Search

Description, Suction-mediated cutaneous DNA uptake yielded high in vivo efficiency and thus provides an alternative transfection platform., This work reports a suction-based cutaneous delivery method for in vivo DNA transfection. Following intradermal Mantoux injection of plasmid DNA in a rat model, a moderate negative pressure is applied to the injection site, a technique similar to Chinese báguàn and Middle Eastern hijama cupping therapies. Strong GFP expression was demonstrated with pEGFP-N1 plasmids where fluorescence was observed as early as 1 hour after dosing. Modeling indicates a strong correlation between focal strain/stress and expression patterns. The absence of visible and/or histological tissue injury contrasts with current in vivo transfection systems such as electroporation. Specific utility was demonstrated with a synthetic SARS-CoV-2 DNA vaccine, which generated host humoral immune response in rats with notable antibody production. This method enables an easy-to-use, cost-effective, and highly scalable platform for both laboratorial transfection needs and clinical applications for nucleic acid–based therapeutics and vaccines.

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.

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.

A new pandemic is ongoing in several parts of the world. The agent responsible is the newly emerged severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). The symptoms associated with this virus are known as the coronavirus disease-2019 (COVID-19). In this review, we summarize the published data on virus specific antibodies in hospitalized patients with COVID-19 disease, patients recovered from the disease and the individuals who are asymptomatic with SARS-CoV-2 infections. The review highlights the following: i) an adjunct role of antibody tests in the diagnosis of COVID-19 in combination with RT-PCR; ii) status of antibodies from COVID-19 convalescent patients to select donors for plasma therapy; iii) the potential confounding effects of other coronaviruses, measles, mumps and rubella in antibody testing due to homology of certain viral genes; and iv) the role of antibody testing for conducting surveillance in populations, incidence estimation, contact tracing and epidemiologic studies., Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2); Diagnosis of COVID-19; Antibody tests; RT-PCR.

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.

In article number https://doi.org/10.1002/advs.201902802, David B. Weiner, Daniel W. Kulp, and co‐workers demonstrate that synthetic DNA delivery and adaptive electroporation can launch de novo assembly of different nanoparticle vaccines in the hosts to elicit significantly improved responses while reducing required doses. Next‐generation designer vaccines can now be rapidly evaluated in vivo to facilitate clinical translation for the betterment of human and animal health.

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

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.

Ovarian cancer is frequently diagnosed as peritoneal carcinomatosis. Unlike other tumor locations, the peritoneal cavity is commonly exposed to gut-breaching and ascending genital microorganisms and has a unique immune environment. IL-33 is a local cytokine that can activate innate and adaptive immunity. We studied the effectiveness of local IL-33 delivery in the treatment of cancer that has metastasized to the peritoneal cavity. Direct peritoneal administration of IL-33 delayed the progression of metastatic peritoneal cancer. Prolongation in survival was not associated with a direct effect of IL-33 on tumor cells, but with major changes in the immune microenvironment of the tumor. IL-33 promoted a significant increase in the leukocyte compartment of the tumor immunoenvironment and an allergic cytokine profile. We observed a substantial increase in the number of activated CD4+ T-cells accompanied by peritoneal eosinophil infiltration, B-cell activation and activation of peritoneal macrophages which displayed tumoricidal capacity. Depletion of CD4+ cells, eosinophils or macrophages reduced the anti-tumor effects of IL-33 but none of these alone were sufficient to completely abrogate its positive benefit. In conclusion, local administration of IL-33 generates an allergic tumor environment resulting in a novel approach for treatment of metastatic peritoneal malignancies, such as advanced ovarian cancer.

Elevated low-density lipoprotein cholesterol (LDL-C) is one of the major contributors to cardiovascular heart disease (CHD), the leading cause of death worldwide. Due to severe side effects of statins, alternative treatment strategies are required for statin-intolerant patients. Monoclonal antibodies (mAbs) targeting proprotein convertase subtilisin/kexin type 9 (PCSK9) have shown great efficacy in LDL-C reduction. Limitations for this approach include the need for multiple injections as well as increased costs associated with patient management. Here, we engineered a DNA-encoded mAb (DMAb) targeting PCSK9 (daPCSK9), as an alternative approach to protein-based lipid-lowering therapeutics, and we characterized its expression and activity. A single intramuscular administration of mouse daPCSK9 generated expression in vivo for over 42 days that corresponded with a substantial decrease of 28.6% in non-high-density lipoprotein cholesterol (non-HDL-C) and 10.3% in total cholesterol by day 7 in wild-type mice. Repeated administrations of the DMAb plasmid led to increasing expression, with DMAb levels of 7.5 μg/mL at day 62. daPCSK9 therapeutics may provide a novel, simple, less frequent, cost-effective approach to reducing LDL-C, either as a stand-alone therapy or in combination with other LDL-lowering therapeutics for synergistic effect.

