK293 cells using transduction with recombinant adenovirus serotype 5 (rAd5) carrying genes expressing heavy and light chains of antibodies on the example of two broadspectrum anti-influenza antibodies 27F3 and CR9114.Methods. Ad5-27F3-H, Ad5-CR9114-H, and Ad5-27F3-L recombinant adenoviruses carrying the 27F3 antibody heavy chain gene, CR9114 antibody heavy chain gene, and 27F3 light chain gene, respectively, were generated using the AdEasy™ Adenoviral vector system. To accumulate preparative amounts of recombinant r27F3 and rCR9114 antibodies, the HEK293 suspension cell line was transduced with recombinant adenoviruses carrying genes for heavy and light chains of antibodies. The cells were cultured in a wave-type bioreactor. Chromatography was used to purify recombinant antibodies from the culture medium. After analyzing the molecular weights of purified antibodies using protein electrophoresis, their ability to interact with influenza A and B viruses was analyzed using the Western blot technique, while their ability to neutralize influenza A and B viruses was evaluated using the virus neutralization assay.Results. A method for the accumulation and purification of recombinant r27F3 and CR9114 antibodies from the culture medium of a suspension culture of human cells following transduction with its recombinant adenoviruses carrying the genes for heavy and light chains of these antibodies was developed. The ability of the r27F3 antibody to interact with and neutralize influenza A viruses of group 1 (except influenza A virus subtype H2) and group 2 was shown. The ability of the rCR9114 antibody to interact with influenza A viruses of group 1 and influenza B viruses, as well as to neutralize influenza A viruses of group 1, was demonstrated.Conclusions. A technology for obtaining recombinant antibodies in a suspension culture of HEK293 cells using transduction with recombinant adenoviruses carrying genes expressing heavy and light chains of antibodies was developed along with a confirmation of their specificity.

Lung-Targeted Transgene Expression of Nanocomplexed Ad5 Enhances Immune Response in the Presence of Preexisting Immunity

Recombinant adenovirus serotype 5 (Ad5) vector has been widely applied in vaccine development targeting infectious diseases, such as Ebola virus disease and coronavirus disease 2019 (COVID-19). However, the high prevalence of preexisting anti-vector immunity compromises the immunogenicity of Ad5-based vaccines. Thus, there is a substantial unmet need to minimize preexisting immunity while improving the insert-induced immunity of Ad5 vectors. Herein, we address this need by utilizing biocompatible nanoparticles to modulate Ad5–host interactions. We show that positively charged human serum albumin nanoparticles ((+)HSAnp), which are capable of forming a complex with Ad5, significantly increase the transgene expression of Ad5 in both coxsackievirus–adenovirus receptor-positive and -negative cells. Furthermore, in charge- and dose-dependent manners, Ad5/(+)HSAnp complexes achieve robust (up to 227-fold higher) and long-term (up to 60 days) transgene expression in the lungs of mice following intranasal instillation. Importantly, in the presence of preexisting anti-Ad5 immunity, complexed Ad5-based Ebola and COVID-19 vaccines significantly enhance antigen-specific humoral response and mucosal immunity. These findings suggest that viral aggregation and charge modification could be leveraged to engineer enhanced viral vectors for vaccines and gene therapies.

Abstract LB097: A simian challenge model to evaluate the CD8-positive T cell response with cancer therapeutics

