Prime-boost vaccination regimens with INO-4800 and INO-4802 augment and broaden immune responses against SARS-CoV-2 in nonhuman primates.

The enhanced transmissibility and immune evasion associated with emerging SARS-CoV-2 variants demands the development of next-generation vaccines capable of inducing superior protection amid a shifting pandemic landscape. Since a portion of the global population harbors some level of immunity from vaccines based on the original Wuhan-Hu-1 SARS-CoV-2 sequence or natural infection, an important question going forward is whether this immunity can be boosted by next-generation vaccines that target emerging variants while simultaneously maintaining long-term protection against existing strains. Here, we evaluated the immunogenicity of INO-4800, our synthetic DNA vaccine candidate for COVID-19 currently in clinical evaluation, and INO-4802, a next-generation DNA vaccine designed to broadly target emerging SARS-CoV-2 variants, as booster vaccines in nonhuman primates. Rhesus macaques primed over one year prior with the first-generation INO-4800 vaccine were boosted with either INO-4800 or INO-4802 in homologous or heterologous prime-boost regimens. Both boosting schedules led to an expansion of T cells and antibody responses which were characterized by improved neutralizing and ACE2 blocking activity across wild-type SARS-CoV-2 as well as multiple variants of concern. These data illustrate the durability of immunity following vaccination with INO-4800 and additionally support the use of either INO-4800 or INO-4802 in prime-boost regimens.

Intradermal-delivered DNA vaccine induces durable immunity mediating a reduction in viral load in a rhesus macaque SARS-CoV-2 challenge model.

Coronavirus disease 2019 (COVID-19), caused by the SARS-CoV-2 virus, has had a dramatic global impact on public health and social and economic infrastructures. Here, we assess the immunogenicity and anamnestic protective efficacy in rhesus macaques of an intradermal (i.d.)-delivered SARS-CoV-2 spike DNA vaccine, INO-4800, currently being evaluated in clinical trials. Vaccination with INO-4800 induced T cell responses and induced spike antigen and RBD binding antibodies with ADCP and ADCD activity. Sera from the animals neutralized both the D614 and G614 SARS-CoV-2 pseudotype viruses. Several months after vaccination, animals were challenged with SARS-CoV-2 resulting in rapid recall of anti-SARS-CoV-2 spike protein T cell and neutralizing antibody responses. These responses were associated with lower viral loads in the lung. These studies support the immune impact of INO-4800 for inducing both humoral and cellular arms of the adaptive immune system, which are likely important for providing durable protection against COVID-19 disease.

Multivalent DNA Vaccines as A Strategy to Combat Multiple Concurrent Epidemics: Mosquito-Borne and Hemorrhagic Fever Viruses.

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 nonhuman 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.

SARS-CoV-2 Assays To Detect Functional Antibody Responses That Block ACE2 Recognition in Vaccinated Animals and Infected Patients.

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.

Immunogenicity of a DNA vaccine candidate for COVID-19.

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.

Protective Efficacy and Long-Term Immunogenicity in Cynomolgus Macaques by Ebola Virus Glycoprotein Synthetic DNA Vaccines.

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.

Synergy of Immune Checkpoint Blockade with a Novel Synthetic Consensus DNA Vaccine Targeting TERT.

Immune checkpoint blockade antibodies are setting a new standard of care for cancer patients. It is therefore important to assess any new immune-based therapies in the context of immune checkpoint blockade. Here, we evaluate the impact of combining a synthetic consensus TERT DNA vaccine that has improved capacity to break tolerance with immune checkpoint inhibitors. We observed that blockade of CTLA-4 or, to a lesser extent, PD-1 synergized with TERT vaccine, generating more robust anti-tumor activity compared to checkpoint alone or vaccine alone. Despite this anti-tumor synergy, none of these immune checkpoint therapies showed improvement in TERT antigen-specific immune responses in tumor-bearing mice. αCTLA-4 therapy enhanced the frequency of T-bet+/CD44+ effector CD8+ T cells within the tumor and decreased the frequency of regulatory T cells within the tumor, but not in peripheral blood. CTLA-4 blockade synergized more than Treg depletion with TERT DNA vaccine, suggesting that the effect of CTLA-4 blockade is more likely due to the expansion of effector T cells in the tumor rather than a reduction in the frequency of Tregs. These results suggest that immune checkpoint inhibitors function to alter the immune regulatory environment to synergize with DNA vaccines, rather than boosting antigen-specific responses at the site of vaccination.