The impact of broad-spectrum antibiotics on antimicrobial resistance and disruption of the beneficial microbiome compels the urgent investigation of bacteria-specific approaches such as antibody-based strategies. Among these, DNA-delivered monoclonal antibodies (DMAbs), produced by muscle cells in vivo, potentially allow the prevention or treatment of bacterial infections circumventing some of the hurdles of protein IgG delivery. Here, we optimize DNA-delivered monoclonal antibodies consisting of two potent human IgG clones, including a non-natural bispecific IgG1 candidate, targeting Pseudomonas aeruginosa. The DNA-delivered monoclonal antibodies exhibit indistinguishable potency compared to bioprocessed IgG and protect against lethal pneumonia in mice. The DNA-delivered monoclonal antibodies decrease bacterial colonization of organs and exhibit enhanced adjunctive activity in combination with antibiotics. These studies support DNA-delivered monoclonal antibodies delivery as a potential strategy to augment the host immune response to prevent serious bacterial infections, and represent a significant advancement toward broader practical delivery of monoclonal antibody immunotherapeutics for additional infectious pathogens., DNA-delivered monoclonal antibodies (DMAbs) can be produced by muscle cells in vivo, potentially allowing prevention or treatment of infectious diseases. Here, the authors show that two DMAbs targeting Pseudomonas aeruginosa proteins confer protection against lethal pneumonia in mice.

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.

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.

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.

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.

Summary VGX-1027 [(S,R)-3-phenyl-4,5-dihydro-5-isoxasole acetic acid] is a small molecule compound with immunomodulatory properties, which favourably influences the development of immuno-inflammatory and autoimmune diseases in different animal models such as type 1 diabetes mellitus, pleurisy, rheumatoid arthritis and inflammatory bowel disease. However, the precise mechanism of action of VGX-1027 remains to be ascertained. With this aim, we have studied the immunomodulatory effects of VGX-1027 in vitro, using a genome-wide oligonucleotide microarray approach, and in vivo, using the NZB/NZW F1 model of systemic lupus erythematosus. Microarray data revealed that the administration of VGX-1027 profoundly affected the immune response to exogenous antigens, by modulating the expression of genes that are primarily involved in antigen processing and presentation as well as genes that regulate immune activation. When administered in vivo VGX-1027 ameliorated the course of the disease in the NZB/NZW F1 mice, which correlated with higher per cent survival and improved clinical and histopathological signs. The data presented herein support the theory that VGX-1027 modulates immunity, probably by inhibiting inflammatory antigen presentation and so limiting immune cell expansion.

DNA vaccine-induced immunity can be enhanced by the co-delivery of synthetic gene-encoding molecular adjuvants. Many of these adjuvants have included cytokines, chemokines or co-stimulatory molecules that have been demonstrated to enhance vaccine-induced immunity by increasing the magnitude or type of immune responses and/or protective efficacy. In this way, through the use of adjuvants, immune responses can be highly customizable and functionally tailored for optimal efficacy against pathogen specific (i.e., infectious agent) or non-pathogen (i.e., cancer) antigens. In the novel study presented here, we examined the use of cellular transcription factors as molecular adjuvants. Specifically the co-delivery of (a) RelA, a subunit of the NF-κB transcription complex or (b) T-bet, a Th1-specific T box transcription factor, along with a prototypical DNA vaccine expressing HIV-1 proteins was evaluated. As well, all of the vaccines and adjuvants were administered to mice using in vivo electroporation (EP), a technology demonstrated to dramatically increase plasmid DNA transfection and subsequent transgene expression with concomitant enhancement of vaccine induced immune responses. As such, this study demonstrated that co-delivery of either adjuvant resulted in enhanced T and B cell responses, specifically characterized by increased T cell numbers, IFN-γ production, as well as enhanced antibody responses. This study demonstrates the use of cellular transcription factors as adjuvants for enhancing DNA vaccine-induced immunity.