Abstract The demonstration that antibodies can be used to treat cancers has revolutionized the field of cancer immunotherapy and gave hope to many patients. Originally targeting tumor-specific antigen, new therapeutic antibodies target now immune checkpoint markers expressed on T cells. The advantage is to generate an active and long-lasting antitumor immunity with the achievement of a broad and polyclonal antitumor immunity, addressing better the heterogeneity of cancers and reducing the chances of immune escape. The development of such highly specific molecules is particularly challenging however when it comes to preclinical safety assessment and efficacy studies. An ideal animal model needs to possess the target so that the drug product is pharmacologically active. Moreover, such model should ideally provide further insight in determining the pharmacological action of the product and the determination of safety to maximize the usefulness in animal studies. Intriguingly, no equivalent of the traditional T-cell-dependent antibody response (TDAR) assay, which evaluates mainly the CD4+ T cells response, is currently available for the CD8+ T cell response. The goal of this study is to fill this gap with a Non-Human Primate (NHP) challenge model that will elicit a potent CD8+ T cell response. MHC I-genotyped Mauritian cynomolgus macaques (MCMs) were immunized with 3 replication incompetent recombinant adenovirus serotype 5 (Ad5) vectors encoding Gag, Nef or Pol SIV proteins. MCMs were distributed into 3 groups and received two intramuscular injections of adenoviruses 4 weeks apart. One group received the anti-PD-L1 Atezolizumab and a second received a molecule in early development targeting another immune checkpoint molecule. T cell activation was monitored with blood samples taken on a weekly basis for 8 weeks. Blood samples were used to characterize the T cell immunophenotypes and to correlate them with some functional assays. Vaccination was well tolerated and could be conducted on standard safety toxicology studies without compromising other endpoints. Adv5-SIVs modulated total T cells 2 weeks after a single vaccination, induced T-cell activation, T-cell proliferation and the expression of several checkpoint molecules. Moreover, functional assays confirmed that T-cell activation profile correlated with an antigen-specific CD8 T-cell response. Compounds administered differently enhanced the profile of T-cell activation, the upregulation of immune checkpoint molecules and antigen specific responses of their T cells. Moreover, both compounds extended the cytolytic activity of the antigen-specific CD8+ T cell up to 8 weeks. Taken together, the vaccination model we developed seems appropriate to study the pharmacology of new protein-based therapeutics targeting CD8+ T cells. Citation Format: Richard Graveline, Morad Haida, Carolyne Dumont, Dominic Poulin, Florence Poitout-Belissent, Rana Samadfan, Sven Kronenberg, Franziska Regenass-Lechner, Rodney Prell, Marie-Soleil Piche. A simian challenge model to evaluate the CD8-positive T cell response with cancer therapeutics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB097.

H101 treatment of hepatic metastasis of colorectal cancer with recombinant human adenovirus 5 injection: A phase I clinical trial-TROJAN 021.

3605 Background: Recombinant human adenovirus serotype 5 injection (H101), obtained through genetic engineering to delete the E1B domain and part of the E3 domain and then selectively replicated in tumor cells, has been approved in 2005 for the local treatment of nasopharyngeal carcinoma and head and neck cancers. This trial aimed to evaluate the safety and the preliminary treatment efficacy of H101 combined with standard treatments in patients with liver metastases from colorectal cancer. Methods: In this phase 1, dose-escalation trial (ChiCTR1900027922), 17-75 years old colorectal cancer patients with unresectable liver metastases that failed to first-line therapy were included at The Tenth People's Hospital Affiliated to Tongji University between 2018.9 and 2020.12. All patients received H101 combined with standard therapy (bevacizumab + mFOLFOX6/FOLFIRI). Ultrasound-guided injection of H101 into the liver metastases was performed for all patients, with one of the following doses: 5×1011 vp/injection for the low, 1×1012 vp/injection for the moderate, 2×1012 vp/injection for the moderate-high, and 3×1012 vp/injection for the high dose groups. Intravenous infusion of bevacizumab (5 mg/kg) and administration of standard-dose mFOLFOX6 or FOLFIRI within 48 h after the intratumor injection was performed. The primary endpoint of the trial was the maximum tolerated dose (MTD). The secondary endpoints included safety, tumor responses, and tumor marker CEA. The adverse events (AEs) were monitored according to the NCI Common Terminology Criteria for Adverse Events (CTCAE, Version 3.0) and evaluated within 1 week after injection. Imaging examinations were performed for all patients to evaluate the tumor responses, according to the irRECIST. Results: Finally, 8 patients were included; the numbers of patients in the 4 groups were 1, 3, 3, and 1, respectively. MTD was not observed in this study. No grade >4 AEs were observed. The major AE included fatigue (5/8), fever (4/8), shiver (3/8), abdominal pain (3/8), and night sweat (3/8). The tumor responses included partial response in one patient (moderate dose group), stable disease in 6 patients (1, 1, 3, and 1 in the low, moderate, moderate-high, and high dose groups, respectively), and progressive disease in 1 patient (moderate group). No dose-effect response was found. The tumor marker CEA was reduced in 6 of the 8 patients, including 1 (1/1), 3 (3/3), 2 (2/3), and 0 patients in the low, moderate, moderate-high, and high-dose groups, respectively. Conclusions: The safety of H101 combined with standard therapy for liver metastases from colorectal cancer is acceptable, which also shows certain preliminary efficacy. The phase 2 trial is now ongoing. Clinical trial information: ChiCTR1900027922. [Table: see text]

