Co-immunization with DNA vaccines encoding yidR and IL-17 augments host immune response against Klebsiella pneumoniae infection in mouse model.

Klebsiella pneumoniae is an important gram-negative bacterium that causes severe respiratory and healthcare-associated infections. Although antibiotic therapy is applied to treat severe infections caused by K. pneumoniae, drug-resistant isolates pose a huge challenge to clinical practices owing to adverse reactions and the mismanagement of antibiotics. Several studies have attempted to develop vaccines against K. pneumoniae, but there are no licensed vaccines available for the control of K. pneumoniae infection. In the current study, we constructed a novel DNA vaccine, pVAX1-YidR, which encodes a highly conserved virulence factor YidR and a recombinant expression plasmid pVAX1-IL-17 encoding Interleukin-17 (IL-17) as a molecular adjuvant. Adaptive immune responses were assessed in immunized mice to compare the immunogenicity of the different vaccine schemes. The results showed that the targeted antigen gene was expressed in HEK293T cells using an immunofluorescence assay. Mice immunized with pVAX1-YidR elicited a high level of antibodies, induced strong cellular immune responses, and protected mice from K. pneumoniae challenge. Notably, co-immunization with pVAX1-YidR and pVAX1-IL-17 significantly augmented host adaptive immune responses and provided better protection against K. pneumoniae infections in vaccinated mice. Our study demonstrates that combined DNA vaccines and molecular adjuvants is a promising strategy to develop efficacious antibacterial vaccines against K. pneumoniae infections.

The XCL1-Mediated DNA Vaccine Targeting Type 1 Conventional Dendritic Cells Combined with Gemcitabine and Anti-PD1 Antibody Induces Potent Antitumor Immunity in a Mouse Lung Cancer Model.

With the advent of cancer immunotherapy, there is a growing interest in vaccine development as a means to activate the cellular immune system against cancer. Despite the promise of DNA vaccines in this regard, their effectiveness is hindered by poor immunogenicity, leading to modest therapeutic outcomes across various cancers. The role of Type 1 conventional dendritic cells (cDC1), capable of cross-presenting vaccine antigens to activate CD8+T cells, emerges as crucial for the antitumor function of DNA vaccines. To address the limitations of DNA vaccines, a promising approach involves targeting antigens to cDC1 through the fusion of XCL1, a ligand specific to the receptor XCR1 on the surface of cDC1. Here, female C57BL/6 mice were selected for tumor inoculation and immunotherapy. Additionally, recognizing the complexity of cancer, this study explored the use of combination therapies, particularly the combination of cDC1-targeted DNA vaccine with the chemotherapy drug Gemcitabine (Gem) and the anti-PD1 antibody in a mouse lung cancer model. The study's findings indicate that fusion antigens with XCL1 effectively enhance both the immunogenicity and antitumor effects of DNA vaccines. Moreover, the combination of the cDC1-targeted DNA vaccine with Gemcitabine and anti-PD1 antibody in the mouse lung cancer model demonstrates an improved antitumor effect, leading to the prolonged survival of mice. In conclusion, this research provides important support for the clinical investigation of cDC1-targeting DNA vaccines in combination with other therapies.

DNA Vaccines: History, Molecular Mechanisms and Future Perspectives.

The history of DNA vaccine began as early as the 1960s with the discovery that naked DNA can transfect mammalian cells in vivo. In 1992, the evidence that such transfection could lead to the generation of antigen-specific antibody responses was obtained and supported the development of this technology as a novel vaccine platform. The technology then attracted immense interest and high hopes in vaccinology, as evidence of high immunogenicity and protection against virulent challenges accumulated from several animal models for several diseases. In particular, the capacity to induce T-cell responses was unprecedented in non-live vaccines. However, the technology suffered its major knock when the success in animals failed to translate to humans, where DNA vaccine candidates were shown to be safe but remained poorly immunogenic, or not associated with clinical benefit. Thanks to a thorough exploration of the molecular mechanisms of action of these vaccines, an impressive range of approaches have been and are currently being explored to overcome this major challenge. Despite limited success so far in humans as compared with later genetic vaccine technologies such as viral vectors and mRNA, DNA vaccines are not yet optimised for human use and may still realise their potential.

Th1 and Th2 epitopes of Cowdria polymorphic gene 1 of Ehrlichia ruminantium.

