Non-standard viral genome-derived RNA activates TLR3 and type I IFN signaling to induce cDC1-dependent CD8+ T-cell responses during vaccination in mice

There is a critical need to develop vaccine adjuvants that induce robust immune responses able to protect against intracellular pathogens, including viruses. Previously, we described defective viral genome-derived oligonucleotides (DDOs) as novel adjuvants that strongly induce type 1 immune responses, including protective Th1 CD4+ T-cells and effector CD8+ T-cells in mice. Here, we unravel the early innate response required for this type 1 immunity induction. Upon DDO subcutaneous injection, type 1 conventional dendritic cells (cDC1s) accumulate rapidly in the draining lymph node in a Toll-like receptor 3 (TLR3)- and type I interferon (IFN)-dependent manner. cDC1 accumulation in the lymph node is required for antigen-specific CD8+ T-cell responses. Notably, in contrast to poly I:C, DDO administration resulted in type I IFN expression at the injection site, but not in the draining lymph node. Additionally, DDOs induced an inflammatory cytokine profile distinct from that induced by poly I:C. Therefore, DDOs represent a powerful new adjuvant to be used during vaccination against intracellular pathogens.     

Immune intervention strategies for HIV-1 infection of humans in the SIV macaque model

Studies in the SIVmac macaque model have demonstrated that the extent of virus-specific CD4+ and CD8+ T-cell responses induced by vaccination prior to virus-challenge exposure correlate with viremia containment following establishment of infection. These findings led to the hypothesis that active immunization with vaccines able to induce virus-specific T-cell responses following the establishment of infection could also ameliorate the virological outcome. Here, we will review the relative effect of ART and vaccination during primary SIVmac infection of macaques.     

Kinetics of the inflammatory response following intramuscular injection of aluminum adjuvant

Aluminum-containing adjuvants are widely used in human and veterinary vaccines, but their mechanism of action is not well understood. Recent evidence suggests an important role for inflammation in the immune response to aluminum-adjuvanted vaccines. To better understand this process, vaccines with aluminum adjuvant were injected into naïve or previously immunized mice and the injection sites were characterized for the corresponding primary and secondary inflammatory response at different time points after immunization. Inflammatory cells appeared at the injection site between 2 h and 6 h after vaccination, dominated by neutrophils at first, followed by macrophages, and later eosinophils and MHCII⁺ cells. The number of cells at the injection site increased over time, except neutrophils, which decreased in number after day 2. There was extensive phagocytosis of aluminum adjuvant particles by macrophages. In secondary immunized mice, a faster and more robust recruitment of eosinophils, macrophages, and antigen presenting cells was observed at the injection site. The enhanced recruitment of inflammatory cells in previously immunized mice coincided with increased expression of relevant chemokines at the injection site. Since neutrophils accumulated first in response to aluminum-adjuvanted vaccines, their role was evaluated by depleting them prior to vaccination. Neutrophil depletion transiently reduced the recruitment of macrophages but it did not change the recruitment of eosinophils and MHCII⁺ cells or the quality and magnitude of the antibody response.     

Antibody-mediated delivery of T-cell epitopes to antigen-presenting cells induce strong CD4 and CD8 T-cell responses

Targeted delivery of antigen to antigen-presenting cells (APCs) enhances antigen presentation and thus, is a potent strategy for making more efficacious vaccines. This can be achieved by use of antibodies with specificity for endocytic surface molecules expressed on the APC. We aimed to compare two different antibody-antigen fusion modes in their ability to induce T-cell responses; first, exchange of immunoglobulin (Ig) constant domain loops with a T-cell epitope (Troybody), and second, fusion of T-cell epitope or whole antigen to the antibody C-terminus. Although both strategies are well-established, they have not previously been compared using the same system. We found that both antibody-antigen fusion modes led to presentation of the T-cell epitope. The strength of the T-cell responses varied, however, with the most efficient Troybody inducing CD4 T-cell proliferation and cytokine secretion at 10–100-fold lower concentration than the antibodies carrying antigen fused to the C-terminus, both in vitro and after intravenous injection in mice. Furthermore, we exchanged this loop with an MHCI-restricted T-cell epitope, and the resulting antibody enabled efficient cross-presentation to CD8 T cells in vivo. Targeting of antigen to APCs by use of such antibody-antigen fusions is thus an attractive vaccination strategy for increased activation of both CD4 and CD8 peptide-specific T cells.     