Ad26.COV2.S protects Syrian hamsters against G614 spike variant SARS-CoV-2 and does not enhance respiratory disease

Previously we have shown that a single dose of recombinant adenovirus serotype 26 (Ad26) vaccine expressing a prefusion stabilized SARS-CoV-2 spike antigen (Ad26.COV2.S) is immunogenic and provides protection in Syrian hamster and non-human primate SARS-CoV-2 infection models. Here, we investigated the immunogenicity, protective efficacy, and potential for vaccine-associated enhanced respiratory disease (VAERD) mediated by Ad26.COV2.S in a moderate disease Syrian hamster challenge model, using the currently most prevalent G614 spike SARS-CoV-2 variant. Vaccine doses of 1 × 109 and 1 × 1010 VP elicited substantial neutralizing antibodies titers and completely protected over 80% of SARS-CoV-2 inoculated Syrian hamsters from lung infection and pneumonia but not upper respiratory tract infection. A second vaccine dose further increased neutralizing antibody titers that was associated with decreased infectious viral load in the upper respiratory tract after SARS-CoV-2 challenge. Suboptimal non-protective immune responses elicited by low-dose A26.COV2.S vaccination did not exacerbate respiratory disease in SARS-CoV-2-inoculated Syrian hamsters with breakthrough infection. In addition, dosing down the vaccine allowed to establish that binding and neutralizing antibody titers correlate with lower respiratory tract protection probability. Overall, these preclinical data confirm efficacy of a one-dose vaccine regimen with Ad26.COV2.S in this G614 spike SARS-CoV-2 virus variant Syrian hamster model, show the added benefit of a second vaccine dose, and demonstrate that there are no signs of VAERD under conditions of suboptimal immunity.

Vaccination With Recombinant Adenoviruses Expressing the Bluetongue Virus Subunits VP7 and VP2 Provides Protection Against Heterologous Virus Challenge.

Bluetongue virus (BTV) is the causative agent of a disease that affects domestic and wild ruminants and leads to critical economic losses. BTV is an arbovirus from the Reoviridae family that is typically transmitted by the bite of infected Culicoides midges. BTV possesses multiple serotypes (up to 28 have been described), and immunity to one serotype offers little cross-protection to other serotypes. The design of vaccines that provide protection across multiple serotypes is therefore highly desirable to control this disease. We previously reported that a recombinant replication-defective human adenovirus serotype 5 (Ad5) that expresses the VP7 inner core protein of BTV serotype 8 (Ad5VP7-8) induced T-cell responses and provided protection. In the present work, we evaluated as BTV vaccine the combination of Ad5VP7-8 with another recombinant Ad5 that expresses the outer core protein VP2 from BTV-1 (Ad5VP2-1). The combination of Ad5VP2-1 and Ad5VP7-8 protected against homologous BTV challenge (BTV-1 and BTV-8) and partially against heterologous BTV-4 in a murine model. Cross-reactive anti-BTV immunoglobulin G (IgG) were detected in immunized animals, but no significant titers of neutralizing antibodies were elicited. The Ad5VP7-8 immunization induced T-cell responses that recognized all three serotypes tested in this study and primed cytotoxic T lymphocytes specific for VP7. This study further confirms that targeting antigenic determinant shared by several BTV serotypes using cellular immunity could help develop multiserotype BTV vaccines.