A Novel DNA Vaccine Platform Enhances Neo-antigen-like T Cell Responses against WT1 to Break Tolerance and Induce Anti-tumor Immunity.

Tumor-associated antigens have emerged as important immunotherapeutic targets in the fight against cancer. Germline tumor antigens, such as WT1, Wilms' tumor gene 1, are overexpressed in many human malignancies but have low expression in somatic tissues. Recent vaccination approaches to target WT1 have been hampered by poor in vivo immune potency, likely due to the conserved self-antigen nature of WT1. In this study, we use a novel synthetic micro-consensus SynCon DNA vaccine approach with the goal of breaking tolerance and increasing vaccine immune potency. This approach induced new, neo-antigen-like responses that were superior to those induced by native WT1 DNA immunogens for driving T cell immunity and breaking tolerance. Non-human primates (NHPs) vaccinated with SynCon WT1 antigens elicited immune responses against native rhesus WT1 peptides. When delivered by electroporation (EP) in mice, SynCon-based WT1 constructs elicited strong CD4 and CD8 T cell responses (including IFN-γ, CD107a, and TNF-α) to both native and consensus peptides. In addition, SynCon WT1 vaccine-induced antibodies recognized native WT1 in vitro. Vaccination with the SynCon WT1 immunogens was capable of slowing tumor growth in therapeutic models in vivo. These data support the further study of synthetic consensus DNA vaccines for breaking tolerance to important germline antigens.

NOTCH Signaling Regulates Asymmetric Cell Fate of Fast- and Slow-Cycling Colon Cancer-Initiating Cells.

Colorectal cancer cells with stem-like properties, referred to as colon cancer-initiating cells (CCIC), have high tumorigenic potential. While CCIC can differentiate to promote cellular heterogeneity, it remains unclear whether CCIC within a tumor contain distinct subpopulations. Here, we describe the co-existence of fast- and slow-cycling CCIC, which can undergo asymmetric division to generate each other, highlighting CCIC plasticity and interconvertibility. Fast-cycling CCIC express markers, such as LGR5 and CD133, rely on MYC for their proliferation, whereas slow-cycling CCIC express markers, such as BMI1 and hTERT, are independent of MYC. NOTCH signaling promotes asymmetric cell fate, regulating the balance between these two populations. Overall, our results illuminate the basis for CCIC heterogeneity and plasticity by defining a direct interconversion mechanism between slow- and fast-cycling CCIC. Cancer Res; 76(11); 3411-21. ©2016 AACR.

DNA vaccines targeting heavy chain C-terminal fragments of Clostridium botulinum neurotoxin serotypes A, B, and E induce potent humoral and cellular immunity and provide protection from lethal toxin challenge.

Botulinum neurotoxins (BoNTs) are deadly, toxic proteins produced by the bacterium Clostridium botulinum that can cause significant diseases in humans. The use of the toxic substances as potential bioweapons has raised concerns by the Centers for Disease Control and Prevention and the United States Military. Currently, there is no licensed vaccine to prevent botulinum intoxication. Here we present an immunogenicity study to evaluate the efficacy of novel monovalent vaccines and a trivalent cocktail DNA vaccine targeting the heavy chain C-terminal fragments of Clostridium botulinum neurotoxin serotypes A, B, and E. These synthetic DNA vaccines induced robust humoral and polyfunctional CD4(+) T-cell responses which fully protected animals against lethal challenge after just 2 immunizations. In addition, naïve animals administered immunized sera mixed with the lethal neurotoxin were 100% protected against intoxication. The data demonstrate the protective efficacy induced by a combinative synthetic DNA vaccine approach. This study has importance for the development of vaccines that provide protective immunity against C. botulinum neurotoxins and other toxins.

Multivalent TB vaccines targeting the esx gene family generate potent and broad cell-mediated immune responses superior to BCG.