Cowdria polymorphic gene 1 (cpg1, Erum2510, ERUM_RS01380) has been shown to induce 30% and 100% protection in sheep immunised by deoxyribonucleic acid (DNA) prime combined with DNA boost and DNA prime combined with protein boost, respectively, against heartwater infection via needle challenge. To localise its antigenic regions for inclusion in a multi-epitope DNA vaccine against heartwater, Erum2510 was cleaved into five overlapping subfragments. These subfragments were expressed individually in an Escherichia coli host expression system and evaluated for their ability to induce proliferative responses, Th1 and Th2 cytokines (interferon gamma [IFN-γ] and interleukin 4 [IL-4]) via enzyme-linked immunospot (ELISpot), quantitative real time polymerase chain reaction (qRT-PCR) and flow cytometry. Recombinant (r)proteins 3 and 4 were shown to induce immunodominant Th1 and Th2 immune responses characterised by the secretion of effector cytokines IFN-γ and IL-4 in addition to differential messenger ribonucleic acid (mRNA) expression of tumour necrosis factor (TNF), IL-2, IL-1, IL-18, IL-10, transforming growth factor (TGF), granulocyte-macrophage colony-stimulating factor (GM-CSF) and inducible nitric oxide synthase (iNOS). Thirty-seven overlapping synthetic peptides (16 mer) spanning the lengths of these immunodominant rproteins were synthesised and assayed. A peptide pool comprising p9 and p10 derived from rprotein 3 induced a Th1-biased immune response. A peptide pool comprising p28 and p29 derived from rprotein 4 induced a mixed Th1 and Th2 immune response characterised by secretion of IFN-γ and differential mRNA expression of IL-1, IL-2, IL-10, IL-12, iNOS, TGF, TNF and GM-CSF. Only one of the peptides (p29) induced secretion of IL-4. Phenotypic analysis showed significant activation of cluster of differentiation 8+ (CD8+), cluster of differentiation 4+ (CD4+) and B+ lymphocyte populations. Findings suggest that Erum2510 rproteins and synthetic peptides can induce both cellular and humoral immune responses, thereby implicating their importance in protection against heartwater.Contribution: This study will facilitate the design of an effective multi-epitope DNA vaccine against heartwater that will contribute to control this economically important disease in sub-Saharan Africa and beyond.

Trimeric, APC-Targeted Subunit Vaccines Protect Mice against Seasonal and Pandemic Influenza.

Viral subunit vaccines contain the specific antigen deemed most important for development of protective immune responses. Typically, the chosen antigen is a surface protein involved in cellular entry of the virus, and neutralizing antibodies may prevent this. For influenza, hemagglutinin (HA) is thus a preferred antigen. However, the natural trimeric form of HA is often not considered during subunit vaccine development. Here, we have designed a vaccine format that maintains the trimeric HA conformation while targeting antigen toward major histocompatibility complex class II (MHCII) molecules or chemokine receptors on antigen-presenting cells (APC) for enhanced immunogenicity. Results demonstrated that a single DNA vaccination induced strong antibody and T-cell responses in mice. Importantly, a single DNA vaccination also protected mice from lethal challenges with influenza viruses H1N1 and H5N1. To further evaluate the versatility of the format, we developed MHCII-targeted HA from influenza A/California/04/2009(H1N1) as a protein vaccine and benchmarked this against Pandemrix and Flublok. These vaccine formats are different, but similar immune responses obtained with lower vaccine doses indicated that the MHCII-targeted subunit vaccine has an immunogenicity and efficacy that warrants progression to larger animals and humans. IMPORTANCE Subunit vaccines present only selected viral proteins to the immune system and allow for safe and easy production. Here, we have developed a novel vaccine where influenza hemagglutinin is presented in the natural trimeric form and then steered toward antigen-presenting cells for increased immunogenicity. We demonstrate efficient induction of antibodies and T-cell responses, and demonstrate that the vaccine format can protect mice against influenza subtypes H1N1, H5N1, and H7N1.

Preclinical evaluation of a precision medicine approach to DNA vaccination in type 1 diabetes.