Polyethylene glycol–modified DOTAP:cholesterol/adenovirus hybrid vectors have improved transduction efficiency and reduced immunogenicity

Despite the success of recombinant adenovirus serotype 5 (Ad5) and dioleoyltrimethylaminopropane:cholesterol (DOTAP:Chol) liposomes as viral and nonviral vectors in clinical trials, these vectors still suffer from drawbacks such as high immunogenicity and low transfection efficiency, respectively. To address these issues, some researchers have synthesized DOTAP:Chol/Ad5 hybrid vectors hoping to combine the unique features of the individual vectors. While this method shows promise, the limitations associated with Ad5 were not completely ameliorated. The objective of the present study was to use PEGylated liposomes to improve upon these previous studies, with the aim of maintaining high transduction efficiency and reducing the immune response. PEGylated DOTAP:Chol liposomes were synthesized at different molar ratios of polyethylene glycol (PEG) (0 to 0.04), and its effects on transduction efficiency and immunogenicity of Ad5 were studied. Coating of Ad5 with PEGylated liposomes was confirmed using transmission electron microscopy and dynamic light scattering, and these techniques indicated that the size of the complexes was ~ 140 nm. Transduction efficiency was enhanced nearly 100-fold in CAR-negative cells treated with PEGylated liposome/Ad5 complexes compared to unmodified Ad5. In addition, PEGylated liposome/Ad5 complexes significantly reduced the humoral and innate immune responses relative to native Ad5 as assessed by antiAd5 antibodies and IL-6 production by macrophage cells. Moreover, PEGylation decreased the cytotoxicity and hemolytic activity of the liposomes. In conclusion, the transduction efficiency, biocompatibility, and immunogenicity of DOTAP:Chol/Ad5 complexes were significantly improved by including an optimum amount of a PEG-lipid.

Heterologous Prime Boost Vaccination Induces Protective Melanoma-Specific CD8+ T Cell Responses

Cancer vaccination aims at inducing an adaptive immune response against tumor-derived antigens. In this study, we utilize recombinant human adenovirus serotype 5 (rAd5) and recombinant lymphocytic choriomeningitis virus (rLCMV)-based vectors expressing the melanocyte differentiation antigen gp100. In contrast to single or homologous vaccination, a heterologous prime boost vaccination starting with a rAd5-gp100 prime immunization followed by a rLCMV-gp100 boost injection induces a high magnitude of polyfunctional gp100-specific CD8+ T cells. Our data indicate that an optimal T cell induction is dependent on the order and interval of the vaccinations. A prophylactic prime boost vaccination with rAd5- and rLCMV-gp100 protects mice from a B16.F10 melanoma challenge. In the therapeutic setting, combination of the vaccination with low-dose cyclophosphamide showed a synergistic effect and significantly delayed tumor growth. Our findings suggest that heterologous viral vector prime boost immunizations can mediate tumor control in a mouse melanoma model.

