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Respiratory Syncytial virus (RSV) is a major threat to many vulnerable populations. There are currently no approved vaccines, and RSV remains a high unmet global medical need. Here we describe the employment of a novel synthetic DNA-encoded antibody technology platform to develop and deliver an engineered human DNA-encoded monoclonal antibody (dMAbTM) targeting the fusion protein (F) of RSV as a new approach to prevention or therapy of at risk populations. In in vivo models, a single administration of synthetic DNA-encoding the single-chain fragment variable-constant fragment (scFv-Fc) RSV-F dMAb resulted in robust and durable circulating levels of a functional antibody systemically and in mucosal tissue. In cotton rats, which are the gold-standard animals to model RSV infection, we observed sustained scFv-Fc RSV-F dMAb in the sera and lung-lavage samples, demonstrating the potential for both long-lasting immunity to RSV and effective biodistribution. The scFv-Fc RSV-F dMAb harbored in the sera exhibited RSV antigen-specific binding and potent viral neutralizing activity. Importantly, in vivo delivery of synthetic DNA-encoding, the scFv-Fc RSV-F dMAb protected animals against viral challenge. Our findings support the significance of dMAbs as a potential platform technology for durable protection against RSV disease.

Hepatitis B virus (HBV) causes a potentially life-threatening liver infection that frequently results in life-long chronic infection. HBV is responsible for 887,000 deaths each year, most resulting from chronic liver diseases and hepatocellular carcinoma. Presently, there are 250 million chronic HBV carriers worldwide who are at a high risk for developing cirrhosis and hepatocellular carcinoma (HCC). HCC is the most common type of liver cancer with a strong association with HBV infection. HBV transmission through blood transfusions and perinatal transfer from infected mother to child have been common routes of infection. In the present study, we describe the development of a synthetic DNA plasmid encoding an anti-HBV human monoclonal antibody specific for the common “a determinant region” of HBsAg of hepatitis B virus and demonstrate the ability of this platform at directing in vivo antibody expression. In vivo delivery of this DNA encoded monoclonal antibody (DMAb) plasmid in mice resulted in expression of human IgG over a period of one month following a single injection. Serum antibody was found to recognize the relevant conformational epitope from plasma purified native HBsAg as well as bound HBV in HepG2.2.15 cells. The serum DMAb efficiently neutralized HBV and prevented infection of HepaRG cells in vitro. Additional study of these HBV-DMAb as a possible therapy or immunoprophylaxis for HBV infection is warranted.

Significant concerns have arisen over the past 3 y from the increased global spread of the mosquito-borne flavivirus, Zika. Accompanying this spread has been an increase in cases of the devastating birth defect microcephaly as well as of Guillain–Barré syndrome in adults in many affected countries. Currently there is no vaccine or therapy for this infection; however, we sought to develop a combination approach that provides more rapid and durable protection than traditional vaccination alone. A novel immune-based prophylaxis/therapy strategy entailing the facilitated delivery of a synthetic DNA consensus prME vaccine along with DNA-encoded anti-ZIKV envelope monoclonal antibodies (dMAb) were developed and evaluated for antiviral efficacy. This immediate and persistent protection strategy confers the ability to overcome shortcomings inherent with conventional active vaccination or passive immunotherapy. A collection of novel dMAbs were developed which were potent against ZIKV and could be expressed in serum within 24–48 h of in vivo administration. The DNA vaccine, from a previous development, was potent after adaptive immunity was developed, protecting against infection, brain and testes pathology in relevant mouse challenge models and in an NHP challenge. Delivery of potent dMAbs protected mice from the same murine viral challenge within days of delivery. Combined injection of dMAb and the DNA vaccine afforded rapid and long-lived protection in this challenge model, providing an important demonstration of the advantage of this synergistic approach to pandemic outbreaks.

Following the recent clinical clearance of an Indian DNA COVID-19 vaccine, India and Africa are potential regions where DNA vaccines may become a major delivery system subject to a range of immunological and regulatory scrutiny. The ongoing COVID pandemic highlights the need to tackle viral variants and expand the number of antigens and assess diverse delivery systems. To address some of these key issues, we have created a Dengue DNA vaccine candidate with the EDIII region as the key antigen given the promise of this segment in not causing ADE, a challenge with this disease. In addition, we have added the NS1 region to broaden the immune response. Following a large Dengue viral sequencing exercise in India, complemented with data from east Africa, our approach was to generate a consensus of four serotypes ED3-NS1 vaccine to explore tackling the issue of diversity. OurIn silicostructural analysis of EDIII consensus vaccine sequence revealed that epitopes are structurally conserved and immunogenic across HLA diversity. Vaccination of mice with this construct induced pan-serotype neutralizing antibodies and antigen-specific T cell responses. Furthermore, the DNA vaccination confers protection against DENV challenge in AG129 mice. Finally, assaying of intracellular staining for IFN-γ, immunoglobulin IgG2(a/c) /IgG1 ratios as well as immune gene profiling suggested a strong Th1-dominant immune response. Our Dengue DNA platform with a focus on Indo-African sequences offers an approach for assessing cross reactive immunity in animal models and lays the foundation for human vaccine roll out either as a stand-alone or mix and match strategy.

Emerging coronaviruses from zoonotic reservoirs, including severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV), and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), have been associated with human-to-human transmission and significant morbidity and mortality. Here, we study both intradermal and intramuscular 2-dose delivery regimens of an advanced synthetic DNA vaccine candidate encoding a full-length MERS-CoV spike (S) protein, which induced potent binding and neutralizing antibodies as well as cellular immune responses in rhesus macaques. In a MERS-CoV challenge, all immunized rhesus macaques exhibited reduced clinical symptoms, lowered viral lung load, and decreased severity of pathological signs of disease compared with controls. Intradermal vaccination was dose sparing and more effective in this model at protecting animals from disease. The data support the further study of this vaccine for preventing MERS-CoV infection and transmission, including investigation of such vaccines and simplified delivery routes against emerging coronaviruses.

Zika virus (ZIKV) infection is endemic to several world regions, and many others are at high risk for seasonal outbreaks. Synthetic DNA-encoded monoclonal antibody (DMAb) is an approach that enables in vivo delivery of highly potent mAbs to control infections. We engineered DMAb-ZK190, encoding the mAb ZK190 neutralizing antibody, which targets the ZIKV E protein DIII domain. In vivo-delivered DMAb-ZK190 achieved expression levels persisting >10 weeks in mice and >3 weeks in non-human primate (NHPs), which is protective against ZIKV infectious challenge. This study is the first demonstration of infectious disease control in NHPs following in vivo delivery of a nucleic acid-encoded antibody, supporting the importance of this new platform., Esquivel et al. deliver DNA-encoded ZK190 antibody (DMAb-ZK190) by intramuscular administration. DMAb-ZK190 targets the E protein of Zika virus, and it controls Zika virus infection in mice and non-human primates when delivered prior to infection, offering an alternative strategy for rapid protection.

Vaccines are considered one of the greatest advances in modern medicine. The global burden of numerous infectious diseases has been significantly reduced, and in some cases, effectively eradicated through the deployment of specific vaccines. However, efforts to develop effective new vaccines against infectious pathogens such as influenza, Human immunodeficiency virus (HIV), dengue virus (DENV), chikungunya virus (CHIKV), Ebola virus, and Zika virus (ZIKV) have proven challenging. Zika virus is a mosquito-vectored flavivirus responsible for periodic outbreaks of disease in Africa, Southeast Asia, and the Pacific Islands dating back over 50 years. Over this period, ZIKV infections were subclinical in most infected individuals and resulted in mild cases of fever, arthralgia, and rash in others. Concerns about ZIKV changed over the past two years, however, as outbreaks in Brazil, Central American countries, and Caribbean islands revealed novel aspects of infection including vertical and sexual transmission modes. Cases have been reported showing dramatic neurological pathologies including microcephaly and other neurodevelopmental problems in babies born to ZIKV infected mothers, as well as an increased risk of Guillain-Barre syndrome in adults. These findings prompted the World Health Organization to declare ZIKV a public health emergency in 2016, which resulted in expanded efforts to develop ZIKV vaccines and immunotherapeutics. Several ZIKV vaccine candidates that are immunogenic and effective at blocking ZIKV infection in animal models have since been developed, with some of these now being evaluated in the clinic. Additional therapeutics under investigation include anti-ZIKV monoclonal antibodies (mAbs) that have been shown to neutralize infection in vitro as well as protect against morbidity in mouse models of ZIKV infection. In this review, we summarize the current understanding of ZIKV biology and describe our efforts to rapidly develop a vaccine against ZIKV.

