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

Chlamydial Type III Secretion System Needle Protein Induces Protective Immunity against Chlamydia muridarum Intravaginal Infection

Chlamydia trachomatis imposes serious health problems and causes infertility. Because of asymptomatic onset, it often escapes antibiotic treatment. Therefore, vaccines offer a better option for the prevention of unwanted inflammatory sequelae. The existence of serologically distinct serovars of C. trachomatis suggests that a vaccine will need to provide protection against multiple serovars. Chlamydia spp. use a highly conserved type III secretion system (T3SS) composed of structural and effector proteins which is an essential virulence factor. In this study, we expressed the T3SS needle protein of Chlamydia muridarum, TC_0037, an ortholog of C. trachomatis CdsF, in a replication-defective adenoviral vector (AdTC_0037) and evaluated its protective efficacy in an intravaginal Chlamydia muridarum model. For better immune responses, we employed a heterologous prime-boost immunization protocol in which mice were intranasally primed with AdTC_0037 and subcutaneously boosted with recombinant TC_0037 and Toll-like receptor 4 agonist monophosphoryl lipid A mixed in a squalene nanoscale emulsion. We found that immunization with TC_0037 antigen induced specific humoral and T cell responses, decreased Chlamydia loads in the genital tract, and abrogated pathology of upper genital organs. Together, our results suggest that TC_0037, a highly conserved chlamydial T3SS protein, is a good candidate for inclusion in a Chlamydia vaccine

Chlamydial Type III Secretion System Needle Protein Induces Protective Immunity against Chlamydia muridarum Intravaginal Infection

Chlamydia trachomatis imposes serious health problems and causes infertility. Because of asymptomatic onset, it often escapes antibiotic treatment. Therefore, vaccines offer a better option for the prevention of unwanted inflammatory sequelae. The existence of serologically distinct serovars of C. trachomatis suggests that a vaccine will need to provide protection against multiple serovars. Chlamydia spp. use a highly conserved type III secretion system (T3SS) composed of structural and effector proteins which is an essential virulence factor. In this study, we expressed the T3SS needle protein of Chlamydia muridarum, TC_0037, an ortholog of C. trachomatis CdsF, in a replication-defective adenoviral vector (AdTC_0037) and evaluated its protective efficacy in an intravaginal Chlamydia muridarum model. For better immune responses, we employed a heterologous prime-boost immunization protocol in which mice were intranasally primed with AdTC_0037 and subcutaneously boosted with recombinant TC_0037 and Toll-like receptor 4 agonist monophosphoryl lipid A mixed in a squalene nanoscale emulsion. We found that immunization with TC_0037 antigen induced specific humoral and T cell responses, decreased Chlamydia loads in the genital tract, and abrogated pathology of upper genital organs. Together, our results suggest that TC_0037, a highly conserved chlamydial T3SS protein, is a good candidate for inclusion in a Chlamydia vaccine

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)

ELISA titration for M2- and NP-binding IgG antibodies in the sera of mice, 8 months post-immunization with Ad5-tet-M2NP.

<p>Mice were immunized intranasally using 10⁸ PFU of Ad5-tet-M2NP and 10⁸ PFU of Ad-null. Mouse sera were collected 8 months post-immunization. Titration of the sera was performed in ELISA plates pre-coated with synthetic M2 (<b>A</b>) or recombinant NP (<b>B</b>) proteins. <i>X</i>-axis–serum dilution; <i>Y</i>-axis–optical density (<i>λ</i> = 450 nm). Data are represented as the mean <b>± SD</b> for 10 mice in the experimental group.</p

Quantification of interferon-γ secreting M2- or NP-specific T cells in the spleens of mice immunized with Ad5-tet-M2NP.

<p>BALB/c mice received a single intranasal immunization with either 10⁷ PFU or 10⁸ PFU doses of Ad5-tet-M2NP or 10⁸ PFU Ad5-null (“empty vector”). Thirty days post-immunization, mouse spleens were aseptically collected and the MNC fraction was obtained using ficoll-plaque gradient centrifugation. 99%-pure CD8 and CD4 cell suspensions were obtained using cell sorting by a BD FACSAria II (A-C). Syngenic dendritic cells (DCs) were differentiated in 7-day bone marrow cultures with GM-CSF. For the presentation of antigens in the context of MHC class I (D, E, F), dendritic cells were transduced with recombinant Ad5-tet-M2 or Ad5-tet-NP, or Ad5-null (100 PFU per cell). For the presentation of antigens in the context of MHC class II (G, H, I), DCs were pre-activated with LPS (1 μg/ml) (LPS-DC) and then loaded with either 50 μg/ml synthetic M2 (sM2) or 5 μg/ml recombinant NP (rNP) antigens. Splenic MNCs (D, G) or purified CD8 (F, I) or CD4 (E, H) cells were co-cultured in duplicate over 18 hrs with antigen-presenting DCs or LPS-DCs before the numbers of interferon-γ-secreting T cells were counted using ELISPOT. Numbers of spots are shown as the mean ± SD (per 1 million splenic MNCs), after deduction of the baseline spots found in the negative control group (mice that received a sham immunization, i.e., intranasal PBS). Asterisks are shown for significant differences (p<0.05) in comparison to the control group of mice immunized with Ad5-null (Mann–Whitney–Wilcoxon nonparametric test).</p

Survival rate in 5 challenge experiments with mice pre-immunized with Ad5-tet-M2NP or Ad5-null.

<p>Survival rate in 5 challenge experiments with mice pre-immunized with Ad5-tet-M2NP or Ad5-null.</p