Иммуногенные и защитные свойства рекомбинантного аденовируса человека 5-го серотипа, экспрессирующего ген гликопротеина G вируса бешенства вакцинного штамма РВ-97

Иммуногенные и защитные свойства рекомбинантного аденовируса человека 5-го серотипа, экспрессирующего ген гликопротеина G вируса бешенства вакцинного штамма РВ-97

P5377A SUMO1-SIM motif within the C-terminal alpha-helical domain of S100A1 prevents its proteasomal degradation in cardiomyocytes

Abstract S100A1, a EF-hand calcium (Ca2+) sensor protein, predominantly expressed in cardiomyocytes, improves contractile performance and energy metabolism targeting activity of downstream key factors such as SERCA2a and F1ATPase. Vice versa, decreased cardiac S100A1 expression in heart failure thereby accelerates the progression to contractile and energetic failure. Although previous studies showed that transcriptional inhibition may contribute to the loss of S100A1 expression, little is known with respect to its post-translational stability in cardiomyocytes. As such, advanced understanding of the molecular mechanisms that may regulate S100A1 degradation may be of interest to target its stability in the failing heart. In search of protein stabilizing motifs, we identified a Small Ubiquitin-related Modifier1 (SUMO1) interaction motif (SIM) in the S100A1 C-terminal domain extending from AA 69–87 as well as multiple ubiquitylatable Lysin-residues within the N-terminal domain by an in silico structural analysis. Given the role of the C-terminal domain of S100A1 in accommodating target binding and activity modulation, we hypothesized that these residues may convey a dual function and also contribute to S100A1 protein stability. To answer this question, we generated different Myc-tagged and non-tagged C-terminal deletion mutants (Myc-S100a1ΔCTs/S100a1ΔCTs) within the SIM motif encompassing AA 1–74 and AA 1–83 besides the full length AA 1–94 (S100A1 -FL). Constructs packaged into bicistronic recombinant Adenovirus Serotype 5 also contained GFP to control for sufficient transduction. Ad5-mediated gene delivery of S100a1-Fl to cell culture resulted in robust S100A1-FL protein expression. In contrast, delivery of the truncated S100a1 mutants did not yield detectable S100A1ΔCT protein variants, whereas the presence of mRNA from all S100a1ΔCT variants by PCR excluded transcriptional inhibition or blockade as a potential cause for the failure to detect S100A1ΔCT protein variants. When a proteasomal inhibitor (MG-132) was then used, S100A1ΔCT variants were detected at protein level in tested models indicating that proteasomal degradation may target SIM deleted S100A1 ΔCT variants. This notion was corroborated by unchanged expression levels of FL-S100A1 as well as deletion C-terminal mutants containing the entire SIM motif. Our novel results indicate that the C-terminal domain of S100A1 may contain a SIM motif which shields S100A1 protein against proteasomal degradation. SUMOylation analysis as well as site-directed mutagenesis of the motif is subsequently required to determine conditions where S100A1 SUMOylation may be decreased (i.e. heart failure) and which specific AA residues may convey this action. Overall, the S100A1 C-terminus may play a role in target recognition as well as in the regulation of S100A1's protein stability. In vivo studies will be required to provide the ultimate proof for this novel hypothesis. Acknowledgement/Funding DZHK

Abstract LB-269: Development of a vaccine model to track the CD8-specific response in Mauritius cynomolgus monkey