Antigen-specific immunotherapy involves the delivery of self-antigens as proteins or peptides (or using nucleic acids encoding them) to reestablish tolerance. The Endotope platform supports the optimal presentation of endogenously expressed epitopes on appropriate major histocompatibility complex (MHC) class I and II molecules. Using specific epitopes that are disease-relevant (including neoepitopes and mimotopes) and restricted to the subject's MHC haplotypes provides a more focused and tailored way of targeting autoreactive T cells. We evaluated the efficacy of an Endotope DNA vaccine tailored to the nonobese diabetic (NOD) mouse in parallel to one expressing the Proinsulin protein, a central autoantigen in NOD mice, and assessed the influence of several parameters (e.g., route, dosing frequency, disease stage) on diabetes prevention. Secretion of encoded peptides and intradermal delivery of DNA offered more effective disease prevention. Long-term weekly treatments were needed to achieve protection that can persist after discontinuation, likely mediated by regulatory T cells induced by at least one epitope. Although epitopes were presented for at least 2 wk, weekly treatments were needed, at least initially, to achieve significant protection. While Endotope and Proinsulin DNA vaccines were effective at both the prediabetic normoglycemic and dysglycemic stages of disease, Proinsulin provided better protection in the latter stage, particularly in animals with slower progression of disease, and Endotope limited insulitis the most in the earlier stage. Thus, our data support the possibility of applying a precision medicine approach based on tailored epitopes for the treatment of tissue-specific autoimmune diseases with DNA vaccines.

Spherical nucleic acids as an infectious disease vaccine platform.

SignificanceUsing SARS-CoV-2 as a relevant case study for infectious disease, we investigate the structure-function relationships that dictate antiviral spherical nucleic acid (SNA) vaccine efficacy. We show that the SNA architecture can be rapidly employed to target COVID-19 through incorporation of the receptor-binding domain, and that the resulting vaccine potently activates human cells in vitro and mice in vivo. Furthermore, when challenged with a lethal viral infection, only mice treated with the SNA vaccine survived. Taken together, this work underscores the importance of rational vaccine design for infectious disease to yield vaccines that elicit more potent immune responses to effectively fight disease.

Enhanced Cross-Reactive and Polyfunctional Effector-Memory T Cell Responses by ICVAX-a Human PD1-Based Bivalent HIV-1 Gag-p41 Mosaic DNA Vaccine.

Vaccine-induced protective T cell immunity is necessary for HIV-1 functional cure. We previously reported that rhesus PD1-Gag-based DNA vaccination sustained simian-human immunodeficiency virus (SHIV) suppression by inducing effector-memory CD8+ T cells. Here, we investigated a human PD1-Gag-based DNA vaccine, namely, ICVAX, for clinical translation. PD1-based dendritic cell targeting and mosaic antigenic designs were combined to generate the ICVAX by fusing the human soluble PD1 domain with a bivalent HIV-1 Gag-p41 mosaic antigen. The mosaic antigen was cross-reactive with patients infected with B, CRF07/08_BC, and CRF01_AE variants. In mice, ICVAX elicited stronger, broader, and more polyfunctional T cell responses than mosaic Gag-p41 alone, and suppressed EcoHIV infection more efficiently. In macaques, ICVAX elicited polyfunctional effector-memory T cell responses that targeted multiple nonoverlapping epitopes of the Gag-p41 antigen. Furthermore, ICVAX manufactured following good manufacturing practices proved potent immunogenicity in macaques after biannual homologous vaccination, warranting clinical evaluation of ICVAX as an immunotherapy against HIV-1. IMPORTANCE This study presents that ICVAX, a PD1-based DNA vaccine against HIV-1, could induce broad and polyfunctional T cell responses against different HIV-1 subtypes. ICVAX encodes a recombinant antigen consisting of the human soluble PD1 domain fused with two mosaic Gag-p41 antigens. The mosaic antigens cover more than 500 HIV-1 strains circulating in China including the subtypes B/B', CRF01_AE, and CRF07/08_BC. In mice, ICVAX elicited stronger, broader, and more polyfunctional T cell responses, with better EcoHIV suppression than the nontargeting mosaic Gag-p41 DNA vaccine. Moreover, both lab-generated and GMP-grade ICVAX also elicited strong polyfunctional effector-memory T cell responses in rhesus macaques with good immunogenicity against multiple nonoverlapping epitopes of the Gag-p41 antigen. This study therefore highlights the great potential to translate the PD1-based DNA vaccine approach into clinical use, and opens up new avenues for alternative HIV-1 vaccine design for HIV-1 preventive and functional cure.

Induction of tier-2 neutralizing antibodies in mice with a DNA-encoded HIV envelope native like trimer.

