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Current therapies for the hepatitis B virus (HBV), a major cause of severe liver disease, suppress viral replication but replication rebounds if therapy is withdrawn. It is widely accepted that immune activation is needed to control replication off-therapy. To specifically activate T cells crossreactive between the hepatitis B core and e antigens (HBcAg/HBeAg) in chronically infected patients, we developed a therapeutic vaccine candidate. The vaccine encompass codon-optimized HBcAg and IL-12 expressing plasmids delivered using targeted high-pressure injection combined with in vivo electroporation. One dose of the vaccine primed a B-cell-independent polyfunctional T-cell response, in wild-type, and in HBeAg-transgenic mice with an impaired ability to respond to HBc/eAg. The response peaked at 2 weeks and contracted at week 6 after vaccination. Coadministration of IL-12 improved antibody levels, and T-cell expansion and functionality. The vaccine primed T cells that, 2 weeks after a single dose, cleared hepatocytes transiently expressing HBcAg in vaccinated wild-type and HBeAg-transgenic mice. However, 4 weeks later, these functional responses were lost. Booster doses after 8–12 weeks effectively restored function and expansion of the rapidly contracting T cells. Thus, this vaccine strategy primes functional HBcAg-specific T cells in a host with dysfunctional response to HBV.

A sustained viral response (SVR) after interferon-based therapy of chronic hepatitis C virus (HCV) infection is regarded to represent a cure. Previous studies have used different markers to clarify whether an SVR truly represents a cure, but no study has combined a clinical work-up with highly sensitive HCV RNA detection, and the determination of immune responses.To determine clinical, histological, virological and immunological markers 5-20 years after SVR.In 54 patients, liver biochemistry, histology and elastography were evaluated. Liver biopsies, plasma and peripheral blood mononuclear cells (PBMCs) were tested for minute amounts of HCV RNA. HCV-specific T-cell responses were monitored by ELISpot and pentamer staining, and humoral responses by measuring HCV nonstructural (NS)3-specific antibodies and virus neutralisation.Liver disease regressed significantly in all patients, and 51 were HCV RNA-negative in all tissues tested. There was an inverse association between liver disease, HCV-specific T-cell responses and HCV antibody levels with time from SVR, supporting that the virus had been cleared. The three patients, who all lacked signs of liver disease, had HCV RNA in PBMCs 5-9 years after SVR. All three had HCV-specific T cells and NS3 antibodies, but no cross-neutralising antibodies.Our combined data confirm that a SVR corresponds to a long-term clinical cure. The waning immune responses support the disappearance of the antigenic stimulus. Transient HCV RNA traces may be detected in some patients up to 9 years after SVR, but no marker associates this with an increased risk for liver disease.

Presently the development of new therapies for hepatitis C virus (HCV) is rapidly moving forward. Almost every week new data appear on how direct acting antivirals (DAAs) succeed or fail in clinical trials. Despite the potency of many of the DAA combinations, the effect exerted by ribavirin (RBV) is still needed for an effective therapy in many new DAA combinations. Due to the strong antiviral effect of DAAs, it is likely that a major complementary therapeutic effect exerted by RBV is immune modulation resulting in an increased barrier to development of resistance. For HCV genotype 1a infections elimination of pegylated interferon, is not possible in many DAA combinations without jeopardizing the results. The host immune response is thus likely to play a key role even during DAA-based therapies. Hence, T cells may recognize and eliminate viral variants with resistance to the DAAs. We herein show several examples where this may be the case, supporting the rationale of including the host response also in the new therapeutic regimens. This review will describe the potential benefits of combining various DAAs with means to activate the specific immune response against HCV.