SUMMARY Synthetically engineered DNA-encoded monoclonal antibodies (DMAbs) are an in vivo platform for evaluation and delivery of human mAb to control against infectious disease. Here, we engineer DMAbs encoding potent anti-Zaire ebolavirus (EBOV) glycoprotein (GP) mAbs isolated from Ebola virus disease survivors. We demonstrate the development of a human IgG1 DMAb platform for in vivo EBOV-GP mAb delivery and evaluation in a mouse model. Using this approach, we show that DMAb-11 and DMAb-34 exhibit functional and molecular profiles comparable to recombinant mAb, have a wide window of expression, and provide rapid protection against lethal mouse-adapted EBOV challenge. The DMAb platform represents a simple, rapid, and reproducible approach for evaluating the activity of mAb during clinical development. DMAbs have the potential to be a mAb delivery system, which may be advantageous for protection against highly pathogenic infectious diseases, like EBOV, in resource-limited and other challenging settings., Graphical Abstract, In Brief Monoclonal antibodies are an important approach for emerging infectious disease prevention. Patel et al. demonstrate engineering and in vivo delivery of DNA-encoded monoclonal antibodies (DMAbs) targeting the Zaire ebolavirus (EBOV) glycoprotein. DMAbs protect against lethal mouse-adapted EBOV and are useful for rapid evaluation of fully human mAbs in live animal models.

The emergence of multiple concurrent infectious diseases localized in the world creates a complex burden on global public health systems. Outbreaks of Ebola, Lassa, and Marburg viruses in overlapping regions of central and West Africa and the co-circulation of Zika, Dengue, and Chikungunya viruses in areas with A. aegypti mosquitos highlight the need for a rapidly deployable, safe, and versatile vaccine platform readily available to respond. The DNA vaccine platform stands out as such an application. Here, we present proof-of-concept studies from mice, guinea pigs, and non-human primates for two multivalent DNA vaccines delivered using in vivo electroporation (EP) targeting mosquito-borne (MMBV) and hemorrhagic fever (MHFV) viruses. Immunization with MMBV or MHFV vaccines via intradermal EP delivery generated robust cellular and humoral immune responses against all target viral antigens in all species. MMBV vaccine generated antigen-specific binding antibodies and IFNγ-secreting lymphocytes detected in NHPs up to six months post final immunization, suggesting induction of long-term immune memory. Serum from MHFV vaccinated NHPs demonstrated neutralizing activity in Ebola, Lassa, and Marburg pseudovirus assays indicating the potential to offer protection. Together, these data strongly support and demonstrate the versatility of DNA vaccines as a multivalent vaccine development platform for emerging infectious diseases.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a global pandemic of COVID-19, resulting in cases of mild to severe respiratory distress and significant mortality. The global outbreak of this novel coronavirus has now infected >20 million people worldwide, with >5 million cases in the United States (11 August 2020). The development of diagnostic and research tools to determine infection and vaccine efficacy is critically needed., Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a global pandemic of COVID-19, resulting in cases of mild to severe respiratory distress and significant mortality. The global outbreak of this novel coronavirus has now infected >20 million people worldwide, with >5 million cases in the United States (11 August 2020). The development of diagnostic and research tools to determine infection and vaccine efficacy is critically needed. We have developed multiple serologic assays using newly designed SARS-CoV-2 reagents for detecting the presence of receptor-binding antibodies in sera. The first assay is surface plasmon resonance (SPR) based and can quantitate both antibody binding to the SARS-CoV-2 spike protein and blocking to the Angiotensin-converting enzyme 2 (ACE2) receptor in a single experiment. The second assay is enzyme-linked immunosorbent assay (ELISA) based and can measure competition and blocking of the ACE2 receptor to the SARS-CoV-2 spike protein with antispike antibodies. The assay is highly versatile, and we demonstrate the broad utility of the assay by measuring antibody functionality of sera from small animals and nonhuman primates immunized with an experimental SARS-CoV-2 vaccine. In addition, we employ the assay to measure receptor blocking of sera from SARS-CoV-2-infected patients. The assay is shown to correlate with pseudovirus neutralization titers. This type of rapid, surrogate neutralization diagnostic can be employed widely to help study SARS-CoV-2 infection and assess the efficacy of vaccines.

The coronavirus family member, SARS-CoV-2 has been identified as the causal agent for the pandemic viral pneumonia disease, COVID-19. At this time, no vaccine is available to control further dissemination of the disease. We have previously engineered a synthetic DNA vaccine targeting the MERS coronavirus Spike (S) protein, the major surface antigen of coronaviruses, which is currently in clinical study. Here we build on this prior experience to generate a synthetic DNA-based vaccine candidate targeting SARS-CoV-2 S protein. The engineered construct, INO-4800, results in robust expression of the S protein in vitro. Following immunization of mice and guinea pigs with INO-4800 we measure antigen-specific T cell responses, functional antibodies which neutralize the SARS-CoV-2 infection and block Spike protein binding to the ACE2 receptor, and biodistribution of SARS-CoV-2 targeting antibodies to the lungs. This preliminary dataset identifies INO-4800 as a potential COVID-19 vaccine candidate, supporting further translational study., There is currently no licensed SARS-CoV-2 vaccine. Here, the authors generate an optimized DNA vaccine candidate encoding the SARS-CoV-2 spike antigen, demonstrating induction of specific T cells and neutralizing antibody responses in mice and guinea pigs. These initial results support further development of this vaccine candidate.

Background There remains an important need for prophylactic anti-Ebola virus vaccine candidates that elicit long-lasting immune responses and can be delivered to vulnerable populations that are unable to receive live-attenuated or viral vector vaccines. Methods We designed novel synthetic anti-Ebola virus glycoprotein (EBOV-GP) DNA vaccines as a strategy to expand protective breadth against diverse EBOV strains and evaluated the impact of vaccine dosing and route of administration on protection against lethal EBOV-Makona challenge in cynomolgus macaques. Long-term immunogenicity was monitored in nonhuman primates for >1 year, followed by a 12-month boost. Results Multiple-injection regimens of the EBOV-GP DNA vaccine, delivered by intramuscular administration followed by electroporation, were 100% protective against lethal EBOV-Makona challenge. Impressively, 2 injections of a simple, more tolerable, and dose-sparing intradermal administration followed by electroporation generated strong immunogenicity and was 100% protective against lethal challenge. In parallel, we observed that EBOV-GP DNA vaccination induced long-term immune responses in macaques that were detectable for at least 1 year after final vaccination and generated a strong recall response after the final boost. Conclusions These data support that this simple intradermal-administered, serology-independent approach is likely important for additional study towards the goal of induction of anti-EBOV immunity in multiple at-risk populations.

More than 100 million people have been infected with severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). Common laboratory mice are not susceptible to wild-type SARS-CoV-2 infection, challenging the development and testing of effective interventions. Here, we describe the development and testing of a mouse model for SARS-CoV-2 infection based on transduction of the respiratory tract of laboratory mice with an adeno-associated virus vector (AAV6) expressing human ACE-2 (AAV6.2FF-hACE2). We validated this model using a previously described synthetic DNA vaccine plasmid, INO-4800 (pS). Intranasal instillation of AAV6.2FF-hACE2 resulted in robust hACE2 expression in the respiratory tract. pS induced robust cellular and humoral responses. Vaccinated animals were challenged with 105 TCID50 SARS-CoV-2 (hCoV-19/Canada/ON-VIDO-01/2020) and euthanized four days post-challenge to assess viral load. One immunization resulted in 50% protection and two immunizations were completely protective. Overall, the AAV6.2FF-hACE2 mouse transduction model represents an easily accessible, genetically diverse mouse model for wild-type SARS-CoV-2 infection and preclinical evaluation of potential interventions., Graphical abstract, Immunology; Virology

The skin is an ideal target tissue for vaccine delivery for a number of reasons. It is highly accessible, and most importantly, enriched in professional antigen presenting cells. Possessing strong similarities to human skin physiology and displaying a defined epidermis, the guinea pig is an appropriate model to study epidermal delivery of vaccine. However, whilst we have characterized the humoral responses in the guinea pig associated with skin vaccine protocols we have yet to investigate the T cell responses. In response to this inadequacy, we developed an IFN-γ ELISpot assay to characterize the cellular immune response in the peripheral blood of guinea pigs. Using a nucleoprotein (NP) influenza pDNA vaccination regimen, we characterized host T cell responses. After delivery of the DNA vaccine to the guinea pig epidermis we detected robust and rapid T cell responses. The levels of IFN-γ spot-forming units averaged approximately 5000 per million cells after two immunizations. These responses were broad in that multiple regions across the NP antigen elicited a T cell response. Interestingly, we identified a number of NP immunodominant T cell epitopes to be conserved across an outbred guinea pig population, a phenomenon which was also observed after immunization with a RSV DNA vaccine. We believe this data enhances our understanding of the cellular immune response elicited to a vaccine in guinea pigs, and globally, will advance the use of this model for vaccine development, especially those targeting skin as a delivery site.