HIV Envelope (Env) is the main vaccine target for induction of neutralizing antibodies. Stabilizing Env into native-like trimer (NLT) conformations is required for recombinant protein immunogens to induce autologous neutralizing antibodies(nAbs) against difficult to neutralize HIV strains (tier-2) in rabbits and non-human primates. Immunizations of mice with NLTs have generally failed to induce tier-2 nAbs. Here, we show that DNA-encoded NLTs fold properly in vivo and induce autologous tier-2 nAbs in mice. DNA-encoded NLTs also uniquely induce both CD4 + and CD8 + T-cell responses as compared to corresponding protein immunizations. Murine neutralizing antibodies are identified with an advanced sequencing technology. The structure of an Env-Ab (C05) complex, as determined by cryo-EM, identifies a previously undescribed neutralizing Env C3/V5 epitope. Beyond potential functional immunity gains, DNA vaccines permit in vivo folding of structured antigens and provide significant cost and speed advantages for enabling rapid evaluation of new HIV vaccines.

Therapeutic DNA Vaccines against HPV-Related Malignancies: Promising Leads from Clinical Trials.

In 2014 and 2021, two nucleic-acid vaccine candidates named MAV E2 and VGX-3100 completed phase III clinical trials in Mexico and U.S., respectively, for patients with human papillomavirus (HPV)-related, high-grade squamous intraepithelial lesions (HSIL). These well-tolerated but still unlicensed vaccines encode distinct HPV antigens (E2 versus E6+E7) to elicit cell-mediated immune responses; their clinical efficacy, as measured by HSIL regression or cure, was modest when compared with placebo or surgery (conization), but both proved highly effective in clearing HPV infection, which should help further optimize strategies for enhancing vaccine immunogenicity, toward an ultimate goal of preventing malignancies in millions of patients who are living with persistent, oncogenic HPV infection but are not expected to benefit from current, prophylactic vaccines. The major roadblocks to a highly efficacious and practical product remain challenging and can be classified into five categories: (i) getting the vaccines into the right cells for efficient expression and presentation of HPV antigens (fusion proteins or epitopes); (ii) having adequate coverage of oncogenic HPV types, beyond the current focus on HPV-16 and -18; (iii) directing immune protection to various epithelial niches, especially anogenital mucosa and upper aerodigestive tract where HPV-transformed cells wreak havoc; (iv) establishing the time window and vaccination regimen, including dosage, interval and even combination therapy, for achieving maximum efficacy; and (v) validating therapeutic efficacy in patients with poor prognosis because of advanced, recurrent or non-resectable malignancies. Overall, the room for improvements is still large enough that continuing efforts for research and development will very likely extend into the next decade.

Self-Assembled Particles Combining SARS-CoV-2 RBD Protein and RBD DNA Vaccine Induce Synergistic Enhancement of the Humoral Response in Mice.

Despite the fact that a range of vaccines against COVID-19 have already been created and are used for mass vaccination, the development of effective, safe, technological, and affordable vaccines continues. We have designed a vaccine that combines the recombinant protein and DNA vaccine approaches in a self-assembled particle. The receptor-binding domain (RBD) of the spike protein of SARS-CoV-2 was conjugated to polyglucin:spermidine and mixed with DNA vaccine (pVAXrbd), which led to the formation of particles of combined coronavirus vaccine (CCV-RBD) that contain the DNA vaccine inside and RBD protein on the surface. CCV-RBD particles were characterized with gel filtration, electron microscopy, and biolayer interferometry. To investigate the immunogenicity of the combined vaccine and its components, mice were immunized with the DNA vaccine pVAXrbd or RBD protein as well as CCV-RBD particles. The highest antigen-specific IgG and neutralizing activity were induced by CCV-RBD, and the level of antibodies induced by DNA or RBD alone was significantly lower. The cellular immune response was detected only in the case of DNA or CCV-RBD vaccination. These results demonstrate that a combination of DNA vaccine and RBD protein in one construct synergistically increases the humoral response to RBD protein in mice.

Dominant Antiviral CD8+ T Cell Responses Empower Prophylactic Antibody-Eliciting Vaccines Against Cytomegalovirus.

