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Despite preventive vaccines for oncogenic human papillomaviruses (HPVs), cervical intraepithelial neoplasia (CIN) is common, and current treatments are ablative and can lead to long-term reproductive morbidity. We assessed whether VGX-3100, synthetic plasmids targeting HPV-16 and HPV-18 E6 and E7 proteins, delivered by electroporation, would cause histopathological regression in women with CIN2/3. Efficacy, safety, and immunogenicity of VGX-3100 were assessed in CIN2/3 associated with HPV-16 and HPV-18, in a randomised, double-blind, placebo-controlled phase 2b study. Patients from 36 academic and private gynaecology practices in seven countries were randomised (3:1) to receive 6 mg VGX-3100 or placebo (1 mL), given intramuscularly at 0, 4, and 12 weeks. Randomisation was stratified by age (<25 vs ≥25 years) and CIN2 versus CIN3 by computer-generated allocation sequence (block size 4). Funder and site personnel, participants, and pathologists were masked to treatment. The primary efficacy endpoint was regression to CIN1 or normal pathology 36 weeks after the first dose. Per-protocol and modified intention-to-treat analyses were based on patients receiving three doses without protocol violations, and on patients receiving at least one dose, respectively. The safety population included all patients who received at least one dose. The trial is registered at ClinicalTrials.gov (number NCT01304524) and EudraCT (number 2012-001334-33). Between Oct 19, 2011, and July 30, 2013, 167 patients received either VGX-3100 (n=125) or placebo (n=42). In the per-protocol analysis 53 (49·5%) of 107 VGX-3100 recipients and 11 (30·6%) of 36 placebo recipients had histopathological regression (percentage point difference 19·0 [95% CI 1·4–36·6]; p=0·034). In the modified intention-to-treat analysis 55 (48·2%) of 114 VGX-3100 recipients and 12 (30·0%) of 40 placebo recipients had histopathological regression (percentage point difference 18·2 [95% CI 1·3–34·4]; p=0·034). Injection-site reactions occurred in most patients, but only erythema was significantly more common in the VGX-3100 group (98/125, 78·4%) than in the placebo group (24/42, 57·1%; percentage point difference 21·3 [95% CI 5·3–37·8]; p=0·007). VGX-3100 is the first therapeutic vaccine to show efficacy against CIN2/3 associated with HPV-16 and HPV-18. VGX-3100 could present a non-surgical therapeutic option for CIN2/3, changing the treatment outlook for this common disease. Inovio Pharmaceuticals.

The AIM2 inflammasome induces maturation of the proinflammatory cytokines IL-1β and IL-18. Using AIM2-deficient mice, Fitzgerald and colleagues and Alnemri and colleagues show that the AIM2 inflammasome is essential for host defense against cytosolic bacteria and DNA viruses. Inflammasomes regulate the activity of caspase-1 and the maturation of interleukin 1β (IL-1β) and IL-18. AIM2 has been shown to bind DNA and engage the caspase-1-activating adaptor protein ASC to form a caspase-1-activating inflammasome. Using Aim2 -deficient mice, we identify a central role for AIM2 in regulating caspase-1-dependent maturation of IL-1β and IL-18, as well as pyroptosis, in response to synthetic double-stranded DNA. AIM2 was essential for inflammasome activation in response to Francisella tularensis , vaccinia virus and mouse cytomegalovirus and had a partial role in the sensing of Listeria monocytogenes . Moreover, production of IL-18 and natural killer cell–dependent production of interferon-γ, events critical in the early control of virus replication, were dependent on AIM2 during mouse cytomegalovirus infection in vivo . Collectively, our observations demonstrate the importance of AIM2 in the sensing of both bacterial and viral pathogens and in triggering innate immunity.