Interventions to prevent HIV-1 infection and alternative tools in HIV cure therapy remain pressing goals. Recently, numerous broadly neutralizing HIV-1 monoclonal antibodies (bNAbs) have been developed that possess the characteristics necessary for potential prophylactic or therapeutic approaches. However, formulation complexities, especially for multiantibody deliveries, long infusion times, and production issues could limit the use of these bNAbs when deployed, globally affecting their potential application. Here, we describe an approach utilizing synthetic DNA-encoded monoclonal antibodies (dmAbs) for direct in vivo production of prespecified neutralizing activity. We designed 16 different bNAbs as dmAb cassettes and studied their activity in small and large animals. Sera from animals administered dmAbs neutralized multiple HIV-1 isolates with activity similar to that of their parental recombinant mAbs. Delivery of multiple dmAbs to a single animal led to increased neutralization breadth. Two dmAbs, PGDM1400 and PGT121, were advanced into nonhuman primates for study. High peak-circulating levels (between 6 and 34 μg/ml) of these dmAbs were measured, and the sera of all animals displayed broad neutralizing activity. The dmAb approach provides an important local delivery platform for the in vivo generation of HIV-1 bNAbs and for other infectious disease antibodies.

Identification of novel molecular adjuvants which can boost and enhance vaccine-mediated immunity and provide dose-sparing potential against complex infectious diseases and for immunotherapy in cancer is likely to play a critical role in the next generation of vaccines. Given the number of challenging targets for which no or only partial vaccine options exist, adjuvants that can address some of these concerns are in high demand. Here, we report that a designed truncated Interleukin-36 gamma (IL-36 gamma) encoded plasmid can act as a potent adjuvant for several DNA-encoded vaccine targets including human immunodeficiency virus (HIV), influenza, and Zika in immunization models. We further show that the truncated IL-36 gamma (opt-36&gamma, t) plasmid provides improved dose sparing as it boosts immunity to a suboptimal dose of a Zika DNA vaccine, resulting in potent protection against a lethal Zika challenge.

Background Vaccination and passive antibody therapies are critical for controlling infectious diseases. Passive antibody administration has limitations, including the necessity for purification and multiple injections for efficacy. Vaccination is associated with a lag phase before generation of immunity. Novel approaches reported here utilize the benefits of both methods for the rapid generation of effective immunity. Methods A novel antibody-based prophylaxis/therapy entailing the electroporation-mediated delivery of synthetic DNA plasmids encoding biologically active anti-chikungunya virus (CHIKV) envelope monoclonal antibody (dMAb) was designed and evaluated for antiviral efficacy, as well as for the ability to overcome shortcomings inherent with conventional active vaccination and passive immunotherapy. Results One intramuscular injection of dMAb produced antibodies in vivo more rapidly than active vaccination with an anti-CHIKV DNA vaccine. This dMAb neutralized diverse CHIKV clinical isolates and protected mice from viral challenge. Combination of dMAb and the CHIKV DNA vaccine afforded rapid and long-lived protection. Conclusions A DNA-based dMAb strategy induced rapid protection against an emerging viral infection. This method can be combined with DNA vaccination as a novel strategy to provide both short- and long-term protection against this emerging infectious disease. These studies have implications for pathogen treatment and control strategies.

Powassan virus (POWV) infection is a tick-borne emerging infectious disease in the United States and North America. Like Zika virus, POWV is a member of the family Flaviviridae. POWV causes severe neurological sequalae, meningitis, encephalitis, and can cause death. Although the risk of human POWV infection is low, its incidence in the U.S. in the past 16 years has increased over 300%, urging immediate attention. Despite the disease severity and its growing potential for threatening larger populations, currently there are no licensed vaccines which provide protection against POWV. We developed a novel synthetic DNA vaccine termed POWV-SEV by focusing on the conserved portions of POWV pre-membrane and envelope (prMEnv) genes. A single immunization of POWV-SEV elicited broad T and B cell immunity in mice with minimal cross-reactivity against other flaviviruses. Antibody epitope mapping demonstrated a similarity between POWV-SEV-induced immune responses and those elicited naturally in POWV-infected patients. Finally, POWV-SEV induced immunity provided protection against POWV disease in lethal challenge experiments., Author summary Powassan virus (POWV) is an emerging RNA virus, belonging to the tick-borne flavivirus family and transmitted to humans through the bite of an infected tick. Infection can produce severe neurological manifestations, including meningitis and encephalitis, leading to death. Despite the potential for its emergence, currently antiviral therapies are not available to treat or prevent this emerging infection. This situation calls for concern and needs to be addressed. In this study, we have designed and produced a consensus, synthetic enhanced vaccine (SEV) against POWV (POWV-SEV) that focuses on regions of the envelope protein. The effectiveness of this vaccine was evaluated murine models. We have tested the antigen-specific humoral responses to the POWV-SEVs including the induction of neutralizing antibody responses. In addition, cellular immunogenicity including identification of dominant epitopes and polyfunctionality of cytokine-producing T-cells were characterized in POWV-SEV administered mice. Finally, we assessed the protective efficacy of POWV-SEV using a murine challenge natural infection model of POWV. These studies are highly novel and support the feasibility of developing an envelope-based synthetic enhanced DNA vaccine to aid in mitigating the public health threat emerging tick-borne viruses may pose to outdoor pets and humans in endemic areas.

Leptospirosis is a bacterial zoonotic disease caused by an infection with a spirochete belonging to the genus Leptospira. In animals, leptospirosis displays a wide range of pathologies, including fever, abortion, icterus, and uveitis. Conversely, infection in humans is associated with multi-organ injury, resulting in an increased rate of fatalities. Pathogenic leptospires are able to translocate through cell monolayers at a rate significantly greater than that of non-pathogenic leptospires. Thus, vaccine approaches have been focused on targeting bacterial motility, lipopolysaccharides (LPSs), lipoproteins, outer-membrane proteins (OMPs) and other potential virulence factors. Previous studies have indicated that leptospiral proteins elicit long-lasting immunological memory in infected humans. In the study reported here, the efficacy of a synthetic consensus DNA vaccine developed against the Leptospira membrane lipoprotein LipL45 was tested. After in vivo electroporation (EP) mediated intramuscular immunization with a synthetic LipL45 DNA vaccine (pLipL45) immunized mice developed a significant cellular response along with the development of anti-LipL45-specific antibodies. Specifically, the pLipL45 vaccine induced a significant Th1 type immune response, indicated by the higher production of IL-12 and IFN-γ cytokines. The results presented here are the first demonstration that a LipL45 based DNA immunogen has potential as a anti-Leptospira vaccine.