Human cytomegalovirus (HCMV) is an ubiquitous herpesvirus that can cause serious morbidity and mortality in immunocompromised or immune-immature individuals. A vaccine that induces immunity to CMV in these target populations is therefore highly needed. Previous attempts to generate efficacious CMV vaccines primarily focused on the induction of humoral immunity by eliciting neutralizing antibodies. Current insights encourage that a protective immune response to HCMV might benefit from the induction of virus-specific T cells. Whether addition of antiviral T cell responses enhances the protection by antibody-eliciting vaccines is however unclear. Here, we assessed this query in mouse CMV (MCMV) infection models by developing synthetic vaccines with humoral immunity potential, and deliberately adding antiviral CD8+ T cells. To induce antibodies against MCMV, we developed a DNA vaccine encoding either full-length, membrane bound glycoprotein B (gB) or a secreted variant lacking the transmembrane and intracellular domain (secreted (s)gB). Intradermal immunization with an increasing dose schedule of sgB and booster immunization provided robust viral-specific IgG responses and viral control. Combined vaccination of the sgB DNA vaccine with synthetic long peptides (SLP)-vaccines encoding MHC class I-restricted CMV epitopes, which elicit exclusively CD8+ T cell responses, significantly enhanced antiviral immunity. Thus, the combination of antibody and CD8+ T cell-eliciting vaccines provides a collaborative improvement of humoral and cellular immunity enabling enhanced protection against CMV.

Self-assembling ferritin nanoparticles coupled with linear sequences from canine distemper virus haemagglutinin protein elicit robust immune responses.

BACKGROUND: Canine distemper virus (CDV), which is highly infectious, has caused outbreaks of varying scales in domestic and wild animals worldwide, so the development of a high-efficiency vaccine has broad application prospects. Currently, the commercial vaccine of CDV is an attenuated vaccine, which has the disadvantages of a complex preparation process, high cost and safety risk. It is necessary to develop a safe and effective CDV vaccine that is easy to produce on a large scale. In this study, sequences of CDV haemagglutinin (HA) from the Yanaka strain were aligned, and three potential linear sequences, termed YaH3, YaH4, and YaH5, were collected. To increase the immunogenicity of the epitopes, ferritin was employed as a self-assembling nanoparticle element. The ferritin-coupled forms were termed YaH3F, YaH4F, and YaH5F, respectively. A full-length HA sequence coupled with ferritin was also constructed as a DNA vaccine to compare the immunogenicity of nanoparticles in prokaryotic expression. RESULT: The self-assembly morphology of the proteins from prokaryotic expression was verified by transmission electron microscopy. All the proteins self-assembled into nanoparticles. The expression of the DNA vaccine YaHF in HEK-293T cells was also confirmed in vitro. After subcutaneous injection of epitope nanoparticles or intramuscular injection of DNA YaHF, all vaccines induced strong serum titres, and long-term potency of antibodies in serum could be detected after 84 days. Strong anti-CDV neutralizing activities were observed in both the YaH4F group and YaHF group. According to antibody typing and cytokine detection, YaH4F can induce both Th1 and Th2 immune responses. The results of flow cytometry detection indicated that compared with the control group, all the immunogens elicited an increase in CD3. Simultaneously, the serum antibodies induced by YaH4F and YaHF could significantly enhance the ADCC effect compared with the control group, indicating that the antibodies in the serum effectively recognized the antigens on the cell surface and induced NK cells to kill infected cells directly. CONCLUSIONS: YaH4F self-assembling nanoparticle obtained by prokaryotic expression has no less of an immune effect than YaHF, and H4 has great potential to become a key target for the easy and rapid preparation of epitope vaccines.

Nasal prevention of SARS-CoV-2 infection by intranasal influenza-based boost vaccination in mouse models.