After acute hepatitis C virus (HCV) infection, 20% of individuals clear the virus, which is dependent on sustained Th1 CD4+ T lymphocyte–mediated responses together with polyfunctional CD8+ T cells [1–4]. HCV is highly efficient in establishing persistent infections, and the specific T-cell responses are impaired during chronic infection [5–8] due to appearance of escape mutants [4, 9], inhibitory effects exerted by viral proteins [10], or expression of coinhibitory receptors resulting in T-cell exhaustion [11, 12]. One way to prime T cells and/or reactivate impaired T cells is to express HCV antigens under a more “immunogenic” setting than natural infection where massive antigen expression appears in the tolerogenic liver environment. Therapeutic vaccination has been tested with some success in HCV-infected patients showing evidence of T-cell activation [13, 14]. Vectored vaccines tailored to generate robust T-cell immunity have recently reached the clinic. MVATG16643, a modified virus Ankara (MVA)–based vaccine [15], has shown in a phase I trial good safety and the capacity to induce interferon γ (IFN-γ)–producing T cells with significant although transient viral load decrease in chronically infected patients [16]. Used in combination with pegylated (PEG)–interferon α (IFN-α)/ribavirin in a phase II clinical trial, MVATG16643 resulted in a significant early viral response [17]. The DNA vaccine ChronVac-C [18] has also shown the capacity to induce T cells, which had transient effects on viral load in a phase I/IIa trial [19], and it has been suggested that it improves cure rates when given before PEG–IFN-α/ribavirin treatment [19]. The combined use of DNA vaccines with viral vectors in a prime/boost regimen has been proven useful for enhancing response levels in clinical studies [20–22]. Similarly, in vivo electroporation (EP)–mediated delivery of DNA vaccines either as stand-alone or in a prime/boost setting with viral vectors has also served to enhance the development of polyfunctional CD8+ T-cell responses [23, 24]. Here we have performed a proof-of-concept study to define the extent of improvement that could result from a prime/boost approach based on ChronVac-C and MVATG16643—2 individual vaccines currently in the clinic. These 2 vaccine regimens have been optimized extensively individually previously, and we wanted to investigate whether the combination of 2 regimens could confer additional benefits over the individual regimens. This study thus combines for the first time in the HCV setting approaches previously shown individually to enhance immunogenicity (ie, DNA vaccine delivery with in vivo EP and prime/boost using viral vectors). We performed an exhaustive evaluation of this concept in wild-type C57BL/6J and human leucocyte antigen (HLA)–A2 transgenic mice. This strategy was found very potent for the improvement of polyfunctional CD4+ and CD8+ HCV-specific responses and resulted in a significant increase of epitope recognition.

Virus-specific CTL with high levels of functional avidity have been associated with viral clearance in hepatitis C virus (HCV) infection and with enhanced protective immunity. In chronic HCV infection, lack of antiviral CTL is frequently observed. In this study, we aim to investigate novel HCV TCRs that differ in Ag specificity. This involved isolating new HCV-specific murine TCRs that recognize a conserved HLA-A2–restricted CTL epitope within the nonstructural protein (NS) 5A viral protein and comparing them with TCRs recognizing another conserved CTL target in the NS3 viral protein. This was done by expressing the TCRs in human T cells and analyzing the function of the resulting TCR-transduced T cells. Our result indicates that these TCRs are efficiently assembled in transduced human T cells. They recognize peptide-loaded targets and demonstrate polyfunctional features such as IL-2, IFN-γ, and TNF-α secretion. However, in contrast to NS3-specific TCRs, the NS5A TCR-transduced T cells consist of a smaller proportion of polyfunctional T cells and require more peptide ligands to trigger the effector functions, including degranulation. Despite the differences, NS5A TCRs show effective inhibition of HCV replication in human hepatoma cells with persistent HCV RNA replication. Moreover, cellular injury demonstrated by aspartate aminotransferase release and cell death is less significant in the hepatoma cells following coincubation with NS5A TCR-transduced T cells, which is a property consistent with noncytotoxic antiviral CTLs. Our results suggest that HCV TCR-transduced T cells may be promising for the treatment of patients with chronic HCV infections.

