Circumventing antivector immunity: potential use of nonhuman adenoviral vectors

Adenoviruses are efficient gene delivery vectors based on their ability to transduce a wide variety of cell types and drive high-level transient transgene expression. While there have been advances in modifying human adenoviral (HAdV) vectors to increase their safety profile, there are still pitfalls that need to be further addressed. Preexisting humoral and cellular immunity against common HAdV serotypes limits the efficacy of gene transfer and duration of transgene expression. As an alternative, nonhuman AdV (NHAdV) vectors can circumvent neutralizing antibodies against HAdVs in immunized mice and monkeys and in human sera, suggesting that NHAdV vectors could circumvent preexisting humoral immunity against HAdVs in a clinical setting. Consequently, there has been an increased interest in developing NHAdV vectors for gene delivery in humans. In this review, we outline the recent advances and limitations of HAdV vectors for gene therapy and describe examples of NHAdV vectors focusing on their immunogenicity, tropism, and potential as effective gene therapy vehicles

Vaccination potential of B and T epitope-enriched NP and M2 against Influenza A viruses from different clades and hosts

To avoid outbreaks of influenza virus epidemics and pandemics among human populations, modern medicine requires the development of new universal vaccines that are able to provide protection from a wide range of influenza A virus strains. In the course of development of a universal vaccine, it is necessary to consider that immunity must be generated even against viruses from different hosts because new human epidemic virus strains have their origins in viruses of birds and other animals. We have enriched conserved viral proteins–nucleoprotein (NP) and matrix protein 2 (M2)—by B and T-cell epitopes not only human origin but also swine and avian origin. For this purpose, we analyzed M2 and NP sequences with respect to changes in the sequences of known T and B-cell epitopes and chose conserved and evolutionarily significant epitopes. Eventually, we found consensus sequences of M2 and NP that have the maximum quantity of epitopes that are 100% coincident with them. Consensus epitope-enriched amino acid sequences of M2 and NP proteins were included in a recombinant adenoviral vector. Immunization with Ad5-tet-M2NP induced strong CD8 and CD4 T cells responses, specific to each of the encoded antigens, i.e. M2 and NP. Eight months after immunization with Ad5-tet-M2NP, high numbers of M2- and NP-responding “effector memory” CD44posCD62neg T cells were found in the mouse spleens, which revealed a long-term T cell immune memory conferred by the immunization. In all, the challenge experiments showed an extraordinarily wide-ranging efficacy of protection by the Ad5-tet-M2NP vaccine, covering 5 different heterosubtypes of influenza A virus (2 human, 2 avian and 1 swine). © 2018 Tutykhina et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited

The differences in immunoadjuvant mechanisms of TLR3 and TLR4 agonists on the level of antigen-presenting cells during immunization with recombinant adenovirus vector

Abstract Background Agonists of TLR3 and TLR4 are effective immunoadjuvants for different types of vaccines. The mechanisms of their immunostimulatory action differ significantly; these differences are particularly critical for immunization with non-replicating adenovirus vectors (rAds) based vaccines. Unlike traditional vaccines, rAd based vaccines are not designed to capture vaccine antigens from the external environment by antigen presenting cells (APCs), but rather they are targeted to the de novo synthesis of vaccine antigens in APCs transfected with rAd. To date, there is no clear understanding about approaches to improve the efficacy of rAd vaccinations with immunoadjuvants. In this study, we investigated the immunoadjuvant effect of TLR3 and TLR4 agonists on the level of activation of APCs during vaccination with rAds. Results We demonstrated that TLR3 and TLR4 agonists confer different effects on the molecular processes in APCs that determine the efficacy of antigen delivery and activation of antigen-specific CD4+ and CD8+ T cells. APCs activated with agonists of TLR4 were characterized by up-regulated production of target antigen mRNA and protein encoded in rAd, as well as enhanced expression of the co-activation receptors CD80, CD86 and CD40, and pro-inflammatory cytokines TNF-α, IL6 and IL12. These effects of TLR4 agonists have provided a significant increase in the number of antigen-specific CD4+ and CD8+ T cells. TLR3 agonist, on the contrary, inhibited transcription and synthesis of rAd-encoded antigens, but improved expression of CD40 and IFN-β in APCs. The cumulative effect of TLR3 agonist have resulted in only a slight improvement in the activation of antigen-specific T cells. Also, we demonstrated that IFN-β and TNF-α, secreted by APCs in response to TLR3 and TLR4 agonists, respectively, have an opposite effect on the transcription of the targeted gene encoded in rAd. Specifically, IFN-β inhibited, and TNF-α stimulated the expression of target vaccine antigens in APCs. Conclusions Our data demonstrate that agonists of TLR4 but not TLR3 merit further study as adjuvants for development of vaccines based on recombinant adenoviral vectors