Influenza virus remains a significant public health threat despite innovative vaccines and antiviral drugs. A major limitation to current vaccinations and therapies against influenza virus is pathogenic diversity generated by shift and drift. A simple, cost-effective passive immunization strategy via in vivo production of cross-protective antibody molecules may augment existing vaccines and antiviral drugs in seasonal and pandemic outbreaks. We engineered synthetic plasmid DNA to encode two novel and broadly cross-protective monoclonal antibodies targeting influenza A and B. We utilized enhanced in vivo delivery of these plasmid DNA-encoded monoclonal antibody (DMAb) constructs and show that this strategy induces robust levels of functional antibodies directed against influenza A and B viruses in mouse sera. Mice receiving a single inoculation with anti-influenza A DMAb survive lethal Group 1 H1 and Group 2 H3 influenza A challenges, while inoculation with anti-influenza B DMAb yields protection against lethal Victoria and Yamagata lineage influenza B morbidity and mortality. Furthermore, these two DMAbs can be delivered coordinately resulting in exceptionally broad protection against both influenza A and B. We demonstrate this protection is similar to that achieved by conventional protein antibody delivery. DMAbs warrant further investigation as a novel immune therapy platform with distinct advantages for sustained immunoprophylaxis against influenza., Nucleic acid delivery: Instant, wide-ranging protection against influenza A and B A novel innoculation technique involving the injection of antibody-producing plasmid DNA has shown to be effective against influenza in mice. The flu is responsible for up to half a million deaths each year and up to five million cases of severe disease, while also posing a substantial pandemic threat, even with our current repertoire of vaccines. A team of researchers led by Sarah Elliott and David Weiner of The Wistar Institute of Anatomy and Biology, Philadelphia, developed potent plasmid-based constructs that, once injected, entered hosts’ cells and utilized cellular machinery to encode antibodies protective against a range of influenza A and B subtypes. DNA inoculation conferred acute protection from disease, with treated individuals also being immune to subsequent exposure. This approach warrants further investigation as an alternative technology for practical delivery of monoclonal antibody therapeutics.

Zika virus (ZIKV) is an emerging pathogen causally associated with serious sequelae in fetuses, inducing fetal microcephaly and other neurodevelopment defects. ZIKV is primarily transmitted by mosquitoes, but can persist in human semen and sperm, and sexual transmission has been documented. Moreover, exposure of type-I interferon knockout mice to ZIKV results in severe damage to the testes, epididymis and sperm. Candidate ZIKV vaccines have shown protective efficacy in preclinical studies carried out in animal models, and several vaccines have entered clinical trials. Here, we report that administration of a synthetic DNA vaccine encoding ZIKV pre-membrane and envelope (prME) completely protects mice against ZIKV-associated damage to the testes and sperm and prevents viral persistence in the testes following challenge with a contemporary strain of ZIKV. These data suggest that DNA vaccination merits further investigation as a potential means to reduce ZIKV persistence in the male reproductive tract., Zika virus (ZIKV) can persist in human semen and sperm, which can result in sexual transmission. Here, Griffin et al. show that a DNA vaccine, expressing ZIKV pre-membrane and envelope proteins, protects mice from infection-associated damage to testes and sperm, and prevents viral persistence in testes.

Respiratory syncytial virus (RSV) is a massive medical burden in infants, children and the elderly worldwide, and an effective, safe RSV vaccine remains an unmet need. Here we assess a novel vaccination strategy based on the intradermal delivery of a SynCon® DNA-based vaccine encoding engineered RSV-F antigen using a surface electroporation device (SEP) to target epidermal cells, in clinically relevant experimental models. We demonstrate the ability of this strategy to elicit robust immune responses. Importantly we demonstrate complete resistance to pulmonary infection at a single low dose of vaccine in the cotton rat RSV/A challenge model. In contrast to the formalin-inactivated RSV (FI-RSV) vaccine, there was no enhanced lung inflammation upon virus challenge after DNA vaccination. In summary the data presented outline the pre-clinical development of a highly efficacious, tolerable and safe non-replicating vaccine delivery strategy.

Significant concerns have been raised owing to the rapid global spread of infection and disease caused by the mosquito-borne Zika virus (ZIKV). Recent studies suggest that ZIKV can also be transmitted sexually, further increasing the exposure risk for this virus. Associated with this spread is a dramatic increase in cases of microcephaly and additional congenital abnormalities in infants of ZIKV-infected mothers, as well as a rise in the occurrence of Guillain Barre’ syndrome in infected adults. Importantly, there are no licensed therapies or vaccines against ZIKV infection. In this study, we generate and evaluate the in vivo efficacy of a novel, synthetic, DNA vaccine targeting the pre-membrane+envelope proteins (prME) of ZIKV. Following initial in vitro development and evaluation studies of the plasmid construct, mice and non-human primates were immunised with this prME DNA-based immunogen through electroporation-mediated enhanced DNA delivery. Vaccinated animals were found to generate antigen-specific cellular and humoral immunity and neutralisation activity. In mice lacking receptors for interferon (IFN)-α/β (designated IFNAR−/−) immunisation with this DNA vaccine induced, following in vivo viral challenge, 100% protection against infection-associated weight loss or death in addition to preventing viral pathology in brain tissue. In addition, passive transfer of non-human primate anti-ZIKV immune serum protected IFNAR−/− mice against subsequent viral challenge. This study in NHP and in a pathogenic mouse model supports the importance of immune responses targeting prME in ZIKV infection and suggests that additional research on this vaccine approach may have relevance for ZIKV control and disease prevention in humans. Zika virus: A DNA vaccine candidate. A Zika virus vaccine candidate has been developed that elicits an immune response and confers protection in animal models. An international team of researchers headed by David Weiner of The Wistar Institute, Philadelphia, USA, generated a DNA-based vaccine candidate using genes encoding for Zika virus surface proteins—with the intent that these genes, when transferred to a host, would produce characteristic proteins allowing the host to recognise and protect against Zika virus infection. Weiner’s team found that their vaccine candidate generated a specific immune cell response in healthy mice, and offered protection to mice genetically engineered to be vulnerable to the virus. Importantly, their vaccine also elicited functional responses in non-human primates, highlighting that further research into this approach is warranted for the immunisation of humans against Zika virus.

Mayaro virus (MAYV) of the genus alphavirus is a mosquito-transmitted emerging infectious disease that causes an acute febrile illness, rash, headaches, and nausea that may turn into incapacitating, persistent arthralgias in some victims. Since its discovery in Trinidad in 1954, cases of MAYV infection have largely been confined there and to the northern countries of South America, but recently, MAYV cases have been reported in some island nations in the Caribbean Sea. Accompanying these reports is evidence that new vectors, including Aedes spp. mosquitos, recently implicated in the global spread of Zika and chikungunya viruses, are competent for MAYV transmission, which, if true, could facilitate the spread of MAYV beyond its current range. Despite its status as an emerging virus, there are no licensed vaccines to prevent MAYV infection nor therapeutics to treat it. Here, we describe the development and testing of a novel DNA vaccine, scMAYV-E, that encodes a synthetically-designed consensus MAYV envelope sequence. In vivo electroporation-enhanced immunization of mice with this vaccine induced potent humoral responses including neutralizing antibodies as well as robust T-cell responses to multiple epitopes in the MAYV envelope. Importantly, these scMAYV-E-induced immune responses protected susceptible mice from morbidity and mortality following a MAYV challenge., Author summary Mayaro virus (MAYV) is a mosquito-transmitted virus that causes fever, headache, chills, nausea and joint pain that can last for months after infection. The rising number of cases, due to increased mosquito circulation, and the threat of an epidemic emphasize its importance as an emerging virus, but there are no licensed vaccines to prevent Mayaro infection nor therapeutics to treat it. In this study, we gathered fundamental knowledge on how the immune system responds to MAYV infection, and we evaluated the efficacy of a novel, synthetic DNA envelope vaccine (scMAYV-E) in mice. Analysis of immune responses in mice demonstrated that this vaccine induces potent T cell immunity and antibodies. Mice who receive the vaccine and then are challenged with live MAYV are protected against Mayaro disease. This data provides evidence that the DNA-based MAYV vaccine may be able to prevent Mayaro disease. Thus, the scMAYV-E vaccine is a promising step forward for MAYV vaccine development. Future testing will evaluate whether this vaccine is a viable means to halt the spread of MAYV and protect individuals from Mayaro disease.