Early immune responses in skin and lymph node after skin delivery of Toll-like receptor agonists in neonatal and adult pigs

The skin is potentially an important vaccine delivery route facilitated by a high number of resident antigen presenting cells (APCs), which are known to be stimulated by different Toll-like receptor agonists (TLRa). In this study, neonatal and adult pigs were vaccinated in the skin using dissolving microneedle patches to investigate the immuno-stimulatory potential of different TLRa and possible age-dependent differences early after vaccination. These patches contained TLR1/2a (Pam3Cys), TLR7/8a (R848) or TLR9a (CpG ODN) combined with inactivated porcine reproductive and respiratory syndrome virus (PRRSV) or with an oil-in-water stable emulsion. Vaccinated skin and draining lymph nodes were analysed for immune response genes using microfluidic high-throughput qPCR to evaluate the early immune response and activation of APCs. Skin pathology and immunohistochemistry were used to evaluate the local immune responses and APCs in the vaccinated skin, respectively.In both neonatal and adult pigs, skin vaccination with TLR7/8a induced the most prominent early inflammatory and immune cell responses, particularly in the skin. Skin histopathology and immunohistochemistry of APCs showed comparable results for neonatal and adult pigs after vaccination with the different TLRa vaccines. However, in vaccinated neonatal pigs in the skin and draining lymph node more immune response related genes were upregulated compared to adult pigs. We showed that both neonatal and adult skin could be stimulated to develop an immune response, particularly after TLR7/8a vaccination, with age-dependent differences in regulation of immune genes. Therefore, age-dependent differences in local early immune responses should be considered when developing skin vaccines.     

CpG oligodeoxynucleotides activate dendritic cells in vivo and induce a functional and protective vaccine immunity against a TERT derived modified cryptic MHC class I-restricted epitope

The use of synthetic peptides derived from tumor-associated Ags is attractive for the development of antitumoral vaccines as far as strong adjuvants are found to render them immunogenic. Here, we investigated the possibility to enhance the CD8 response against the human and mouse shared TERT(572Y) HLA-A*0201 restricted modified cryptic peptide by using ODN-CpG as adjuvant. Humanized transgenic mice were immunized with the TERT(572Y) modified cryptic peptide in the presence of ODN-CpG and compared to mice immunized in IFA. By contrast with IFA, we first showed that, in vivo, ODN-CpG leads to the recruitment of dendritic cells in the lymph nodes draining the injection site. Those cells and especially the CD11c+ CD11b- CD8a+ lymphoid and the CD11c+ B220+ plasmacytoid dendritic cells were activated as shown by up-regulation of CD40 at their cell surface. Immunization against TERT(572Y) peptide in the presence of ODN-CpG rather than IFA led to a strong CD8 response and can delayed mortality in an induced tumor model. Study of the CD8 response obtained after antigenic challenge suggested that a functional memory response is induced upon vaccination with ODN-CpG. Thus, MHC class I-restricted epitope in combination of ODN-CpG is a promising and rather simple cancer vaccine formulation.     

A novel therapeutic hepatitis B vaccine induces cellular and humoral immune responses and breaks tolerance in hepatitis B virus (HBV) transgenic mice

Therapeutic vaccines are currently being developed for chronic hepatitis B and C. As an alternative to long-term antiviral treatment or to support only partially effective therapy, they should activate the patient's immune system effectively to fight and finally control the virus. A paradigm of therapeutic vaccination is the potent induction of T-cell responses against key viral antigens – besides activation of a humoral immune response. We have evaluated the potential of a novel vaccine formulation comprising particulate hepatitis B surface (HBsAg) and core antigen (HBcAg), and the saponin-based ISCOMATRIX™ adjuvant for its ability to stimulate T and B cell responses in C57BL/6 mice and its ability to break tolerance in syngeneic HBV transgenic (HBVtg) mice. In C57BL/6 mice, the vaccine induced multifunctional HBsAg- and HBcAg-specific CD8+ T cells detected by staining for IFNγ, TNFα and IL-2, as well as high antibody titers against both antigens. Vaccination of HBVtg animals induced potent HBsAg- and HBcAg-specific CD8+ T-cell responses in spleens and HBcAg-specific CD8+ T-cell responses in livers as well as anti-HBs seroconversion two weeks post injection. Vaccination further reduced HBcAg expression in livers of HBVtg mice without causing liver damage.     