BACKGROUND: Vaccines in emergency use are efficacious against COVID-19, yet vaccine-induced prevention against nasal SARS-CoV-2 infection remains suboptimal. METHODS: Since mucosal immunity is critical for nasal prevention, we investigated the efficacy of an intramuscular PD1-based receptor-binding domain (RBD) DNA vaccine (PD1-RBD-DNA) and intranasal live attenuated influenza-based vaccines (LAIV-CA4-RBD and LAIV-HK68-RBD) against SARS-CoV-2. FINDINGS: Substantially higher systemic and mucosal immune responses, including bronchoalveolar lavage IgA/IgG and lung polyfunctional memory CD8 T cells, were induced by the heterologous PD1-RBD-DNA/LAIV-HK68-RBD as compared with other regimens. When vaccinated animals were challenged at the memory phase, prevention of robust SARS-CoV-2 infection in nasal turbinate was achieved primarily by the heterologous regimen besides consistent protection in lungs. The regimen-induced antibodies cross-neutralized variants of concerns. Furthermore, LAIV-CA4-RBD could boost the BioNTech vaccine for improved mucosal immunity. INTERPRETATION: Our results demonstrated that intranasal influenza-based boost vaccination induces mucosal and systemic immunity for effective SARS-CoV-2 prevention in both upper and lower respiratory systems. FUNDING: This study was supported by the Research Grants Council Collaborative Research Fund, General Research Fund and Health and Medical Research Fund in Hong Kong; Outbreak Response to Novel Coronavirus (COVID-19) by the Coalition for Epidemic Preparedness Innovations; Shenzhen Science and Technology Program and matching fund from Shenzhen Immuno Cure BioTech Limited; the Health@InnoHK, Innovation and Technology Commission of Hong Kong; National Program on Key Research Project of China; donations from the Friends of Hope Education Fund; the Theme-Based Research Scheme.

Techniques for Developing and Assessing Immune Responses Induced by Synthetic DNA Vaccines for Emerging Infectious Diseases.

Vaccines are one of mankind's greatest medical advances, and their use has drastically reduced and in some cases eliminated (e.g., smallpox) disease and death caused by infectious agents. Traditional vaccine modalities including live-attenuated pathogen vaccines, wholly inactivated pathogen vaccines, and protein-based pathogen subunit vaccines have successfully been used to create efficacious vaccines against measles, mumps, rubella, polio, and yellow fever. These traditional vaccine modalities, however, take many months to years to develop and have thus proven less effective for use in creating vaccines to emerging or reemerging infectious diseases (EIDs) including influenza, Human immunodeficiency virus (HIV), dengue virus (DENV), chikungunya virus (CHIKV), West Nile virus (WNV), Middle East respiratory syndrome (MERS), and the severe acute respiratory syndrome coronaviruses 1 and 2 (SARS-CoV and SARS-CoV-2). As factors such as climate change and increased globalization continue to increase the pace of EID development, newer vaccine modalities are required to develop vaccines that can prevent or attenuate EID outbreaks throughout the world. One such modality, DNA vaccines, has been studied for over 30 years and has numerous qualities that make them ideal for meeting the challenge of EIDs including; (1) DNA vaccine candidates can be designed within hours of publishing of a pathogens genetic sequence; (2) they can be manufactured cheaply and rapidly in large quantities; (3) they are thermostable and have reduced requirement for a cold-chain during distribution, and (4) they have a remarkable safety record in the clinic. Optimizations made in plasmid design as well as in DNA vaccine delivery have greatly improved the immunogenicity of these vaccines. Here we describe the process of making a DNA vaccine to an EID pathogen and describe methods used for assessing the immunogenicity and protective efficacy of DNA vaccines in small animal models.

A Nucleic Acid-Based Orthopoxvirus Vaccine Targeting the Vaccinia Virus L1, A27, B5, and A33 Proteins Protects Rabbits against Lethal Rabbitpox Virus Aerosol Challenge.

In the age of COVID, nucleic acid vaccines have garnered much attention, at least in part, because of the simplicity of construction, production, and flexibility to adjust and adapt to an evolving outbreak. Orthopoxviruses remain a threat on multiple fronts, especially as emerging zoonoses. In response, we developed a DNA vaccine, termed 4pox, that protected nonhuman primates against monkeypox virus (MPXV)-induced severe disease. Here, we examined the protective efficacy of the 4pox DNA vaccine delivered by intramuscular (i.m.) electroporation (EP) in rabbits challenged with aerosolized rabbitpox virus (RPXV), a model that recapitulates the respiratory route of exposure and low dose associated with natural smallpox exposure in humans. We found that 4pox-vaccinated rabbits developed immunogen-specific antibodies, including neutralizing antibodies, and did not develop any clinical disease, indicating protection against aerosolized RPXV. In contrast, unvaccinated animals developed significant signs of disease, including lesions, and were euthanized. These findings demonstrate that an unformulated, nonadjuvanted DNA vaccine delivered i.m. can protect against an aerosol exposure. IMPORTANCE The eradication of smallpox and subsequent cessation of vaccination have left a majority of the population susceptible to variola virus or other emerging poxviruses. This is exemplified by human monkeypox, as evidenced by the increase in reported endemic and imported cases over the past decades. Therefore, a malleable vaccine technology that can be mass produced and does not require complex conditions for distribution and storage is sought. Herein, we show that a DNA vaccine, in the absence of a specialized formulation or adjuvant, can protect against a lethal aerosol insult of rabbitpox virus.