Monoclonal antibody preparations have demonstrated considerable clinical utility in the treatment of specific malignancies, as well as inflammatory and infectious diseases. Antibodies are conventionally delivered by passive administration, typically requiring costly large-scale laboratory development and production. Additional limitations include the necessity for repeat administrations, and the length of in vivo potency. Therefore, the development of methods to generate therapeutic antibodies and antibody like molecules in vivo, distinct from an active antigen-based immunization strategy, would have considerable clinical utility. In fact, adeno-associated viral (AAV) vector mediated delivery of immunoglobulin genes with subsequent generation of functional antibodies has recently been developed. As well, anon-viral vector mediated nucleic acid based delivery technology could permit the generation of therapeutic/prophylactic antibodies in vivo, obviating potential safety issues associated with viral vector based gene delivery. This delivery strategy has limitations as well, mainly due to very low in vivo production and expression of protein from the delivered gene. In the study reported here we have constructed an “enhanced and optimized” DNA plasmid technology to generate immunoglobulin heavy and light chains (i.e., Fab fragments) from an established neutralizing anti-HIV envelope glycoprotein monoclonal antibody (VRC01). This “enhanced” DNA (E-DNA) plasmid technology includes codon/RNA optimization, leader sequence utilization, as well as targeted potentiation of delivery and expression of the Fab immunoglobulin genes through use of “adaptive” in vivo electroporation. The results demonstrate that delivery by this method of a single administration of the optimized Fab expressing constructs resulted in generation of Fab molecules in mouse sera possessing high antigen specific binding and HIV neutralization activity for at least 7 d after injection, against diverse HIV isolates. Importantly, this delivery strategy resulted in a rapid increase (i.e., in as little as 48 h) in Fab levels when compared with protein-based immunization. The active generation of functional Fab molecules in vivo has important conceptual and practical advantages over conventional ex vivo generation, purification and passive delivery of biologically active antibodies. Further study of this technique for the rapid generation and delivery of immunoglobulin and immunoglobulin like molecules is highly relevant and timely.

A sudden upsurge of fever cases with joint pain was observed in the outpatient department, Community Health Centre, Rangat during July-August 2010 in Rangat Middle Andaman, India. The aetiological agent responsible for the outbreak was identified as chikungunya virus (CHIKV), by using RT-PCR and IgM ELISA. The study investigated the association of polymorphisms in the human leucocyte antigen class II genes with susceptibility or protection against CHIKV. One hundred and one patients with clinical features suggestive of CHIKV infection and 104 healthy subjects were included in the study. DNA was extracted and typed for HLA-DRB1 and DQB1 alleles. Based on the amino acid sequences of HLA-DQB1 retrieved from the IMGT/HLA database, critical amino acid differences in the specific peptide-binding pockets of HLA-DQB1 molecules were investigated. The frequencies of HLA-DRB1 alleles were not significantly different, whereas lower frequency of HLA-DQB1*03:03 was observed in CHIKV patients compared with the control population [P = 0·001, corrected P = 0·024; odds ratio (OR) = 0, 95% confidence interval (95% CI) 0·0-0·331; Peto's OR = 0·1317, 95% CI 0·0428-0·405). Significantly lower frequency of glutamic acid at position 86 of peptide-binding pocket 1 coding HLA-DQB1 genotypes was observed in CHIKV patients compared with healthy controls (P = 0·004, OR = 0·307, 95% CI 0·125-0·707). Computational binding predictions of CD4 epitopes of CHIKV by NetMHCII revealed that HLA-DQ molecules are known to bind more CHIKV peptides than HLA-DRB1 molecules. The results suggest that HLA-DQB1 alleles and critical amino acid differences in the peptide-binding pockets of HLA-DQB1 alleles might have role in influencing infection and pathogenesis of CHIKV.

There is no licensed vaccine or cure for human cytomegalovirus (CMV), a ubiquitous β-herpesvirus infecting 60–95% of adults worldwide. Infection can cause congenital abnormalities, result in severe disease in immunocompromised patients, and is a major impediment during successful organ transplantation. In addition, it has been associated with numerous inflammatory diseases and cancers, as well as being implicated in the development of essential hypertension, a major risk factor for heart disease. To date, limited data regarding the identification of immunogenic viral targets has frustrated CMV vaccine development. Based upon promising clinical data suggesting an important role for T cells in protecting against disease in the transplantation setting, we designed a novel panel of highly-optimized synthetic vaccines encoding major CMV proteins and evaluated their immune potential in murine studies. Vaccination induced robust CD8+ and CD4+ T cells of great epitopic breadth as extensively analyzed using a novel modified T cell assay described herein. Together with improved levels of CMV-specific T cells as driven by a vaccine, further immune evaluation of each target is warranted. The present model provides an important tool for guiding future immunization strategies against CMV.

To date, there have been several million infections by the Chikungunya virus (CHIKV), a mosquito-transmitted emerging pathogen that is considered to be taxonomically an Old World RNA virus. Although original CHIKV outbreaks were restricted to India, East Asian countries, Northern Italy, and France, a recent sharp rise had been identified in 41 countries or territories in the Caribbean, Central America, South America, and North America. A total of 1,012,347 suspected and 22,579 laboratory-confirmed CHIKV cases have been reported from these areas, which signals an increasing risk to the US mainland. Unlike past epidemics that were usually associated with Ae. aegypti transmission, the Caribbean outbreak was associated with Ae. albopictus transmission as the principal mosquito vector. In addition, the substantial increase in the number of deaths during this epidemic, as well as incidence of neurologic disease, suggests that CHIKV may have become more virulent. Currently, there are no licensed vaccines or therapeutics available for CHIKV or its associated disease pathologies. Therefore, development of new vaccines and therapies that could confer immunity and/or treat clinical symptoms of CHIKV is greatly desired. This chapter describes the use of entirely cutting edge technologies/methodologies developed by our group for the development and evaluation of novel DNA vaccines against CHIKV.

Background While Zika virus (ZIKV) infection is typically self-limited, congenital birth defects and Guillain-Barré syndrome are well-described. There are no therapies or vaccines against ZIKV infection. Methods ZIKA-001 is a phase I, open label, clinical trial designed to evaluate the safety, side effect profile, and immunogenicity of a synthetic, DNA vaccine (GLS-5700) targeting the pre-membrane+envelope proteins (prME) of the virus. Two groups of 20 participants received GLS-5700 at one of two dose levels: 1 mg or 2 mg DNA/dose at 0, 4, and 12 weeks. Vaccine was administered as 0.1 or 0.2 ml (1 or 2 mg) intradermal (ID) injection followed by electroporation (EP) with the CELLECTRA®-3P device Results The median age of the 40 participants was 38 (IQR 30–54) years; 60% were female 30% Latino and 78% white. No SAEs have been reported to date. Local minor AEs were injection site pain, redness, swelling and itching that occurred in half of the participants. Systemic adverse events were rare and included headache, myalgias, upper respiratory infections, fatigue/malaise and nausea. Four weeks after the first dose 25% vs. 60% of the participants in the 1 mg and 2 mg dose seroconverted. By week 6, 2 weeks after the second dose, the response was 65 and 84% respectively and 2 weeks after the third dose all participants in both dosing groups developed antibodies. At the end of the vaccination period over 60% of vaccinated person neutralized Zika virus in a vero cell assay and greater than 80% on neuronal cell targets. The protective efficacy of the antibodies generated by the vaccine was evaluated in the lethal IFNAR−/− mouse model. After the intraperitoneal administration of 0.1 ml of either baseline, week 14 serum or PBS the animals were challenged with 106 PFUs of ZIKV PR209 isolate. Whereas animals administered PBS (control) or baseline serum succumbed after a median of 5 days, those pretreated with week 14 serum from study participants survived suggesting that the humoral response generated by the vaccine is protective in this model. Conclusion Our trial shows for the first time in humans the safety and immunogenicity of an engineered DNA encoding consensus viral protein against ZIKV. Future studies will evaluate the effectiveness of the vaccine. Disclosures C. C. Roberts, GeneOne: Member, Salary. S. White, GeneOne: Member, Salary. A. S. Khan, Inovio: Employee and Shareholder, Salary and Stock. J. Boyer, Inovio: Employee and Shareholder, Salary and Stock. Y. K. Park, GeneOne: Board Member, CEO and Employee, Salary and Stock. S. Trottier, Canadian Institutes of Health Research: Investigator, Research grant. C. Remigio, GeneOne: Employee and Shareholder, Salary and Stock. G. P. Kobinger, GeneOne: Grant Investigator and Scientific Advisor, Grant recipient and Research support. D. Weiner, GeneOne: Grant Investigator and Scientific Advisor, Grant recipient, Licensing agreement or royalty and Stock. J. Maslow, GeneOne: Employee, Salary and Stock.