Vaccination of aged mice with adjuvanted recombinant influenza nucleoprotein enhances protective immunity

Elderly individuals are highly susceptible to influenza virus (IAV) infection and respond poorly to influenza vaccines. Although the generally accepted correlate of protection following influenza vaccination is neutralizing antibody titers, cytotoxic T cell activity has been found to be a better correlate in the elderly. This suggests that vaccines designed to protect against influenza in the elderly should induce both humoral and cellular immunity. The co-induction of T cell immunity is additionally advantageous, as virus-specific T cells are frequently cross-reactive against different strains of IAV. Here, we tested the capacity of a synthetic TLR-4 adjuvant, SLA-SE (second-generation lipid adjuvant formulated in a squalene-based oil-in-water emulsion) to elicit T cell immunity to a recombinant influenza nucleoprotein (rNP), in both young and aged mice. IAV challenge of vaccinated mice resulted in a modest increase in the numbers of NP-specific CD4 and CD8 effector T cells in the spleen, but did not increase numbers of memory phenotype CD8 T cells generated following viral clearance (compared to control vaccinated mice). Cytotoxic activity of CD8, but not CD4 T cells was increased. In addition, SLA-SE adjuvanted vaccination specifically enhanced the production of NP-specific IgG2c antibodies in both young and aged mice. Although NP-specific antibodies are not neutralizing, they can cooperate with CD8 T cells and antigen-presenting cells to enhance protective immunity. Importantly, SLA-SE adjuvanted rNP-vaccination of aged mice resulted in significantly enhanced viral clearance. In addition, vaccination of aged mice resulted in enhanced survival after lethal challenge compared to control vaccination, that approached statistical significance. These data demonstrate the potential of SLA-SE adjuvanted rNP vaccines to (i) generate both cellular and humoral immunity to relatively conserved IAV proteins and (ii) elicit protective immunity to IAV in aged mice.     

Branched and linear lipopeptide vaccines have different effects on primary CD4+ and CD8+ T-cell activation but induce similar tumor-protective memory CD8+ T-cell responses

We compared murine T-cell responses to synthetic lipopeptide vaccines in which the TLR2 ligand Pam(2)Cys was attached to co-linear CD4+ and CD8+ T-cell epitopes of ovalbumin (OVA) in a linear or branched configuration. Mice received OVA-specific transgenic CD8+ and CD4+ T-cells followed by one injection of vaccine. Although the branched lipopeptide was more potent in activating OVA-specific CD4+ and CD8+ T-cells in the primary response, both vaccines induced cytolytic T lymphocytes (CTL) that expressed perforin, granzyme A-C, and IFN-gamma mRNAs and conferred long-term protection of most mice against challenge with OVA-expressing tumor cells. OVA epitope display was reduced in tumors that developed in some mice, suggesting CD8+ T-cell dependent selection.     

HSP70 gene fused with Hantavirus S segment DNA significantly enhances the DNA vaccine potency against hantaviral nucleocapsid protein in vivo