The Zika virus epidemic and associated congenital infections have prompted rapid vaccine development. We assessed two new DNA vaccines expressing premembrane and envelope Zika virus structural proteins. We did two phase 1, randomised, open-label trials involving healthy adult volunteers. The VRC 319 trial, done in three centres, assessed plasmid VRC5288 (Zika virus and Japanese encephalitis virus chimera), and the VRC 320, done in one centre, assessed plasmid VRC5283 (wild-type Zika virus). Eligible participants were aged 18–35 years in VRC19 and 18–50 years in VRC 320. Participants were randomly assigned 1:1 by a computer-generated randomisation schedule prepared by the study statistician. All participants received intramuscular injection of 4 mg vaccine. In VRC 319 participants were assigned to receive vaccinations via needle and syringe at 0 and 8 weeks, 0 and 12 weeks, 0, 4, and 8 weeks, or 0, 4, and 20 weeks. In VRC 320 participants were assigned to receive vaccinations at 0, 4, and 8 weeks via single-dose needle and syringe injection in one deltoid or split-dose needle and syringe or needle-free injection with the Stratis device (Pharmajet, Golden, CO, USA) in each deltoid. Both trials followed up volunteers for 24 months for the primary endpoint of safety, assessed as local and systemic reactogenicity in the 7 days after each vaccination and all adverse events in the 28 days after each vaccination. The secondary endpoint in both trials was immunogenicity 4 weeks after last vaccination. These trials are registered with ClinicalTrials.gov, numbers NCT02840487 and NCT02996461. VRC 319 enrolled 80 participants (20 in each group), and VRC 320 enrolled 45 participants (15 in each group). One participant in VRC 319 and two in VRC 320 withdrew after one dose of vaccine, but were included in the safety analyses. Both vaccines were safe and well tolerated. All local and systemic symptoms were mild to moderate. In both studies, pain and tenderness at the injection site was the most frequent local symptoms (37 [46%] of 80 participants in VRC 319 and 36 [80%] of 45 in VRC 320) and malaise and headache were the most frequent systemic symptoms (22 [27%] and 18 [22%], respectively, in VRC 319 and 17 [38%] and 15 [33%], respectively, in VRC 320). For VRC5283, 14 of 14 (100%) participants who received split-dose vaccinations by needle-free injection had detectable positive antibody responses, and the geometric mean titre of 304 was the highest across all groups in both trials. VRC5283 was well tolerated and has advanced to phase 2 efficacy testing. Intramural Research Program of the Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health.

DNA double-strand breaks (DSBs) are harmful lesions leading to genomic instability or diversity. Non-homologous end-joining (NHEJ) is a prominent DSB repair pathway, which has long been considered to be error-prone. However, recent data have pointed to the intrinsic precision of NHEJ. Three reasons can account for the apparent fallibility of NHEJ: 1) the existence of a highly error-prone alternative end-joining process; 2) the adaptability of canonical C-NHEJ (Ku- and Xrcc4/ligase IV-dependent) to imperfect complementary ends; and 3) the requirement to first process chemically incompatible DNA ends that cannot be ligated directly. Thus, C-NHEJ is conservative but adaptable, and the accuracy of the repair is dictated by the structure of the DNA ends rather than by the C-NHEJ machinery. We present data from different organisms that describe the conservative/versatile properties of C-NHEJ. The advantages of the adaptability/versatility of C-NHEJ are discussed for the development of the immune repertoire and the resistance to ionizing radiation, especially at low doses, and for targeted genome manipulation.