The safety, stability, and ability for repeat homologous vaccination makes the DNA vaccine platform an excellent candidate for an effective HIV-1 vaccine. However, the immunogenicity of early DNA vaccines did not translate from small animal models into larger non-human primates and was markedly lower than viral vectors. In addition to improvements to the DNA vector itself, delivery with electroporation, the inclusion of molecular adjuvants, and heterologous prime-boost strategies have dramatically improved the immunogenicity of DNA vaccines for HIV and currently makes them a leading platform with many areas warranting further research and clinical development.

Chikungunya virus (CHIKV) has caused large outbreaks worldwide in recent years. Acute-phase CHIKV infection has been reported to cause mild to severe febrile illness, and in some patients, this may be followed by long-lasting polyarthritis. The mainstay of treatment includes nonsteroidal anti-inflammatory drugs and other disease-modifying agents, the use of which is based on the assumption of an immunological interference mechanism in the pathogenesis. The present study has been designed to generate preliminary evidence to test this hypothesis. The levels of 30 cytokines were estimated in serum samples of acute CHIKV-infected patients, fully-recovered patients, patients with chronic CHIKV arthritis, and controls, using a quantitative multiplex bead ELISA. The levels of the proinflammatory cytokines IL-1 and IL-6 were elevated in acute patients, but IFN-γ/β and TNF-α levels remained stable. IL-10, which might have an anti-inflammatory effect, was also elevated, indicating a predominantly anti-inflammatory response in the acute phase of infection. Elevation of MCP-1, IL-6, IL-8, MIP-1α, and MIP-1β was most prominent in the chronic phase. These cytokines and chemokines have been shown to play important roles in other arthritides, including epidemic polyarthritis (EPA) caused by Ross River virus (RRV) and rheumatoid arthritis (RA).The immunopathogenesis of chronic CHIKV arthritis might have similarities to these arthritides. The novel intervention strategies being developed for EPA and RA, such as IL-6 and IL-8 signaling blockade, may also be considered for chronic CHIKV arthritis.

Introduction: Re-emergence of Chikungunya is a major public health problem in the southern states of India. Objectives: This study was undertaken to investigate an outbreak of Chikungunya, in June-August 2008 using PCR and determine the prevalent genotypes of Chikungunya virus (CHIKV) associated with the outbreak. Materials and Methods: Samples of blood were collected (in heparinized vacutainer tubes) from suspected patients of CHIKV infection from both Government Taluk Hospital in Kerala and a tertiary care hospital in Chennai, Tamil Nadu. A one-step RT-PCR was carried out on a block thermo-cycler targeting the E2 gene that codes for the viral envelope protein. The amplicons were verified for 305 bp size by standard agarose gel electrophoresis. The PCR products were purified, sequenced, and compared with other CHIKV strains reported from different geographical regions. A phylogenetic tree was constructed using MEGA 4. Results: Altogether 118 samples were collected from patients who presented with sudden onset of fever and/or joint pain, myalgia, and headache. CHIKV infection was confirmed by RT-PCR in 14 patients and all these cases were from Kerala. The positivity correlated with the early stage of the disease as all these patients had fever of less than seven days duration. The study isolates have been allotted the GenBank accession nos. GQ272368-GQ272381. Phylogenetic analysis of recent CHIKV isolates by partial sequencing of E2 region shows that isolates are closely related to strains from neighboring states and the African type. Conclusion: RT-PCR is a useful technique for the early detection of CHIKV infection during outbreaks. Molecular characterization of the strains indicates that majority of the strains have originated from the Central/East African strains of CHIKV.

DNA vaccination is a novel immunization strategy that has great potential for the development of vaccines and immune therapeutics. This strategy has been highly effective in mice, but is less immunogenic in non-human primates and in humans. Enhancing DNA vaccine potency remains a challenge. It is likely that antigen-presenting cells (APCs), and especially dendritic cells (DCs), play a significant role in the presentation of the vaccine antigen to the immune system. A new study reports the synergistic recruitment, expansion and activation of DCs in vivo by high-mobility group box 1 (HMGB1) protein. Such combinational strategies for delivering vaccine in a single, simple platform will hypothetically bolster the cellular immunity in vivo. Here, we combined plasmid encoding human immunodeficiency virus-1 (HIV-1) Gag and Env with an HMGB1 plasmid as a DNA adjuvant in BALB/c mice (by intramuscular immunization via electroporation), and humoral and cellular responses were measured. Co-administration of this potent immunostimulatory adjuvant strongly enhanced the cellular interferon-gamma (IFN-gamma) and humoral immune response compared with that obtained in mice immunized with vaccine only. Our results show that co-immunization with HMGB1 can have a strong adjuvant activity, driving strong cellular and humoral immunity that may be an effective immunological adjuvant in DNA vaccination against HIV-1.

The HIV-1 Vpr protein is a viral accessory protein that plays a number of important roles during HIV infection. The activities of Vpr are numerous and include the induction of apoptosis, the modulation of cell cycle arrest, as well as control of viral transcription. Study of HIV clones lacking Vpr in vitro and analysis of HIV variants isolated from long-term nonprogressors in vivo highlight the importance of Vpr for viral replication as well as immune suppression and cell death. Vpr may therefore be considered among the most important accessory proteins encoded by HIV.

Chronic viral infection is characterized by the functional impairment of virus-specific T-cell responses. Recent evidence has suggested that the inhibitory receptor programmed death 1 (PD-1) is specifically upregulated on antigen-specific T cells during various chronic viral infections. Indeed, it has been reported that human immunodeficiency virus (HIV)-specific T cells express elevated levels of PD-1 and that this expression correlates with the viral load and inversely with CD4 + T-cell counts. More importantly, antibody blockade of the PD-1/PD-L1 pathway was sufficient to both increase and stimulate virus-specific T-cell proliferation and cytokine production. However, the mechanisms that mediate HIV-induced PD-1 upregulation are not known. Here, we provide evidence that the HIV type 1 (HIV-1) accessory protein Nef can transcriptionally induce the expression of PD-1 during infection in vitro. Nef-induced PD-1 upregulation requires its proline-rich motif and the activation of the downstream kinase p38. Further, inhibition of Nef activity by p38 MAPK inhibitor effectively blocked PD-1 upregulation, suggesting that p38 MAPK activation is an important initiating event in Nef-mediated PD-1 expression in HIV-1-infected cells. These data demonstrate an important signaling event of Nef in HIV-1 pathogenesis.

Dengue virus (DENV) is the most important mosquito-borne viral infection in humans. In recent years, the number of cases and outbreaks has dramatically increased worldwide. While vaccines are being developed, none are currently available that provide balanced protection against all DENV serotypes. Advances in human antibody isolation have uncovered DENV neutralizing antibodies (nAbs) that are capable of preventing infection from multiple serotypes. Yet delivering monoclonal antibodies using conventional methods is impractical due to high costs. Engineering novel methods of delivering monoclonal antibodies could tip the scale in the fight against DENV. Here we demonstrate that simple intramuscular delivery by electroporation of synthetic DNA plasmids engineered to express modified human nAbs against multiple DENV serotypes confers protection against DENV disease and prevents antibody-dependent enhancement (ADE) of disease in mice. This synthetic nucleic acid antibody prophylaxis/immunotherapy approach may have important applications in the fight against infectious disease.