Heat shock proteins (HSPs) have been shown to act as adjuvants when coadministered with peptide antigens or given as fusion proteins and enhance the vaccination efficiency. To evaluate the enhancement of the potency of Hantaan virus (HTNV) nucleocapsid protein (NP) immunogenicity by heat shock protein 70 (HSP70), we developed a novel chimeric HTNV S-HSP70 DNA vaccine plasmid by genetically linking HSP70 gene to the full-length HTNV S segment DNA (HTNV S DNA). C57BL/6 mice were immunized with this plasmid followed by a subsequent boost with homologous recombinant protein. The levels of HTNV NP-specific antibody and cellular immune response were measured by use of ELISA, fluorescence activated cell sorter (FACS) analysis, cytotoxicity assay, and IFN-gamma ELISPOT assay. We found that HTNV S-HSP70 DNA vaccination significantly increased the levels of HTNV NP-specific antibody, IgG2a/IgG1 ratio, IFN-gamma producing CD8+ T-cell precursor frequencies, and cytotoxic T lymphocyte (CTL) response when compared with immunization with HTNV S DNA alone or HTNV S DNA physically mixed with HSP70 DNA. By contrast, HSP70 DNA or vector DNA immunization could not induce appreciable levels of specific antibodies and CTL response. Thus, we demonstrate for the first time that HSP70-based HTNV S DNA can induce both humoral and cellular immune response specific for HTNV NP and is a promising candidate DNA vaccine for HTNV infection.     

Vaccination with dendritic cells charged with apoptotic/necrotic B16 melanoma induces the formation of subcutaneous lymphoid tissue

Antigen presentation by dendritic cells (DC) is of key importance for the initiation of the primary immune response. Mice vaccinated with DC charged with apoptotic/necrotic B16 cells (DC-Apo/Nec) are protected against B16 challenge. The aim of this study was to assess vaccine cell migration in our system and to find out if there is an immunological response taking place at the vaccination site. The formation of a pseudocapsule, peripheral node addresin expression in small venules, and the recruitment of a wide variety of cellular populations, including macrophages, polymorphonuclear lymphocytes, and CD8+ and CD4+ T lymphocytes found in association with DC, evidenced the formation of tertiary lymphoid tissue in the vaccination site in our experimental system.     

Enhancement of T cell-mediated immune responses to whole inactivated influenza virus by chloroquine treatment in vivo

Current influenza vaccines induce poor cross-reactive CD8+ T cell responses. Cellular immunity is generally specific for epitopes that are remarkably conserved among different subtypes, suggesting that strategies to improve the cross-presentation of viral antigens by dendritic cells (DC) could elicit a broadly protective immune response. Previous studies have shown that limited proteolysis within the endocytic pathway can favorably influence antigen processing and thus immune responses. Herein, we demonstrate that chloroquine improves the cross-presentation of non-replicating influenza virus in vitro and T cell responses in mice following a single administration of inactivated HI-X31 virus. CD8+ T cells were also recruited to lymph nodes draining the site of infection and able to reduce viral load following pulmonary challenge with the heterologous PR8 virus. These findings may have implications for vaccination strategies aimed at improving the cross-presentation capacity of DCs and thus the size of effector and memory CD8+ T cells against influenza vaccines.     

The comparative efficacy of CTLA-4 and L-selectin targeted DNA vaccines in mice and sheep

The access of antigens to antigen presenting cells (APCs) appears to be a rate-limiting step in the generation of immune responses to DNA vaccines. The cytotoxic T lymphocyte antigen 4 (CTLA-4) and L-selectin represent attractive ligands for use in the targeting of antigen to APCs and lymph nodes. CTLA-4 binds with high affinity to the B7 membrane antigen on APCs, while L-selectin functions as a lymphocyte homing marker and binds to CD34 on the surface of high endothelial venule cells. DNA vaccines encoding human immunoglobulin (HIg), fused to either CTLA-4 or L-selectin, have been shown to generate up to 10,000-fold higher anti-HIg antibody responses than DNA vaccines encoding HIg alone. In this study, the ability of CTLA-4 or L-selectin mediated targeting to enhance the humoral immune response to an alternate vaccine antigen was investigated. DNA vaccines encoding CTLA-4-HIg and L-selectin-HIg fused to the host-protective 45W antigen from Taenia ovis were constructed. In BALB/c mice, the L-selectin targeted vaccine did not improve either the magnitude or speed of antibody responses of vaccinated mice. In contrast, the CTLA-4 targeted DNA vaccine generated 45W-specific antibody responses which were up to 30-fold higher than those achieved with non-targeted DNA vaccination. The kinetic of the antibody response generated following CTLA-4 targeted DNA vaccination was also significantly faster than that achieved with non-targeted DNA vaccination, or with adjuvanted protein vaccination. Vaccination of outbred sheep with DNA vaccines expressing either murine or ovine CTLA-4 targeted antigen failed to enhance immune responses. These findings indicate that CTLA-4 targeting may find application in the improvement of DNA vaccines, but requires further development for applications in large animal species.     