The Combined Expression of the Nonstructural Protein NS1 and the N-Terminal Half of NS2 (NS21-180) by ChAdOx1 and MVA Confers Protection against Clinical Disease in Sheep upon Bluetongue Virus Challenge.

Bluetongue, caused by bluetongue virus (BTV), is a widespread arthropod-borne disease of ruminants that entails a recurrent threat to the primary sector of developed and developing countries. In this work, we report modified vaccinia virus Ankara (MVA) and ChAdOx1-vectored vaccines designed to simultaneously express the immunogenic NS1 protein and/or NS2-Nt, the N-terminal half of protein NS2 (NS21-180). A single dose of MVA or ChAdOx1 expressing NS1-NS2-Nt improved the protection conferred by NS1 alone in IFNAR(-/-) mice. Moreover, mice immunized with ChAdOx1/MVA-NS1, ChAdOx1/MVA-NS2-Nt, or ChAdOx1/MVA-NS1-NS2-Nt developed strong cytotoxic CD8+ T-cell responses against NS1, NS2-Nt, or both proteins and were fully protected against a lethal infection with BTV serotypes 1, 4, and 8. Furthermore, although a single immunization with ChAdOx1-NS1-NS2-Nt partially protected sheep against BTV-4, the administration of a booster dose of MVA-NS1-NS2-Nt promoted a faster viral clearance, reduction of the period and level of viremia and also protected from the pathology produced by BTV infection. IMPORTANCE Current BTV vaccines are effective but they do not allow to distinguish between vaccinated and infected animals (DIVA strategy) and are serotype specific. In this work we have develop a DIVA multiserotype vaccination strategy based on adenoviral (ChAdOx1) and MVA vaccine vectors, the most widely used in current phase I and II clinical trials, and the conserved nonstructural BTV proteins NS1 and NS2. This immunization strategy solves the major drawbacks of the current marketed vaccines.

Safety and immunogenicity of the two-dose heterologous Ad26.ZEBOV and MVA-BN-Filo Ebola vaccine regimen in children in Sierra Leone: a randomised, double-blind, controlled trial.

BACKGROUND: 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. METHODS: 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. FINDINGS: 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]). INTERPRETATION: 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. FUNDING: Innovative Medicines Initiative 2 Joint Undertaking and Janssen Vaccines & Prevention BV.

Safety and long-term immunogenicity of the two-dose heterologous Ad26.ZEBOV and MVA-BN-Filo Ebola vaccine regimen in adults in Sierra Leone: a combined open-label, non-randomised stage 1, and a randomised, double-blind, controlled stage 2 trial.

BACKGROUND: 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. METHODS: 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. FINDINGS: 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. INTERPRETATION: 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. FUNDING: Innovative Medicines Initiative 2 Joint Undertaking and Janssen Vaccines & Prevention BV.

Preexisting memory CD4+ T cells contribute to the primary response in an HIV-1 vaccine trial.

Naive and memory CD4+ T cells reactive with human immunodeficiency virus type 1 (HIV-1) are detectable in unexposed, unimmunized individuals. The contribution of preexisting CD4+ T cells to a primary immune response was investigated in 20 HIV-1-seronegative volunteers vaccinated with an HIV-1 envelope (Env) plasmid DNA prime and recombinant modified vaccinia virus Ankara (MVA) boost in the HVTN 106 vaccine trial (clinicaltrials.gov NCT02296541). Prevaccination naive or memory CD4+ T cell responses directed against peptide epitopes in Env were identified in 14 individuals. After priming with DNA, 40% (8/20) of the elicited responses matched epitopes detected in the corresponding preimmunization memory repertoires, and clonotypes were shared before and after vaccination in 2 representative volunteers. In contrast, there were no shared epitope specificities between the preimmunization memory compartment and responses detected after boosting with recombinant MVA expressing a heterologous Env. Preexisting memory CD4+ T cells therefore shape the early immune response to vaccination with a previously unencountered HIV-1 antigen.