Background The non-structural (NS) 3/4A protease/helicase of the hepatitis C virus is known to modulate signalling pathways in the infected hepatocyte by cleaving CARD adaptor inducing IFNβ (Cardif), T-cell protein tyrosine phosphatase (TC-PTP) and TIR domain-containing adaptor inducing IFNβ (TRIF), but the effects of NS3/4A in vivo still remain unclear. Aim To investigate the influence of NS3/4A on intracellular and intercellular signalling in vivo by analysing the intrahepatic inflammatory response of naive, lipopolysaccharide (LPS)/d-galactosamine (d-galN) or tumour necrosis factor-α (TNFα)/d-galN-treated NS3/4A-transgenic (Tg) mice. Methods The intrahepatic immunity of naive and LPS/d-galN- or TNFα/d-galN-treated NS3/4A-Tg mice was determined using western blot, ELISA, real-time PCR, flow cytometry and survival monitoring. The injection of cytokines or antibodies against signalling components was performed to analyse the relevance of the respective pathways for the investigated issues. A Tg mouse lineage expressing an inactivated NS3/4A protease (NS3/4A Ile1073Ala -Tgs) was generated to examine if protective effects were NS3/4A protease dependent. Results The activation of hepatic signal transducer and activator of transcription 1 and 2 was impaired in NS3/4A-Tg mice after treatment with LPS/d-galN or TNFα/d-galN. This was paralleled by a reduction in hepatic interferon-γ (IFNγ). Reconstitution of IFNγ reverted the resistance to LPS/TNFα in NS3/4A-Tg mice. Subsequently, blocking IFNγ in vivo rendered wild-type mice resistant against treatment with LPS/TNFα. A new Tg mouse expressing an inactivated NS3/4A protease had the same phenotype as wild-type mice with respect to hepatic IFNγ levels and sensitivity to LPS/d-galN. Finally, the chemokine profile was altered in the NS3/4A-Tg mice towards an anti-inflammatory state, which helps to explain the altered immune cell subsets and reduction in hepatic IFNγ production. Conclusions Our data demonstrate that the NS3/4A protease reduces the intrahepatic production of IFNγ and alters TNFα-mediated effects, thereby impairing the hepatic inflammatory response. This may contribute to viral persistence.

Hepatitis B virus core antigen (HBcAg) is thought to be a major target for specific cytotoxic T cells (CTLs) in hepatitis B virus infections. A single dose of hepatitis C virus nonstructural 3/4A DNA (

Extracellular NAD induces the ATP-independent activation of the ionotropic P2X7 purinergic receptor (P2X7R) in murine T lymphocytes via a novel covalent pathway involving ADP-ribosylation of arginine residues on the P2X7R ectodomain. This modification is catalyzed by ART2.2, a GPI-anchored ADP-ribosyltransferase (ART) that is constitutively expressed in murine T cells. We previously reported that ART2.1, a related ecto-ART, is up-regulated in inflammatory murine macrophages that constitutively express P2X7R. Thus, we tested the hypothesis that extracellular NAD acts via ART2.1 to regulate P2X7R function in murine macrophages. Coexpression of the cloned murine P2X7R with ART2.1 or ART2.2 in HEK293 cells verified that P2X7R is an equivalent substrate for ADP-ribosylation by either ART2.1 or ART2.2. However, in contrast with T cells, the stimulation of macrophages or HEK293 cells with NAD alone did not activate the P2X7R. Rather, NAD potentiated ATP-dependent P2X7R activation as indicated by a left shift in the ATP dose-response relationship. Thus, extracellular NAD regulates the P2X7R in both macrophages and T cells but via distinct mechanisms. Although ADP-ribosylation is sufficient to gate a P2X7R channel opening in T cells, this P2X7R modification in macrophages does not gate the channel but decreases the threshold for gating in response to ATP binding. These findings indicate that extracellular NAD and ATP can act synergistically to regulate P2X7R signaling in murine macrophages and also suggest that the cellular context in which P2X7R signaling occurs differs between myeloid and lymphoid leukocytes.