Dengue virus (DENV) is the most important mosquito-borne viral infection in humans, with nearly 400 million infections occurring each year and a continually expanding geographic reach. While vaccines are being developed, none are currently available that provide balanced protection against all DENV serotypes. Advances in human antibody isolation have uncovered DENV neutralizing antibodies (nAbs) that are capable of preventing infection from multiple serotypes. Yet delivering monoclonal antibodies using conventional methods is impractical due to high costs. Engineering nucleic acid-based strategies for delivering monoclonal antibodies could tip the scale in the fight against DENV, as well as other emerging infectious diseases.Here, we describe an approach to delivering cross-reactive neutralizing antibodies against DENV into the host circulation using DNA plasmid-mediated antibody gene transfer. This approach, which we term DNA mAb (DMAB) delivery, generates biologically relevant levels of mAbs after a single intramuscular injection of antibody-encoding DNA followed by in vivo electroporation (EP). Since this approach allows for genetic tailoring of the exact features of the desired antibody, we incorporated Fc region modifications to a naturally occurring human anti-DENV neutralizing antibody to enhance antibody function in vivo. We demonstrate that intramuscular delivery in mice of pDVSF-3 LALA, which encodes a human anti-DENV1-3 IgG1 neutralizing antibody modified with a mutation that abrogates FcgR binding, produces anti-DENV antisera capable of binding and neutralizing DENV1-3. Importantly, mice receiving pDVSF-3 LALA, but not the unmodified pDVSF-3 WT, were protected from both virus-only disease and antibody-enhanced lethal disease. These data establish this novel platform as a safe, effective means of delivering protective monoclonal antibodies to a host.This work was supported by grants funded to DBW through the National Institutes of Health, the DARPA-PROTECT award, and Inovio Pharmaceuticals Inc.

DNA vaccines expressing the envelope (env) of the human immunodeficiency virus type 1 (HIV-1) have been relatively ineffective at generating strong immune responses. In this study, we described the development of a recombinant plasmid DNA (pEK2P-B) expressing an engineered codon-optimized envelope gp140 gene of primary (nonrecombinant) HIV-1 subtype B isolate 6101. Codon usage and RNA optimization of HIV-1 structural genes has been shown to increase protein expression in vitro as well as in the context of DNA vaccines in vivo. To further increase the expression, a synthetic IgE leader with kozak sequences were fused into the env gene. The cytoplasmic tail of the gene was also truncated to prevent recycling. The expression of env by the recombinant pEK2P-B was evaluated using T7 coupled transcription/translation. The construct demonstrated high expression of the HIV-1 env gene in eukaryotic cells as demonstrated in transfected 293-T and RD cells. Immunogenicity of pEK2P-B was evaluated in mice using IFN-gamma ELISpot assay, and the construct was found to be highly immunogenic and crossreactive with HIV-1 clade C env peptides. Three immunodominant peptides were also mapped out. Furthermore, by performing a CFSE flow cytometry-based proliferation assay, 2.4 and 1.5% proliferation was observed in CD4+, CD8+, and CCR+ memory T cells, respectively. Therefore, this engineered synthetic optimized env DNA vaccine may be useful in DNA vaccine and other studies of HIV-1 immunogenicity.

The development of vaccines against arthropod-borne infectious diseases has been wrought with difficulties. Recent advances in human antibody isolation have uncovered neutralizing monoclonal antibodies (mAbs) that are capable of providing protection against pathogen challenge in various animal models. Yet generating and delivering biologically-relevant levels of such antibodies using conventional monoclonal antibody methodology is impractical, often requiring huge expenses and repeated administrations for clinical benefit. Creating new methods of delivering monoclonal antibodies could drastically tip the scale in the fight against a number of devastating pathogens.Here, we describe an approach to delivering neutralizing mAbs in vivo using DNA plasmid-mediated antibody gene transfer. This approach, which we term DNA mAb (DMAb) delivery, generates biologically relevant levels of mAbs after a single intramuscular injection of antibody-encoding DNA followed by in vivo electroporation (EP). First, we demonstrate the ability of DMAb technology to deliver cross-reactive neutralizing antibodies against DENV into the host circulation. Since this approach allows for genetic tailoring of the exact features of the desired antibody, we incorporated Fc region modifications to a naturally occurring human anti-DENV neutralizing antibody to enhance antibody function in vivo. We show that intramuscular delivery in mice of pDVSF-3 LALA, which encodes a human anti-DENV1-3 IgG1 neutralizing antibody modified with a mutation that abrogates FcγR binding, produces anti-DENV antisera capable of binding and neutralizing DENV1-3. Importantly, mice receiving pDVSF-3 LALA, but not the unmodified pDVSF-3 WT, were protected from both virus-only disease and antibody-enhanced lethal disease.Using a similar, targeted genetic approach to antibody modifications, we also show that DMAbs encoding antibodies against Borrelia burgdorferi (the causative agent of Lyme disease) can undergo extensive amino acid modifications that substantially increase in vivo mAb production levels compared to wild-type DMAb sequences. These data illustrate a subset of the functional optimizations made possible with the DMAb platform.This work was supported by grants funded to DBW through the National Institutes of Health, the DARPA-PROTECT award, and Inovio Pharmaceuticals Inc.

HIV-1 sequences are highly diverse due to the inaccuracy of the viral reverse transcriptase. This diversity has been studied and used to categorize HIV isolates into subtypes or clades, which are geographically distinct. To develop effective vaccines against HIV-1, immunogens representing different subtypes may be important for induction of cross-protective immunity, but little data exist describing and comparing the immunogenicity induced by different subtype-based vaccines. This issue is further complicated by poor expression of HIV structural antigens due to rev dependence. One costly approach is to codon optimize each subtype construct to be examined. Interestingly, cis-acting transcriptional elements (CTE) can also by pass rev restriction by a rev independent export pathway. We reasoned that rev+CTE constructs might have advantages for such expression studies. A subtype A envelope sequence from a viral isolate from east Africa was cloned into a eukaryotic expression vector under the control of the CMV-IE promoter. The utility of inclusion of the Mason–Pfizer monkey virus (MPV)-CTE with/without rev for driving envelope expression and immunogenicity was examined. Expression of envelope (gp120) was confirmed by immunoblot analysis and by pseudotype virus infectivity assays. The presence of rev and the CTE together increased envelope expression and viral infection. Furthermore the CTE+rev construct was significantly more immunogenic then CTE alone vector. Isotype analysis and cytokine profiles showed strong Th1 response in plasmid-immunized mice, which also demonstrated the superior nature of the rev+CTE construct. These responses were of similar or greater magnitude to a codon-optimized construct. The resulting cellular immune responses were highly cross-reactive with a HIV-1 envelope subtype B antigen. This study suggests a simple strategy for improving the expression and immunogenicity of HIV subtype-specific envelope antigens as plasmid or vector-borne immunogens.

West Nile virus (WNV) is a member of the Flaviviridae family of vector-borne pathogens. Clinical signs of WNV infection include neurologic symptoms, limb weakness, and encephalitis, which can result in paralysis or death. We report that the WNV-capsid (Cp) by itself induces rapid nuclear condensation and cell death in tissue culture. Apoptosis is induced through the mitochondrial pathway resulting in caspase-9 activation and downstream caspase-3 activation. Capsid gene delivery into the striatum of mouse brain or interskeletal muscle resulted in cell death and inflammation, likely through capsid-induced apoptosis in vivo. These studies demonstrate that the capsid protein of WNV may be responsible for aspects of viral pathogenesis through induction of the apoptotic cascade.

We compared the immunogenicity of plasmid vaccines containing multiple human immunodeficiency virus (HIV) antigens and found that covaccination with plasmids expressing HIV-1 14 kDa vpr gene product profoundly reduces antigen-specific CD8-mediated cytotoxic T-cell activity (CTL). Interestingly, Th1 type responses against codelivered antigens (pGag-Pol, pNef, etc.) encoded by the plasmid vaccines were suppressed. This suggested that vpr might compromise CD8 T-cell immunity in vivo during infection. A pilot primate vaccine study was designed to test the hypothesis to compare the following groups: unvaccinated controls, animals vaccinated without simean immunodeficiency virus (SIV)-Nef antigen plasmid, and animals covaccinated with the identical plasmid antigen and a plasmid construct encoding SIV Vpr/Vpx. Animals were subsequently challenged intrarectally with pathogenic SIVmac251 after the final vaccination of a multiple immunization protocol. Control animals were all infected and exhibited high viral loads and rapid CD4 + T-cell loss. In contrast, the Nef plasmid-vaccinated animals were also infected but exhibited preservation of CD4 + T-cells and a multilog reduction in viral load compared with controls. Animals covaccinated multiple times with the Nef vaccine and pVpr/Vpx plasmid suffered rapid and profound loss of CD4 + T-cells. These results have important implications for the design of multicomponent and particle vaccines for HIV-1 as well as for our understanding of HIV/SIV pathogenesis in vivo.