Abstract Introduction Manipulating the immune system to achieve therapeutic efficacy in cancer treatment has led to a new class of drugs that brought lasting remissions to many patients who had run out of options. The development of new immunotherapy aimed at targeting cancer cells requires the ability to monitor specifically the immune response. Although CD8 T cells play a crucial role in this process, no equivalent of the traditional T-cell-dependent antibody response (TDAR) assay, which evaluates the T-helper immune response, is currently available. Since cancer immunotherapy agents are often tested in non-tumor bearing NHP for safety assessment, the animal models currently available do not allow monitoring of potential exaggerated pharmacology and efficacy of these new drug candidates. Objectives The goal of this study was to develop an NHP vaccination model that specifically elicits a CTL response, in order to evaluate the efficacy in exacerbating the Cytotoxic T Lymphocyte (CTL) response. Experimental Procedures To reach this goal, MHC-genotyped Mauritian cynomolgus macaques (MCMs) were immunized with 3 replication incompetent recombinant adenovirus serotype 5 (Ad5) vectors, each containing the coding sequence for Gag, Nef or Pol SIV proteins. Such model allowed monitoring of the CD8 T cell activation in lieu of a tumor or live virus model. MCMs were distributed into 3 groups: one control group and 2 groups which received two intramuscular injection of the adenovirusrs spaced by 4 or 8 weeks. The immune response was monitored with blood samples taken on a weekly basis for up to 12 weeks. Blood samples were used to 1) characterize the different CD8-positive sub-populations by Tetramer staining and Immunophenotyping; 2) to correlate the immunophenotyping profile obtained with functional assays such as ex vivo recall response and IFNγ ELIspot. Results Of the three groups tested, MCM monkeys receiving 2 injections 8 weeks apart showed the more robust CD8 T cell response. This response was mainly characterized by a proliferation profile (Ki67-positive cells), the expression of activation markers at the surface of CD8 T cells and antigen specific responses of the CD8 T cells. Of the three Gag, Pol and Nef proteins, immunization with Nef gave the most robust response, as observed by the IFNγ ELIspot results and by the presence of a Nef-specific CD8-specific subpopulation observed by tetramer staining. No changes in the distribution of T, B, NK, Treg or Memory T cells was observed between the 3 groups. Conclusion The results obtained during the course of this study suggests that the described CD8-specific vaccination model using Mauritius cynomolgus macaques is a promising model to evaluate the efficacy and potential exaggerated pharmacology of new drug candidates targeting CD8 T cells. Citation Format: Richard Graveline, Morad Haida, Carolyne Dumont, Rana Samadfam, Dominic Poulin, Marie-Soleil Piche. Development of a vaccine model to track the CD8-specific response in Mauritius cynomolgus monkey [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-269.

Boosting BCG with proteins or rAd5 does not enhance protection against tuberculosis in rhesus macaques

Tuberculosis (TB) is the leading cause of death from infection worldwide. The only approved vaccine, BCG, has variable protective efficacy against pulmonary TB, the transmissible form of the disease. Therefore, improving this efficacy is an urgent priority. This study assessed whether heterologous prime-boost vaccine regimens in which BCG priming is boosted with either (i) protein and adjuvant (M72 plus AS01E or H56 plus CAF01) delivered intramuscularly (IM), or (ii) replication-defective recombinant adenovirus serotype 5 (Ad5) expressing various Mycobacterium tuberculosis (Mtb) antigens (Ad5(TB): M72, ESAT-6/Ag85b, or ESAT-6/Rv1733/Rv2626/RpfD) administered simultaneously by IM and aerosol (AE) routes, could enhance blood- and lung-localized T-cell immunity and improve protection in a nonhuman primate (NHP) model of TB infection. Ad5(TB) vaccines administered by AE/IM routes following BCG priming elicited ~10–30% antigen-specific CD4 and CD8 T-cell multifunctional cytokine responses in bronchoalveolar lavage (BAL) but did not provide additional protection compared to BCG alone. Moreover, AE administration of an Ad5(empty) control vector after BCG priming appeared to diminish protection induced by BCG. Boosting BCG by IM immunization of M72/AS01E or H56:CAF01 elicited ~0.1–0.3% antigen-specific CD4 cytokine responses in blood with only a transient increase of ~0.5–1% in BAL; these vaccine regimens also failed to enhance BCG-induced protection. Taken together, this study shows that boosting BCG with protein/adjuvant or Ad-based vaccines using these antigens, by IM or IM/AE routes, respectively, do not enhance protection against primary infection compared with BCG alone, in the highly susceptible rhesus macaque model of tuberculosis.