Evaluation of Direct and Cell-Mediated Lactoferrin Gene Therapy for the Maxillofacial Area Abscesses in Rats

Resistance to antibacterial therapy requires the discovery of new methods for the treatment of infectious diseases. Lactoferrin (LTF) is a well-known naïve first-line defense protein. In the present study, we suggested the use of an adenoviral vector (Ad5) carrying the human gene encoding LTF for direct and cell-mediated gene therapy of maxillofacial area phlegmon in rats. Abscesses were developed by injection of the purulent peritoneal exudate in the molar region of the medial surface of the mandible. At 3–4 days after phlegmon maturation, all rats received ceftriaxone and afterward were subcutaneously injected around the phlegmon with: (1) Ad5 carrying reporter gfp gene encoding green fluorescent protein (Ad5-GFP control group), (2) Ad5 carrying LTF gene (Ad5-LTF group), (3) human umbilical cord blood mononuclear cells (UCBC) transduced with Ad5-GFP (UCBC + Ad5-GFP group), and (4) UCBC transduced with Ad5-LTF (UCBC + Ad5-LTF group). Control rats developed symptoms considered to be related to systemic inflammation and were euthanized at 4–5 days from the beginning of the treatment. Rats from therapeutic groups demonstrated wound healing and recovery from the fifth to seventh day based on the type of therapy. Histological investigation of cervical lymph nodes revealed purulent lymphadenitis in control rats and activated lymphatic tissue in rats from the UCBC + Ad5-LTF group. Our results propose that both approaches of LTF gene delivery are efficient for maxillofacial area phlegmon recovery in rats. However, earlier wound healing and better outcomes in cervical lymph node remodeling in the UCBC + Ad5-LTF group, as well as the lack of direct exposure of the viral vector to the organism, which may cause toxic and immunogenic effects, suggest the benefit of cell-mediated gene therapy

Additional file 1: of The differences in immunoadjuvant mechanisms of TLR3 and TLR4 agonists on the level of antigen-presenting cells during immunization with recombinant adenovirus vector

Figure S1. Effect of TLR agonists on the expression of rAd in DCs. DCs were transduced with rAdTet-off H1 (100 PFU per cell) in the presence of 10 μg/ml agonists of TLR3 (Poly I:C) or TLR4 (LPS, IMM); 24 h after transfection cells were stained with primary (H1-specific) and secondary fluorochrome labeled antibodies. The mean fluorescence (MFI) of H1-positive DCs in the test samples was detected by flow cytometry. Shown are M ± SD, statistically significant (p < 0.05) differences are indicated by asterisks. Figure S2. Effect of TLR3- and TLR4-activated APCs on the reactivation of CD4+ and CD8+ T-cells. Balb/c mice were immunized (i.m.) with 108 PFU rAdTet-off H1. Forty days after immunization, the pool of CD8+ (a, c) and CD4+ (b, d) T cells from the spleen of euthanized immune mice was re-activated in vitro. Sorted CD8+ and CD4+ T cells were co-cultured with bone marrow derived DCs (c, d) or macrophages (MF) (a, b) preloaded with 20 PFU/cell rAdTet-off H1 in the presence of 0–10 μg/ml agonists of TLR3 (Poly I:C) or TLR4 (LPS, IMM). The number of reactivated IFNγ-producing T-cells were detected by ELISPOT and calculated for 1 million spleen cells. Shown are M ± SD, statistically significant differences (p < 0.05) are indicated by asterisks. (PDF 123 kb

Vaccination potential of B and T epitope-enriched NP and M2 against Influenza A viruses from different clades and hosts - Fig 3

<p><b>Schematic diagram of expression system Tet-off (A) and developed genetic construction (B).</b> TRE–tetracycline response element, mCMV–minimal CMV promoter, M2 –M2 protein gene, 2A –nucleotide sequence that codes foot-and-mouth disease virus 2A peptide, NP–NP protein gene, CMV–human cytomegalovirus promoter, tTA–tetracycline-controlled transactivator. Tet-off system consists of two expression cassettes. The first of them contain gene of interest under the control of mCMV. Additionally, this cassette contains TRE. For the activation of expression of gene of interest tTA should bind to the TRE. The second expression cassette contain tTA gene under the control of CMV. Tetracycline and doxycycline bind to tTA and block expression of gene of interest.</p

Le Réveil des combattants : organe de l'Association républicaine des anciens combattants, victimes de la guerre et anciens soldats (I.A.C.)

mai 19311931/05 (N5)