Long-term persistence of vaccination and HAART to human immunodeficiency virus (HIV)

The aim of this study was to monitor the immune responses in HIV-infected patients previously immunized with gp160 or DNA vaccines to analyze whether the introduction of highly active antiretroviral treatment (HAART) would affect the persistence of immunity. The immune responses were evaluated in patients who had participated in randomized trials of therapeutic vaccination. Immunization in conjunction with antiretroviral therapy was effective in inducing HIV-specific T-cell responses. Therapeutic immunizations with recombinant gp160 had a modest effect on CD4-cell counts, the treatment alone lead to a transient clinical benefit in the form of an improved survival after two years of immunization. Immunizations with HIV DNA during HAART treatment permitted persistence or development of innate (NK), CD4+ and/or CD8+ immune responses. HIV specific T-helper cell responses induced by immunization with gp160 were maintained at high levels up to 7 years after the last injection. Cells with HIV-specific interferon-gamma (IFN-gamma) production were retained or increased in long-term HAART treated patients. The impact of a single structured therapy interruption (STI) was analyzed in a small group of patients showing no obvious increase or decrease in the HIV-specific immune response during or after STI. The possibility to induce very long-term strong and persistent immune responses in HIV-infected individuals raises hopes that vaccination preceding therapy interruption might prolong the symptom-free period without HAART.     

Generation of an immune microenvironment as a novel mechanism for myotoxins to potentiate genetic vaccines

We recently reported that administration of low doses of myotoxins at vaccination sites potentiated antigen-specific T-cell immunity induced by genetic cancer vaccines in mice, an effect which was superior to TLR agonists. In the current study, we found unexpectedly that the mechanism of this potent adjuvant effect was immune-mediated. Myotoxins induced sterile inflammation at vaccination sites, associated with a predominant infiltration of dendritic cells (DC). Inhibition of DC recruitment abrogated the immune stimulation effect of myotoxins, suggesting the requirement for DC. Genetic profiling of myotoxin-treated tissues revealed characteristics of an immune microenvironment with up-regulation of chemokines, proinflammatory cytokines, Toll-like receptors (TLR) and their endogenous ligands, and activation of innate immunity. Mechanistic experiments in vivo also elucidated the requirement for genes triggering DC maturation including TLR signaling and CD40. These studies suggest that myotoxins-induced sterile inflammation generates a favorable microenvironment that promotes multiple stages in the development of adaptive immunity. This novel mechanism of immune potentiation may be exploited for development of adjuvants for genetic vaccines against infectious pathogens and cancer.     

Gene gun administration of therapeutic HPV DNA vaccination restores the efficacy of prolonged defrosted viral based vaccine

Freshly defrosted vaccines generate promising antitumor immunity by raising both robust CD8 and CD4 responses with a TC1/Th1-dominant cytokine profile. However, prolonged (overnight) defrosted Sindbis virus-E7/HSP70 priming and Vaccinia-E7/HSP70 booster in mouse model only elicited 20% long-term tumor-free survival in comparison with the fresh vaccines. The present study is to search the possible cause of its potency loss, and to evaluate the ability of pcDNA-E7/HSP70 DNA vaccination via gene gun in restoring the efficacy of E7-specific immune responses and antitumor properties. We used prolonged defrosted SINrep5-E7/HSP70 prime and defrostedVac-E7/HSP70 boost subcutaneously, and administered intradermally cluster (3-day interval) gene gun plasmid E7–HSP70DNA vaccine twice, and evaluated its ability to generate antigen-specific cytotoxic CD8+ T-cell responses using flow cytometry as well as antitumor responses using animal positron-emission tomography (PET) imaging. The prolonged defrosted vaccines showed a significant reduction in the infectivity and a significant decrease of CD8+ and CD4+ T-cells immune responses. Administration of cluster gene gun plasmid E7–HSP70DNA twice was also found to lead to restoration of immunity that elicits a full recovery of the antitumor efficacy of the prolonged defrosted vaccines. Our study suggested that adding cluster gene gun plasmid E7–HSP70DNA vaccine twice offered a simple solution in restoring the efficacy of the prime-boost vaccination with viral vectors and has potentially significant clinical applications.     