Innate immunity is the first immunological defence against pathogens. During virus infection detection of nucleic acids is crucial for the inflammatory response. Here we identify DNA-dependent protein kinase (DNA-PK) as a DNA sensor that activates innate immunity. We show that DNA-PK acts as a pattern recognition receptor, binding cytoplasmic DNA and triggering the transcription of type I interferon (IFN), cytokine and chemokine genes in a manner dependent on IFN regulatory factor 3 (IRF-3), TANK-binding kinase 1 (TBK1) and stimulator of interferon genes (STING). Both cells and mice lacking DNA-PKcs show attenuated cytokine responses to both DNA and DNA viruses but not to RNA or RNA virus infection. DNA-PK has well-established functions in the DNA repair and V(D)J recombination, hence loss of DNA-PK leads to severe combined immunodeficiency (SCID). However, we now define a novel anti-microbial function for DNA-PK, a finding with implications for host defence, vaccine development and autoimmunity. For multicellular organisms, the innate immune system is the first immunological defence against infection, rapidly recognizing and responding to the presence of any pathogen. Many different cell types contribute to the innate immunity, including fibroblasts, epithelial cells, dendritic cells and macrophages. Once alerted to injury or infection, these cells release proteins called cytokines, interferons and chemokines into the blood or directly into tissue. These proteins act as messengers and interact with receptors on the surfaces of other cells in the immune system, stimulating them to join the battle against the infection. Detecting nucleic acids such as DNA is an important part of recognizing pathogens and infectious agents, particularly viruses, and activating the innate immune system. However, while the presence of DNA in the cytoplasm is known to initiate an innate immune response, we do not fully understand how this foreign DNA is sensed, or how the innate immune system is activated once foreign DNA has been detected. Here Ferguson et al. report that a well-known complex of three proteins, collectively called DNA-dependent protein kinase, is able to activate an innate immune response when it detects foreign DNA. This enzyme, called DNA-PK for short, is best known for its ability to repair broken DNA inside the nucleus. Now Ferguson et al. have found that it is also present at high levels within fibroblasts, cells that are often primary targets of viral infection, and they go on to explain how the detection of DNA by DNA-PK triggers a sequence of events that leads to the innate immune response being activated. These events include the transcription of type I interferon, chemokines and cytokines in a manner that depends on the presence IRF-3, a transcription factor that has a central role in the response of the immune system to viral infection. By identifying a role for DNA-PK in the cytoplasm as a DNA sensor, the work of Ferguson et al. increases our understanding of innate immunity. It may also, in the future, lead to an improved understanding of autoimmunity, and might also assist in the development of more immunogenic vaccines based on DNA or microbes that contain DNA.

Multivalent presentation of ligands often enhances receptor activation and downstream signalling. DNA origami offers a precise nanoscale spacing of ligands, a potentially useful feature for therapeutic nanoparticles. Here we use a square-block DNA origami platform to explore the importance of the spacing of CpG oligonucleotides. CpG engages Toll-like receptors and therefore acts to activate dendritic cells. Through in vitro cell culture studies and in vivo tumour treatment models, we demonstrate that square blocks induce Th1 immune polarization when CpG is spaced at 3.5 nm. We observe that this DNA origami vaccine enhances DC activation, antigen cross-presentation, CD8 T-cell activation, Th1-polarized CD4 activation and natural-killer-cell activation. The vaccine also effectively synergizes with anti-PD-L1 for improved cancer immunotherapy in melanoma and lymphoma models and induces long-term T-cell memory. Our results suggest that DNA origami may serve as a platform for controlling adjuvant spacing and co-delivering antigens in vaccines. The spacing of ligands presented to cells can have a huge impact on cellular responses. DNA origami is used to block structures to control the distribution of Toll-like receptor ligands and optimize presentation in the activation of dendritic cells in cancer immunotherapy.