ADP-ribosylation of cell surface proteins in mammalian cells is a post-translational modification by which ecto-ADP-ribosyltransferases (ARTs) transfer ADP-ribose from extracellular NAD to protein targets. The ART2 locus at murine chromosome 7 encompasses the tandem Art2a and Art2b genes that encode the distinct ART2.1 and ART2.2 proteins. Although both ecto-enzymes share 80% sequence identity, ART2.1 activity is uniquely regulated by an allosteric disulfide bond that is reducible in the presence of extracellular thiols, such as cysteine and glutathione, that accumulate in hypoxic and ischemic tissues. Previous studies have characterized the expression of ART2.1 and ART2.2 in murine T lymphocytes but not in other major classes of lymphoid and myeloid leukocytes. Here, we describe the expression of ART2.1 activity in a wide range of freshly isolated or tissue-cultured murine myeloid and lymphoid leukocytes. Spleen-derived macrophages, dendritic cells (DC), and B cells constitutively express ART2.1 as their predominant ART while spleen T cells express both ART2.1 and the thiol-independent ART2.2 isoform. Although bone-marrow-derived macrophages (BMDM) and dendritic cells (BMDC) constitutively express ART2.1 at low levels, it is markedly up-regulated when these cells are stimulated in vitro with IFNbeta or IFNgamma. ART2.1 expression and activity in splenic B cells is modestly up-regulated during incubation in vitro for 24 h, a condition that promotes B cell apoptosis. This increase in ART2.1 is attenuated by IL-4 (a B cell survival factor), but is not affected by IFNbeta/gamma, suggesting a possible induction of ART2.1 as an ancillary response to B cell apoptosis. In contrast, ART2.1 and ART2.2, which are highly expressed in freshly isolated splenic T cells, are markedly down-regulated when purified T cells are incubated in vitro for 12-24 h. Studies with the BW5147 mouse thymocyte line verified basal expression of ART2.1 and ART2.2, as in primary spleen T cells, and demonstrated that both isoforms can be up-regulated when T cells are maintained in the presence of IFNs. Comparison of the surface proteins which are ADP-ribosylated by ART2.1 in the different leukocyte subtypes indicated both shared and cell-specific proteins as ART2.1 substrates. The LFA-1 integrin, a major target for ART2.2 in T cells, is also ADP-ribosylated by the ART2.1 expressed in macrophages. Thus, ART2.1, in contrast to ART2.2, is expressed in a broad range of myeloid and lymphoid leukocytes. The thiol redox-sensitive nature of this ecto-enzyme suggests an involvement in purinergic signaling that occurs in the combined context of inflammation and hypoxia/ischemia.

Ribavirin has proven to be a key component of hepatitis C therapies both involving IFNs and new direct-acting antivirals. The hepatitis C virus–mediated interference with intrahepatic immunity by cleavage of mitochondrial antiviral signaling protein (MAVS) and T cell protein tyrosine phosphatase (TCPTP) suggests an avenue for compounds that may counteract these effects. We therefore studied the effects of ribavirin, with or without inhibition of the nonstructural (NS)3/4A protease, on intrahepatic immunity. The intrahepatic immunity of wild-type and NS3/4A-transgenic mice was determined by Western blot, ELISA, flow cytometry, and survival analysis. Various MAVS or TCPTP constructs were injected hydrodynamically to study their relevance. Ribavirin pretreatment was performed in mice expressing a functional or inhibited NS3/4A protease to analyze its effect on NS3/4A-mediated changes. Intrahepatic NS3/4A expression made mice resistant to TNF-α–induced liver damage and caused an alteration of the intrahepatic cytokine (IFN-γ and IL-10) and chemokine (CCL3, CCL17, CCL22, CXCL9, and CXCL11) profiles toward an anti-inflammatory state. Consistent with this, the number of intrahepatic Th1 cells and IFN-γ+ T cells in NS3/4A-transgenic mice decreased, whereas the amount of Th2 cells increased. These effects could be reversed by injection of uncleavable TCPTP but not uncleavable MAVS and were absent in a mouse expressing a nonfunctional NS3/4A protease. Importantly, the NS3/4A-mediated effects were reversed by ribavirin treatment. Thus, cleavage of TCPTP by NS3/4A induces a shift of the intrahepatic immune response toward a nonantiviral Th2-dominated immunity. These effects are reversed by ribavirin, supporting that ribavirin complements the effects of direct-acting antivirals as an immunomodulatory compound.

The hepatitis C virus (HCV) nonstructural (NS) 5A protein has been shown to promote viral persistence by interfering with both innate and adaptive immunity. At the same time, the HCV NS5A protein has been suggested as a target for antiviral therapy. In this study, we performed a detailed characterization of HCV NS5A immunogenicity in wild-type (wt) and immune tolerant HCV NS5A-transgenic (Tg) C57BL/6J mice. We evaluated how efficiently HCV NS5A-based genetic vaccines could activate strong T cell responses. Truncated and full-length wt and synthetic codon-optimized NS5A genotype 1b genes were cloned into eukaryotic expression plasmids, and the immunogenicity was determined after i.m. immunization in combination with in vivo electroporation. The NS5A-based genetic vaccines primed high Ab levels, with IgG titers of >104 postimmunization. With respect to CD8+ T cell responses, the coNS5A gene primed more potent IFN-γ–producing and lytic cytotoxic T cells in wt mice compared with NS5A-Tg mice. In addition, high frequencies of NS5A-specific CD8+ T cells were found in wt mice after a single immunization. To test the functionality of the CTL responses, the ability to inhibit growth of NS5A-expressing tumor cells in vivo was analyzed after immunization. A single dose of coNS5A primed tumor-inhibiting responses in both wt and NS5A-Tg mice. Finally, immunization with the coNS5A gene primed polyfunctional NS5A-specific CD8+ T cell responses. Thus, the coNS5A gene is a promising therapeutic vaccine candidate for chronic HCV infections.