Considerations in the design of vaccines that induce CD8 T cell mediated immunity

The protective capacity of many currently used vaccines is based on induction of neutralizing antibodies. Many pathogens, however, have adapted themselves in different ways to escape antibody-based immune protection. In particular, for those infections against which conventional neutralizing antibody-based vaccinations appear challenging, CD8 T-cells are considered to be promising candidates for vaccine targeting. The design of vaccines that induce robust and long-lasting protective CD8 T-cell responses however imposes new challenges, as many factors such as kinetics and efficiency of antigen-processing and presentation by antigen presenting cells, T-cell repertoire and cytokine environment during T cell priming contribute to the specificity and functionality of CD8 T-cell responses. In the following, we review the most prominent aspects that underlie CD8 T-cell induction and discuss how this knowledge may help to improve the design of efficient CD8 T-cell inducing vaccines.     

Imiquimod and resiquimod in a mouse model: adjuvants for DNA vaccination by particle-mediated immunotherapeutic delivery

Imiquimod, an immune response modifier and inducer of cytokines in vitro and in vivo, has been shown to have potent antiviral and antitumour activity and to act as an adjuvant for protein vaccination. We have undertaken studies in mice to investigate the potential of imiquimod and resiquimod to adjuvant DNA vaccination. These imidazoquinolines were administered by subcutaneous injection at the vaccination site immediately after particle-mediated immunotherapeutic delivery of plasmid DNA using a gene gun. Imiquimod was found to increase the number and maturation status of dendritic cells in draining lymph nodes, and to enhance antigen-specific CD4(+) and CD8(+) T cell responses, as assessed by analyses of clonal expansion, and the quantity and kinetics of cytokine production from these cells in lymph nodes and spleens collected after vaccination. A more substantial increase in IFN-gamma-producing, compared with IL-4-producing CD4(+) T cells suggested that imiquimod biased the immune response towards a predominance of Th1 cells. The analogue resiquimod was found to be to produce a similar Th1 biased immune response with a 10-fold reduced dose compared with imiquimod. Collectively, these studies suggest that both imiquimod and resiquimod may be suitable adjuvants for therapeutic DNA vaccines requiring induction of potent cytotoxic T cell responses.     

Avipox-based simian immunodeficiency virus (SIV) vaccines elicit a high frequency of SIV-specific CD4+ and CD8+ T-cell responses in vaccinia-experienced SIVmac251-infected macaques

The ability of ALVAC- or fowlpox-based simian immunodeficiency virus (SIV) vaccines to boost SIV-specific CD4+ and CD8+ T-cell responses was tested in 10 vaccinia-experienced macaques infected with SIVmac251. The CD8+ T-cell response to the dominant Gag(181-189) CM9 was quantitated in seven Mamu-A*01-positive macaques by tetramer staining, by ex vivo cytotoxic T-lymphocyte (CTL) activity, and by intracellular cytokine staining (ICS) with the specific Gag(181-189) CM9 peptide. The overall CD8+ T-cell response to Gag was assessed using a peptide pool encompassing the entire Gag protein followed by measurement of TNF-alpha production in ICS assay. Similarly, virus-specific CD4+ T-cell responses were measured by ICS for TNF-alpha following stimulation with the Gag-overlapping peptide and by proliferative response following stimulation with purified p27 Gag. The two vaccine modalities effectively boosted both CD4+ and CD8+ SIV-specific T-cell response despite prior exposure to the vaccinia-derivative NYVAC vector, suggesting that sequential boosting with either avipox-based vector vaccine candidate is a realistic approach in immune therapy of human immunodeficiency virus type 1 (HIV-1)-infected individuals.