An adenovirus serotype 2-vectored ebolavirus vaccine generates robust antibody and cell-mediated immune responses in mice and rhesus macaques

Ebolavirus vaccines based on several adenoviral vectors have been investigated in preclinical studies and clinical trials. The use of adenovirus serotype 2 as a vector for ebolavirus vaccine has not been reported. Herein, we generated rAd2-ZGP, a recombinant replication-incompetent adenovirus serotype 2 expressing codon-optimized Zaire ebolavirus glycoprotein, and evaluated its immunogenicity in mice and rhesus macaques. rAd2-ZGP induced significant antibody and cell-mediated immune responses at 2 weeks after a single immunization. The glycoprotein (GP)-specific immune responses could be further enhanced with a booster immunization. Compared to protein antigens, Zaire ebolavirus GP and Zaire ebolavirus-like particles, rAd2-ZGP could induce stronger cross-reactive antibody and cell-mediated immune responses to heterologous Sudan ebolavirus in mice and rhesus macaques. In rAd2-ZGP-immunized macaques, GP-specific CD8+ T cells could secret IFN-γ and IL-2, indicating a Th1-biased response. In adenovirus serotype 5 seropositive macaques, rAd2-ZGP could induce robust antibody and cell-mediated immune responses, suggesting that the efficacy of rAd2-ZGP is not affected by pre-existing immunity to adenovirus serotype 5. These results demonstrated that rAd2-ZGP can be considered an alternative ebolavirus vaccine for use in adenovirus serotype 5 seropositive subjects or as a sequential booster vaccine after the subjects have been immunized with a recombinant adenovirus serotype 5-based vaccine.

Heterologous prime–boost vaccination with adenoviral vector and protein nanoparticles induces both Th1 and Th2 responses against Middle East respiratory syndrome coronavirus

The Middle East respiratory syndrome coronavirus (MERS-CoV) is a highly pathogenic and zoonotic virus with a fatality rate in humans of over 35%. Although several vaccine candidates have been developed, there is still no clinically available vaccine for MERS-CoV. In this study, we developed two types of MERS-CoV vaccines: a recombinant adenovirus serotype 5 encoding the MERS-CoV spike gene (Ad5/MERS) and spike protein nanoparticles formulated with aluminum (alum) adjuvant. Next, we tested a heterologous prime–boost vaccine strategy, which compared priming with Ad5/MERS and boosting with spike protein nanoparticles and vice versa, with homologous prime–boost vaccination comprising priming and boosting with either spike protein nanoparticles or Ad5/MERS. Although both types of vaccine could induce specific immunoglobulin G against MERS-CoV, neutralizing antibodies against MERS-CoV were induced only by heterologous prime–boost immunization and homologous immunization with spike protein nanoparticles. Interestingly, Th1 cell activation was induced by immunization schedules including Ad5/MERS, but not by those including only spike protein nanoparticles. Heterologous prime–boost vaccination regimens including Ad5/MERS elicited simultaneous Th1 and Th2 responses, but homologous prime–boost regimens did not. Thus, heterologous prime–boost may induce longer-lasting immune responses against MERS-CoV because of an appropriate balance of Th1/Th2 responses. However, both heterologous prime–boost and homologous spike protein nanoparticles vaccinations could provide protection from MERS-CoV challenge in mice. Our results demonstrate that heterologous immunization by priming with Ad5/MERS and boosting with spike protein nanoparticles could be an efficient prophylactic strategy against MERS-CoV infection.

A subunit vaccine based on fiber-2 protein provides full protection against fowl adenovirus serotype 4 and induces quicker and stronger immune responses than an inactivated oil-emulsion vaccine

As the number of hepatitis hydropericardium syndrome (HHS) cases has increased in recent years in China, development of a safe and effective vaccine is now urgent. To address this problem a subunit vaccine is a good option, we here systematically investigated the minimum immune dose of a subunit vaccine against HHS based on recombinant fowl adenovirus serotype 4 (FAdV-4) fiber-2 protein and compared the effects between this subunit vaccine and an inactivated oil-emulsion FAdV-4 vaccine in a vaccination trial. The results revealed that the lowest dose of recombinant fiber-2 protein that could provide 100% protection against challenge with virulent FAdV-4 strain HB1501 as well as elicit protective immunity was 2.5 μg/bird. Neither clinical signs nor gross lesions were observed in chickens. In addition, immunization of specific-pathogen-free (SPF) chickens with recombinant fiber-2 protein (≥2.5 μg/bird) could induce quicker and stronger immune responses than the inactivated oil-emulsion FAdV-4 vaccine. These findings provide important information about the development of subunit vaccines for the control of HHS.