Hepatitis C virus (HCV) is a major cause of severe liver disease, and one major contributing factor is thought to involve a dysfunction of virus-specific T-cells. T-cell receptor (TCR) gene therapy with HCV-specific TCRs would increase the number of effector T-cells to promote virus clearance. We therefore took advantage of HLA-A2 transgenic mice to generate multiple TCR candidates against HCV using DNA vaccination followed by generation of stable T-cell–BW (T-BW) tumour hybrid cells. Using this approach, large numbers of non-structural protein 3 (NS3)-specific functional T-BW hybrids can be generated efficiently. These predominantly target the genetically stable HCV genotype 1 NS31073–1081 CTL epitope, frequently associated with clearance of HCV in humans. These T-BW hybrid clones recognized the NS31073 peptide with a high avidity. The hybridoma effectively recognized virus variants and targeted cells with low HLA-A2 expression, which has not been reported previously. Importantly, high-avidity murine TCRs effectively redirected human non-HCV-specific T-lymphocytes to recognize human hepatoma cells with HCV RNA replication driven by a subgenomic HCV replicon. Taken together, TCR candidates with a range of functional avidities, which can be used to study immune recognition of HCV-positive targets, have been generated. This has implications for TCR-related immunotherapy against HCV.

A major problem in chronic hepatitis B virus (HBV) infection is that treatment with specific antivirals is life-long since they rarely induce a sustained response. An attractive option is therefore to combine antiviral therapy with some type of immune stimulator, such as a therapeutic vaccine. Several lines of evidence suggest that a key target for the cellular immune response is the HBV core antigen (HBcAg). However, it may also be of advantage to simultaneously improve the neutralizing antibody response to the surface (S) region of HBV. We therefore generated chimeric HBcAg particles expressing preS1 residues 1–42 at the tip of the spike region. We could show that this chimeric HBcAg–preS1 protein primed both HBcAg-specific T cells and antibodies to preS1. This strongly suggests that this may be a viable approach to develop an effective bi-functional therapeutic vaccine as an add-on for the treatment of chronic HBV infections.

The hepatitis C virus (HCV)-specific T cell response in patients with chronic HCV is dysfunctional. In this study, we aimed at restoring immunological function through therapeutic vaccination in a transgenic mouse model with impaired HCV-specific T cell responses due to a persistent presence of hepatic HCV nonstructural (NS)3/4A Ags. The HCV-specific T cells have an actively maintained dysfunction reflected in reduced frequency, impaired cytokine production, and impaired effector function in vivo, which can be partially restored by blocking regulatory T cells or programmed cell death ligand 1. We hypothesized that the impairment could be corrected by including sequences that created a normal priming environment by recruiting “healthy” heterologous T cells and by activating innate signaling. Endogenously expressed hepatitis B core Ag (HBcAg) can recruit heterologous T cells and activate TLR (TLR7) signaling. Hence, by combining HCV NS3/4A with different forms of HBcAg we found that heterologous sequences somewhat improved activation and expansion of NS3/4A-specific T cells in a wild-type host. Importantly, the signals provided by HBcAg effectively restored the activation of HCV-specific T cells in a tolerant NS3/4A-transgenic mouse model. The adjuvant effect could also be transferred to the priming of dysfunctional HLA-A2–restricted NS3-specific T cells in vivo. Thus, recruiting healthy heterologous T cells to the site of priming may also help restore HCV-specific responses present in a chronically infected host.