The Ebola epidemics in west Africa and the Democratic Republic of the Congo highlight an urgent need for safe and effective vaccines to prevent Ebola virus disease. We aimed to assess the safety and long-term immunogenicity of a two-dose heterologous vaccine regimen, comprising the adenovirus type 26 vector-based vaccine encoding the Ebola virus glycoprotein (Ad26.ZEBOV) and the modified vaccinia Ankara vector-based vaccine, encoding glycoproteins from Ebola virus, Sudan virus, and Marburg virus, and the nucleoprotein from the Tai Forest virus (MVA-BN-Filo), in Sierra Leone, a country previously affected by Ebola. The trial comprised two stages: an open-label, non-randomised stage 1, and a randomised, double-blind, controlled stage 2. The study was done at three clinics in Kambia district, Sierra Leone. In stage 1, healthy adults (aged ≥18 years) residing in or near Kambia district, received an intramuscular injection of Ad26.ZEBOV (5 × 1010 viral particles) on day 1 (first dose) followed by an intramuscular injection of MVA-BN-Filo (1 × 108 infectious units) on day 57 (second dose). An Ad26.ZEBOV booster vaccination was offered at 2 years after the first dose to stage 1 participants. The eligibility criteria for adult participants in stage 2 were consistent with stage 1 eligibility criteria. Stage 2 participants were randomly assigned (3:1), by computer-generated block randomisation (block size of eight) via an interactive web-response system, to receive either the Ebola vaccine regimen (Ad26.ZEBOV followed by MVA-BN-Filo) or an intramuscular injection of a single dose of meningococcal quadrivalent (serogroups A, C, W135, and Y) conjugate vaccine (MenACWY; first dose) followed by placebo on day 57 (second dose; control group). Study team personnel, except those with primary responsibility for study vaccine preparation, and participants were masked to study vaccine allocation. The primary outcome was the safety of the Ad26.ZEBOV and MVA-BN-Filo vaccine regimen, which was assessed in all participants who had received at least one dose of study vaccine. Safety was assessed as solicited local and systemic adverse events occurring in the first 7 days after each vaccination, unsolicited adverse events occurring in the first 28 days after each vaccination, and serious adverse events or immediate reportable events occurring up to each participant's last study visit. Secondary outcomes were to assess Ebola virus glycoprotein-specific binding antibody responses at 21 days after the second vaccine in a per-protocol set of participants (ie, those who had received both vaccinations within the protocol-defined time window, had at least one evaluable post-vaccination sample, and had no major protocol deviations that could have influenced the immune response) and to assess the safety and tolerability of the Ad26.ZEBOV booster vaccination in stage 1 participants who had received the booster dose. This study is registered at ClinicalTrials.gov, NCT02509494. Between Sept 30, 2015, and Oct 19, 2016, 443 participants (43 in stage 1 and 400 in stage 2) were enrolled; 341 participants assigned to receive the Ad26.ZEBOV and MVA-BN-Filo regimen and 102 participants assigned to receive the MenACWY and placebo regimen received at least one dose of study vaccine. Both regimens were well tolerated with no safety concerns. In stage 1, solicited local adverse events (mostly mild or moderate injection-site pain) were reported in 12 (28%) of 43 participants after Ad26.ZEBOV vaccination and in six (14%) participants after MVA-BN-Filo vaccination. In stage 2, solicited local adverse events were reported in 51 (17%) of 298 participants after Ad26.ZEBOV vaccination, in 58 (24%) of 246 after MVA-BN-Filo vaccination, in 17 (17%) of 102 after MenACWY vaccination, and in eight (9%) of 86 after placebo injection. In stage 1, solicited systemic adverse events were reported in 18 (42%) of 43 participants after Ad26.ZEBOV vaccination and in 17 (40%) after MVA-BN-Filo vaccination. In stage 2, solicited systemic adverse events were reported in 161 (54%) of 298 participants after Ad26.ZEBOV vaccination, in 107 (43%) of 246 after MVA-BN-Filo vaccination, in 51 (50%) of 102 after MenACWY vaccination, and in 39 (45%) of 86 after placebo injection. Solicited systemic adverse events in both stage 1 and 2 participants included mostly mild or moderate headache, myalgia, fatigue, and arthralgia. The most frequent unsolicited adverse event after the first dose was headache in stage 1 and malaria in stage 2. Malaria was the most frequent unsolicited adverse event after the second dose in both stage 1 and 2. No serious adverse event was considered related to the study vaccine, and no immediate reportable events were observed. In stage 1, the safety profile after the booster vaccination was not notably different to that observed after the first dose. Vaccine-induced humoral immune responses were observed in 41 (98%) of 42 stage 1 participants (geometric mean binding antibody concentration 4784 ELISA units [EU]/mL [95% CI 3736–6125]) and in 176 (98%) of 179 stage 2 participants (3810 EU/mL [3312–4383]) at 21 days after the second vaccination. The Ad26.ZEBOV and MVA-BN-Filo vaccine regimen was well tolerated and immunogenic, with persistent humoral immune responses. These data support the use of this vaccine regimen for Ebola virus disease prophylaxis in adults. Innovative Medicines Initiative 2 Joint Undertaking and Janssen Vaccines & Prevention BV.