The HIV-1 vpr gene encodes a 14-kDa virion-packaged protein that has been implicated in viral pathogenesis. Vpr exhibits profound effects on human primary cells influencing proliferation, differentiation, apoptosis, and cytokine production, in part through NF-κB-mediated transcription. NF-κB, a potent transcription factor, activates many proinflammatory cytokines/chemokines upon infection. Here, we analyzed the effect of extracellular Vpr as well as the virion-associated Vpr on β chemokines (MIP-1α, MIP-1β, and RANTES) production in human macrophages and primary lymphocytes (PBLs). Macrophages and PBLs exposed to HIV-1 vpr+ viruses or to recombinant Vpr protein produced significantly less β chemokines compared with cells infected with HIV-1 vpr− viruses or irrelevant control protein (Gag)-exposed cells. These results suggest that a Vpr-mediated increase in virus replication could be in part through down-regulation of chemokine production.

Chikungunya virus (CHIKV) is an important emerging mosquito-borne alphavirus, indigenous to tropical Africa and Asia. It can cause epidemic fever and acute illness characterized by fever and arthralgias. The epidemic cycle of this infection is similar to dengue and urban yellow fever viral infections. The generation of an efficient vaccine against CHIKV is necessary to prevent and/or control the disease manifestations of the infection. In this report, we studied immune response against a CHIKV-envelope DNA vaccine (pEnv) and the role of the CHIKV nonstructural gene 2 (nsP2) as an adjuvant for the induction of protective immune responses in a relevant mouse challenge model. When injected with the CHIKV pEnv alone, 70% of the immunized mice survived CHIKV challenge, whereas when co-injected with pEnv+pnsP2, 90% of the mice survived viral challenge. Mice also exhibited a delayed onset signs of illness, and a marked decrease in morbidity, suggesting a nsP2 mediated adjuvant effect. Co-injection of the pnsP2 adjuvant with pEnv also qualitatively and quantitatively increased antigen specific neutralizing antibody responses compared to vaccination with pEnv alone. In sum, these novel data imply that the addition of nsP2 to the pEnv vaccine enhances anti-CHIKV-Env immune responses and maybe useful to include in future CHIKV clinical vaccination strategies.

The present invention relates to compositions comprising two or more DNA plasmids encoding consensus and transmitted founder HIV envelope glycoproteins which expressed and induce a potent immune response.

DNA-based vaccines, while highly immunogenic in mice, generate significantly weaker responses in primates. Therefore, current efforts are aimed at increasing their immunogenicity, which include optimizing the plasmid/gene, the vaccine formulation and method of delivery. For example, co-immunization with molecular adjuvants encoding an immunomodulatory protein has been shown to improve the antigen (Ag)-specific immune response. Thus, the incorporation of enhancing elements, such as these, may be particularly important in the influenza model in which high titered antibody (Ab) responses are critical for protection. In this regard, we compared the ability of plasmid-encoded high-mobility group box 1 protein (HMGB1), a novel cytokine in which we have previously mutated in order to increase DNA vaccine immunogenicity, with boost Ag-specific immune responses during DNA vaccination with influenza A/PR/8/34 nucleoprotein or the hemagglutinin of A novel H1N1/09. We show that the HMGB1 adjuvant is capable of enhancing adaptive effector and memory immune responses. Although Ag-specific antibodies were detected in all vaccinated animals, a greater neutralizing Ab response was associated with the HMGB1 adjuvant. Furthermore, these responses improved CD8 T+-cell effector and memory responses and provided protection against a lethal mucosal influenza A/PR/8/34 challenge. Thus, co-immunization with HMGB1 has strong in vivo adjuvant activity during the development of immunity against plasmid-encoded Ag.

Protection against infection is the hallmark of immunity and the basis of effective vaccination. For a variety of reasons there is a great demand to develop new, safer and more effective vaccine platforms. In this regard, while ‘first-generation’ DNA vaccines were poorly immunogenic, new genetic ‘optimization’ strategies and the application of in vivo electroporation (EP) have dramatically boosted their potency. We developed a highly optimized plasmid DNA vaccine that expresses the lymphocytic choriomeningitis virus (LCMV) nucleocapsid protein (NP) and evaluated it using the LCMV challenge model, a gold standard for studying infection and immunity. When administered intramuscularly with EP, robust NP-specific cellular and humoral immune responses were elicited, the magnitudes of which approached those following acute LCMV infection. Furthermore, these responses were capable of providing 100% protection against a high-dose, normally lethal virus challenge. This is the first non-infectious vaccine conferring complete protective immunity up to eight weeks after vaccination and demonstrates the potential utility of ‘next-generation’ DNA vaccines.

Chikungunya virus is a mosquito-borne emerging pathogen that has a major health impact in humans and causes fever disease, headache, rash, nausea, vomiting, myalgia, and arthralgia. Indigenous to tropical Africa, recent large outbreaks have been reported in parts of South East Asia and several of its neighboring islands in 2005-07 and in Europe in 2007. Furthermore, positive cases have been confirmed in the United States in travelers returning from known outbreak areas. Currently, there is no vaccine or antiviral treatment. With the threat of an emerging global pandemic, the peculiar problems associated with the more immediate and seasonal epidemics warrant the development of an effective vaccine. In this review, we summarize the evidence supporting these concepts.

Chikungunya virus (CHIKV) is an emerging mosquito-borne alphavirus indigenous to tropical Africa and Asia. Acute illness is characterized by fever, arthralgias, conjunctivitis, rash, and sometimes arthritis. Relatively little is known about the antigenic targets for immunity, and no licensed vaccines or therapeutics are currently available for the pathogen. While the Aedes aegypti mosquito is its primary vector, recent evidence suggests that other carriers can transmit CHIKV thus raising concerns about its spread outside of natural endemic areas to new countries including the U.S. and Europe. Considering the potential for pandemic spread, understanding the development of immunity is paramount to the development of effective counter measures against CHIKV. In this study, we isolated a new CHIKV virus from an acutely infected human patient and developed a defined viral challenge stock in mice that allowed us to study viral pathogenesis and develop a viral neutralization assay. We then constructed a synthetic DNA vaccine delivered by in vivo electroporation (EP) that expresses a component of the CHIKV envelope glycoprotein and used this model to evaluate its efficacy. Vaccination induced robust antigen-specific cellular and humoral immune responses, which individually were capable of providing protection against CHIKV challenge in mice. Furthermore, vaccine studies in rhesus macaques demonstrated induction of nAb responses, which mimicked those induced in convalescent human patient sera. These data suggest a protective role for nAb against CHIKV disease and support further study of envelope-based CHIKV DNA vaccines., Author Summary Chikungunya fever epidemics are sustained by a cycle of human-mosquito-human transmission, with the epidemic cycle being similar to those of dengue and urban yellow fever. While the threat of a pandemic continues to engage the public's attention, the peculiar problems associated with the more immediate and very real seasonal epidemics are also worthy of consideration. Specifically, there are limited viral strains that have been characterized and available for laboratory study as well as limited knowledge of immune responses induced to the virus. In this study, we isolated CHIKV virus from an acutely infected human patient and used this new virus to develop a neutralization assay and a challenge stock, which is effective in a mouse model. Furthermore, we analyzed the ability of an envelope-based synthetic DNA-based vaccine to impact viral disease in the mouse model and to generate protective levels of immune responses in nonhuman primates. We observed that this novel vaccine approach generated protective levels of immune responses in both mouse and non-human primate models. We believe that these studies advance the field of Chikungunya vaccine research as well as the study of immune protection to CHIKV.

A hurdle facing DNA vaccine development is the ability to generate strong immune responses systemically and at local immune sites. We report a novel systemically administered DNA vaccination strategy using intramuscular codelivery of CCL27 or CCL28, which elicited elevated peripheral IFN-gamma and antigen-specific IgG while driving antigen-specific T-cell secretion of cytokine and antibody production in the gut-associated lymphoid tissue and lung. This strategy resulted in induction of long-lived antibody responses that neutralized influenza A/PR8/34 and protected mice from morbidity and mortality associated with a lethal intranasal viral challenge. This is the first example of the use of CCL27 and CCL28 chemokines as adjuvants to influence a DNA vaccine strategy, suggesting further examination of this approach for manipulation of vaccine-induced immunity impacting both quality and phenotype of responses.