Ad26/MVA therapeutic vaccination with TLR7 stimulation in SIV-infected rhesus monkeys.

The development of immunologic interventions that can target the viral reservoir in HIV-1-infected individuals is a major goal of the HIV-1 cure field1,2. However, little evidence exists that the viral reservoir can be sufficiently targeted to improve virologic control following discontinuation of antiretroviral therapy (ART). Here we show that Ad26/MVA3,4 therapeutic vaccination with toll-like receptor 7 (TLR7) stimulation improves virologic control and delays viral rebound following ART discontinuation in SIV-infected rhesus monkeys that initiated ART during acute infection. Ad26/MVA therapeutic vaccination resulted in a dramatic increase in the magnitude and breadth of SIV-specific cellular immune responses in virologically suppressed, SIV-infected monkeys. TLR7 agonist administration led to innate immune stimulation and cellular immune activation. The combination of Ad26/MVA vaccination and TLR7 stimulation resulted in decreased levels of viral DNA in lymph nodes and peripheral blood, as well as improved virologic control and delayed viral rebound following ART discontinuation. Cellular immune breadth correlated inversely with setpoint viral loads and correlated directly with time to viral rebound. These data demonstrate the potential of therapeutic vaccination with innate immune stimulation as a strategy aimed at an HIV-1 functional cure.

An Adenovirus Vaccine Expressing Ebola Virus Variant Makona Glycoprotein Is Efficacious in Guinea Pigs and Nonhuman Primates.

A licensed vaccine against Ebola virus (EBOV) remains unavailable, despite >11 000 deaths from the 2014-2016 outbreak of EBOV disease in West Africa. Past studies have shown that recombinant vaccine viruses expressing EBOV glycoprotein (GP) are able to protect nonhuman primates (NHPs) from a lethal EBOV challenge. However, these vaccines express the viral GP-based EBOV variants found in Central Africa, which has 97.3% amino acid homology to the Makona variant found in West Africa. Our previous study showed that a recombinant adenovirus serotype 5 (Ad5)-vectored vaccine expressing the Makona EBOV GP (MakGP) was safe and immunogenic during clinical trials in China, but it is unknown whether the vaccine protects against EBOV infection. Here, we demonstrate that guinea pigs immunized with Ad5-MakGP developed robust humoral responses and were protected against exposure to guinea pig-adapted EBOV. Ad5-MakGP also elicited specific B- and T-cell immunity in NHPs and conferred 100% protection when animals were challenged 4 weeks after immunization. These results support further clinical development of this candidate and highlight the utility of Ad5-MakGP as a prophylactic measure in future outbreaks of EBOV disease.

Protective efficacy of multiple vaccine platforms against Zika virus challenge in rhesus monkeys.

Zika virus (ZIKV) is responsible for a major ongoing epidemic in the Americas and has been causally associated with fetal microcephaly. The development of a safe and effective ZIKV vaccine is therefore an urgent global health priority. Here we demonstrate that three different vaccine platforms protect against ZIKV challenge in rhesus monkeys. A purified inactivated virus vaccine induced ZIKV-specific neutralizing antibodies and completely protected monkeys against ZIKV strains from both Brazil and Puerto Rico. Purified immunoglobulin from vaccinated monkeys also conferred passive protection in adoptive transfer studies. A plasmid DNA vaccine and a single-shot recombinant rhesus adenovirus serotype 52 vector vaccine, both expressing ZIKV premembrane and envelope, also elicited neutralizing antibodies and completely protected monkeys against ZIKV challenge. These data support the rapid clinical development of ZIKV vaccines for humans.