Where is thy STING? The adaptor protein STING ('stimulator of interferon genes', also known as MITA and ERIS) is emerging as an important component of the innate immune system's response to microbial DNA. Ishikawa et al . show that in the absence of STING the sensitivity of mice to infection by several DNA and RNA viruses is enhanced. STING-mediated interferon induction requires STING to relocalize with TANK-binding kinase 1 from the endoplasmic reticulum to Sec5-containing endosome vesicles. This work implies that STING is essential for host defence against DNA pathogens such as herpes simplex virus. Although the innate immune system is known to be critical for the early detection of invading pathogens and for initiating host defence systems, little is known about how it is galvanized to respond to DNA-based microbes. STING (stimulator of interferon genes) is now shown to be necessary for the initiation of effective type I interferon production and, accordingly, there is an increase in the susceptibility of Sting -knockout mice to infection by the DNA virus HSV-1. The innate immune system is critical for the early detection of invading pathogens and for initiating cellular host defence countermeasures, which include the production of type I interferon (IFN) 1 , 2 , 3 . However, little is known about how the innate immune system is galvanized to respond to DNA-based microbes. Here we show that STING (stimulator of interferon genes) is critical for the induction of IFN by non-CpG intracellular DNA species produced by various DNA pathogens after infection 4 . Murine embryonic fibroblasts, as well as antigen presenting cells such as macrophages and dendritic cells (exposed to intracellular B-form DNA, the DNA virus herpes simplex virus 1 (HSV-1) or bacteria Listeria monocytogenes ), were found to require STING to initiate effective IFN production. Accordingly, Sting -knockout mice were susceptible to lethal infection after exposure to HSV-1. The importance of STING in facilitating DNA-mediated innate immune responses was further evident because cytotoxic T-cell responses induced by plasmid DNA vaccination were reduced in Sting -deficient animals. In the presence of intracellular DNA, STING relocalized with TANK-binding kinase 1 (TBK1) from the endoplasmic reticulum to perinuclear vesicles containing the exocyst component Sec5 (also known as EXOC2). Collectively, our studies indicate that STING is essential for host defence against DNA pathogens such as HSV-1 and facilitates the adjuvant activity of DNA-based vaccines.

Background. Human immunodeficiency virus (HIV) vaccine development remains a global priority. We describe the safety and immunogenicity of a multiclade DNA vaccine prime with a replication-defective recombinant adenovirus serotype 5 (rAd5) boost. Methods. The vaccine is a 6-plasmid mixture encoding HIV envelope (env) subtypes A, B, and C and subtype B gag, pol, and nef, and an rAd5 expressing identical genes, with the exception of nef. Three hundred and twentyfour participants were randomized to receive placebo (n = 138), a single dose of rAd5 at 1010 (n = 24) or 1011 particle units (n = 24), or DNA at 0, 1, and 2 months, followed by rAd5 at either 1010 (n = 114) or 1011 particle units (n = 24) boosting at 6 months. Participants were followed up for 24 weeks after the final vaccination. Results. The vaccine was safe and well tolerated. HIV-specific T cell responses were detected in 63% of vaccinees. Titers of preexisting Ad5 neutralizing antibody did not affect the frequency and magnitude of T cell responses in prime-boost recipients but did affect the response rates in participants that received rAd5 alone (P = .037). Conclusion. The DNA/rAd5 vaccination regimen was safe and induced HIV type 1 multi-clade T cell responses, which were not significantly affected by titers of preexisting rAd5 neutralizing antibody. Trial Registration. ClinicalTrials.gov identifier: NCT00123968.