Extracellular NAD and ATP exert multiple, partially overlapping effects on immune cells. Catabolism of both nucleotides by extracellular enzymes keeps extracellular concentrations low under steady-state conditions and generates metabolites that are themselves signal transducers. ATP and its metabolites signal through purinergic P2 and P1 receptors, whereas extracellular NAD exerts its effects by serving as a substrate for ADP-ribosyltransferases (ARTs) and NAD glycohydrolases/ADPR cyclases like CD38 and CD157. Both nucleotides activate the P2X7 purinoceptor, although by different mechanisms and with different characteristics. While ATP activates P2X7 directly as a soluble ligand, activation via NAD occurs by ART-dependent ADP-ribosylation of cell surface proteins, providing an immobilised ligand. P2X7 activation by either route leads to phosphatidylserine exposure, shedding of CD62L, and ultimately to cell death. Activation by ATP requires high micromolar concentrations of nucleotide and is readily reversible, whereas NAD-dependent stimulation begins at low micromolar concentrations and is more stable. Under conditions of cell stress or inflammation, ATP and NAD are released into the extracellular space from intracellular stores by lytic and non-lytic mechanisms, and may serve as "danger signals" to alert the immune response to tissue damage. Since ART expression is limited to naïve/resting T cells, P2X7-mediated NAD-induced cell death (NICD) specifically targets this cell population. In inflamed tissue, NICD may inhibit bystander activation of unprimed T cells, reducing the risk of autoimmunity. In draining lymph nodes, NICD may eliminate regulatory T cells or provide space for the preferential expansion of primed cells, and thus help to augment an immune response.

positive at baseline and those with HCV antibody seroconversion during follow-up were tested for HCVRNA and sequenced (CoreHVR1/NS5B). Year of infection was estimated as one year after selfreported initiation of injecting. Trends in HCV genotype distribution were evaluated. Factors associated with genotype 3a infection were assessed using logistic regression. Results: Among 818 participants, HCV genotype prevalence was: G1a: 52% (n =422), G1b: 6% (n =46), G2a: 3% (n =22), G2b: 6% (n =53), G3a: 32% (n =263), G4a

Concentrations of TNF-a and IL-6 in supernatants and patient sera were assayed by ELISA. We designed a 5-plex Phosflow FACS protocol to determine activation of key transcription factors in innate immune signalling pathways (NF-kB, ERK1/2, STAT1 and STAT3) in CD14+ monocytes following stimulation with LPS and flagellin. Results: We included 28 patients and 6 normal controls. Patients with compensated cirrhosis had a selective defect in flagellininduced IL-6 production (330±117pg/ml) compared to patients with non-cirrhotic ALD or NAFLD (896±146pg/ml; p = 0.01) or healthy controls (764±96pg/ml). There were no significant differences in flagellin-induced TNFa, nor any differences between cirrhotics and non-cirrhotics in LPS-induced cytokines. There were no differences in serum TNF-a, IL-6 and RANTES, nor in cytokine production when monocytes from healthy volunteers were incubated with patients’ sera before stimulation with LPS or flagellin. Flagellin-induced ERK1/2 phosphorylation in CD14+ monocytes is greater in non-cirrhotics than in cirrhotics (32.8% vs 10.5%, p = 0.056), but there were no differences in LPS-induced ERK1/2 phosphorylation and LPSand flagellin-induced NF-kB phosphorylation. Conclusions: We find selective impairment of TLR5-mediated IL-6 production in patients with compensated cirrhosis compared to non-cirrhotic patients that may be related to defects in ERK1/2 phosphorylation. These data identify potential signalling pathways that may be involved in liver disease progression or in susceptibility of cirrhotic patients to bacterial infections.

The without a doubt major obstacle for making DNA vaccines a commercial success is delivery. If delivery cannot be made simple, cheap and effective, DNA vaccines may not become a viable option for human use. Numerous clinical trials have confirmed that a standard needle and syringe simply do not do the job, i.e., delivering the DNA payload inside the cell. Having recognized this shortcoming, investigators have developed several new approaches for DNA vaccine delivery. In particular, new types of delivery devices, originally intended for in vitro use, have been applied for in vivo delivery. These include particle bombardment or biolistic delivery, and in vivo electroporation (EP). Importantly, both techniques seem to overcome the size barrier, meaning that they work in both mice and larger animals. In vivo EP has the key features of improved DNA uptake, increased antigen expression and a local inflammation. These factors are essential to make DNA vaccines effective in a larger host. Early data from clinical trials with DNA vaccines delivered by in vivo EP are cautiously promising. Thus, we may be entering a new era of DNA vaccination where we start to see clinical effects in humans; however, these may also be accompanied by side effects, as the vaccines become more effective.