In recent years, DNA vaccines have undergone a number of technological advancements that have incited renewed interest and heightened promise in the field. Two such improvements are the use of genetically engineered cytokine adjuvants and plasmid delivery via in vivo electroporation (EP), the latter of which has been shown to increase antigen delivery by nearly 1000-fold compared to naked DNA plasmid delivery alone. Both strategies, either separately or in combination, have been shown to augment cellular and humoral immune responses in not only mice, but also in large animal models. These promising results, coupled with recent clinical trials that have shown enhanced immune responses in humans, highlight the bright prospects for DNA vaccines to address many human diseases.

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.

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.

The Vpr protein of HIV-1 functions as a vital accessory gene by regulating various cellular functions, including cell differentiation, apoptosis, nuclear factor of kappaB (NF-kappaB) suppression and cell-cycle arrest of the host cell. Several reports have indicated that Vpr complexes with the glucocorticoid receptor (GR), but it remains unclear whether the GR pathway is required for Vpr to function. Here, we report that Vpr uses the GR pathway as a recruitment vehicle for the NF-kappaB co-activating protein, poly(ADP-ribose) polymerase-1 (PARP-1). The GR interaction with Vpr is both necessary and sufficient to facilitate this interaction by potentiating the formation of a Vpr-GR-PARP-1 complex. The recruitment of PARP-1 by the Vpr-GR complex prevents its nuclear localization, which is necessary for Vpr to suppress NF-kappaB. The association of GR with PARP-1 is not observed with steroid (glucocorticoid) treatment, indicating that the GR association with PARP-1 is a gain of function that is solely attributed to HIV-1 Vpr. These data provide important insights into Vpr biology and its role in HIV pathogenesis.

The human immunodeficiency virus (HIV) has been reported to target noninfected CD4 and CD8 cells for destruction. This effect is manifested in part through up-regulation of the death receptor Fas ligand (FasL) by HIV-1 negative factor (Nef), leading to bystander damage. However, the signal transduction and transcriptional regulation of this process remains elusive. Here, we provide evidence that p38 mitogen-activated protein kinase (MAPK) is required for this process. Loss-of-function experiments through dominant-negative p38 isoform, p38 siRNA, and chemical inhibitors of p38 activation suggest that p38 is necessary for Nef-induced activator protein-1 (AP-1) activation, as inhibition leads to an attenuation of AP-1-dependent transcription. Furthermore, mutagenesis of the FasL promoter reveals that its AP-1 enhancer element is required for Nef-mediated transcriptional activation. Therefore, a linear pathway for Nef-induced FasL expression that encompasses p38 and AP-1 has been elucidated. Furthermore, chemical inhibition of the p38 pathway attenuates HIV-1-mediated bystander killing of CD8 cells in vitro. (Blood. 2005;106:2059-2068)

A significant hurdle in effective vaccine design for infectious pathogens is the ability to mount immune responses at the portal of entry, namely mucosal sites. The development of a systemic-delivered vaccine approach driving secretory IgA and mucosal T cell immune responses against pathogens would fundamentally change many vaccine strategies. Using mucosal pathogens HIV-1 and Influenza A as models, we demonstrate that a novel systemic administered DNA vaccination strategy utilizing co-delivery of specific mucosal-derived chemokine adjuvants, can produce distal mucosal immune responses. This strategy resulted in redirection of a4b7 and CCR9 or CCR10 positive immune cells to the site of immunization with mucosal retrafficking phenotype. The immune phenotype included enhanced cytokine and cytolytic markers expressed by antigen-specific T cells from the spleen, lung and lamina propria, as well as elevated IgG and secretory IgA responses in secondary lymphoid organs, B cells from the gut, peripheral blood and fecal extracts. Systemic immunization controls, as expected, failed to induce mucosal immunity. These studies have great significance for basic understanding of gut lymphocyte homing, mucosal phenotype commitment and development of vaccine strategies for mucosal pathogens. This work is supported by F32AI054152 to MAK, and N01-A1-15429, NIAID-HVDDT to DBW.