Children account for a substantial proportion of cases and deaths from Ebola virus disease. We aimed to assess the safety and immunogenicity of a two-dose heterologous vaccine regimen, comprising the adenovirus type 26 vector-based vaccine encoding the Ebola virus glycoprotein (Ad26.ZEBOV) and the modified vaccinia Ankara vector-based vaccine, encoding glycoproteins from the Ebola virus, Sudan virus, and Marburg virus, and the nucleoprotein from the Tai Forest virus (MVA-BN-Filo), in a paediatric population in Sierra Leone. This randomised, double-blind, controlled trial was done at three clinics in Kambia district, Sierra Leone. Healthy children and adolescents aged 1–17 years were enrolled in three age cohorts (12–17 years, 4–11 years, and 1–3 years) and randomly assigned (3:1), via computer-generated block randomisation (block size of eight), to receive an intramuscular injection of either Ad26.ZEBOV (5 × 1010 viral particles; first dose) followed by MVA-BN-Filo (1 × 108 infectious units; second dose) on day 57 (Ebola vaccine group), or a single dose of meningococcal quadrivalent (serogroups A, C, W135, and Y) conjugate vaccine (MenACWY; first dose) followed by placebo (second dose) on day 57 (control group). Study team personnel (except for those with primary responsibility for study vaccine preparation), participants, and their parents or guardians were masked to study vaccine allocation. The primary outcome was safety, measured as the occurrence of solicited local and systemic adverse symptoms during 7 days after each vaccination, unsolicited systemic adverse events during 28 days after each vaccination, abnormal laboratory results during the study period, and serious adverse events or immediate reportable events throughout the study period. The secondary outcome was immunogenicity (humoral immune response), measured as the concentration of Ebola virus glycoprotein-specific binding antibodies at 21 days after the second dose. The primary outcome was assessed in all participants who had received at least one dose of study vaccine and had available reactogenicity data, and immunogenicity was assessed in all participants who had received both vaccinations within the protocol-defined time window, had at least one evaluable post-vaccination sample, and had no major protocol deviations that could have influenced the immune response. This study is registered at ClinicalTrials.gov, NCT02509494. From April 4, 2017, to July 5, 2018, 576 eligible children or adolescents (192 in each of the three age cohorts) were enrolled and randomly assigned. The most common solicited local adverse event during the 7 days after the first and second dose was injection-site pain in all age groups, with frequencies ranging from 0% (none of 48) of children aged 1–3 years after placebo injection to 21% (30 of 144) of children aged 4–11 years after Ad26.ZEBOV vaccination. The most frequently observed solicited systemic adverse event during the 7 days was headache in the 12–17 years and 4–11 years age cohorts after the first and second dose, and pyrexia in the 1–3 years age cohort after the first and second dose. The most frequent unsolicited adverse event after the first and second dose vaccinations was malaria in all age cohorts, irrespective of the vaccine types. Following vaccination with MenACWY, severe thrombocytopaenia was observed in one participant aged 3 years. No other clinically significant laboratory abnormalities were observed in other study participants, and no serious adverse events related to the Ebola vaccine regimen were reported. There were no treatment-related deaths. Ebola virus glycoprotein-specific binding antibody responses at 21 days after the second dose of the Ebola virus vaccine regimen were observed in 131 (98%) of 134 children aged 12–17 years (9929 ELISA units [EU]/mL [95% CI 8172–12 064]), in 119 (99%) of 120 aged 4–11 years (10 212 EU/mL [8419–12 388]), and in 118 (98%) of 121 aged 1–3 years (22 568 EU/mL [18 426–27 642]). The Ad26.ZEBOV and MVA-BN-Filo Ebola vaccine regimen was well tolerated with no safety concerns in children aged 1–17 years, and induced robust humoral immune responses, suggesting suitability of this regimen for Ebola virus disease prophylaxis in children. Innovative Medicines Initiative 2 Joint Undertaking and Janssen Vaccines & Prevention BV.