Development of a broad-spectrum synthetic vaccine against TB would represent an important advance to the limited vaccine armamentarium against TB. It is believed that the esx family of TB antigens may represent important vaccine candidates. However, only 4 esx antigens have been studied as potential vaccine antigens. The challenge remains to develop a vaccine that simultaneously targets all 23 members of the esx family to induce enhanced broad-spectrum cell-mediated immunity. We sought to investigate if broader cellular immune responses could be induced using a multivalent DNA vaccine representing the esx family protein members delivered via electroporation. In this study, 15 designed esx antigens were created to cross target all members of the esx family. They were distributed into groups of 3 self-processing antigens each, resulting in 5 trivalent highly optimized DNA plasmids. Vaccination with all 5 constructs elicited robust antigen-specific IFN-γ responses to all encoded esx antigens and induced multifunctional CD4 Th1 and CD8 T cell responses. Importantly, we show that when all constructs are combined into a cocktail, the RSQ-15 vaccine, elicited substantial broad Ag-specific T cell responses to all esx antigens as compared with vaccination with BCG. Moreover, these vaccine-induced responses were highly cross-reactive with BCG encoded esx family members and were highly immune effective in a BCG DNA prime-boost format. Furthermore, we demonstrate the vaccine potential and immunopotent profile of several novel esx antigens never previously studied. These data highlight the likely importance of these novel immunogens for study as preventative or therapeutic synthetic TB vaccines in combination or as stand alone antigens.

Protective immunity to H7N9 influenza viruses elicited by synthetic DNA vaccine.

Despite an intensive vaccine program influenza infections remain a major health problem, due to the viruses' ability to change its envelope glycoprotein hemagglutinin (HA), through shift and drift, permitting influenza to escape protection induced by current vaccines or natural immunity. Recently a new variant, H7N9, has emerged in China causing global concern. First, there have been more than 130 laboratory-confirmed human infections resulting in an alarmingly high death rate (32.3%). Second, genetic changes found in H7N9 appear to be associated with enabling avian influenza viruses to spread more effectively in mammals, thus transmitting infections on a larger scale. Currently, no vaccines or drugs are effectively able to target H7N9. Here, we report the rapid development of a synthetic consensus DNA vaccine (pH7HA) to elicit potent protective immunity against the H7N9 viruses. We show that pH7HA induces broad antibody responses that bind to divergent HAs from multiple new members of the H7N9 family. These antibody responses result in high-titer HAI against H7N9. Simultaneously, this vaccine induces potent polyfunctional effector CD4 and CD8T cell memory responses. Animals vaccinated with pH7HA are completely protected from H7N9 virus infection and any morbidity associated with lethal challenge. This study establishes that this synthetic consensus DNA vaccine represents a new tool for targeting emerging infection, and more importantly, its design, testing and development into seed stock for vaccine production in a few days in the pandemic setting has significant implications for the rapid deployment of vaccines protecting against emerging infectious diseases.

Alarmin IL-33 acts as an immunoadjuvant to enhance antigen-specific tumor immunity.

Studies of interleukin (IL)-33 reveal a number of pleiotropic properties. Here, we report that IL-33 has immunoadjuvant effects in a human papilloma virus (HPV)-associated model for cancer immunotherapy where cell-mediated immunity is critical for protection. Two biologically active isoforms of IL-33 exist that are full-length or mature, but the ability of either isoform to function as a vaccine adjuvant that influences CD4 T helper 1 or CD8 T-cell immune responses is not defined. We showed that both IL-33 isoforms are capable of enhancing potent antigen-specific effector and memory T-cell immunity in vivo in a DNA vaccine setting. In addition, although both IL-33 isoforms drove robust IFN-γ responses, neither elevated secretion of IL-4 or immunoglobulin E levels. Further, both isoforms augmented vaccine-induced antigen-specific polyfunctional CD4(+) and CD8(+) T-cell responses, with a large proportion of CD8(+) T cells undergoing plurifunctional cytolytic degranulation. Therapeutic studies indicated that vaccination with either IL-33 isoform in conjunction with an HPV DNA vaccine caused rapid and complete regressions in vivo. Moreover, IL-33 could expand the magnitude of antigen-specific CD8(+) T-cell responses and elicit effector-memory CD8(+) T cells. Taken together, our results support the development of these IL-33 isoforms as immunoadjuvants in vaccinations against pathogens, including in the context of antitumor immunotherapy.

A microRNA miR-34a-regulated bimodal switch targets Notch in colon cancer stem cells.

microRNAs regulate developmental cell-fate decisions, tissue homeostasis, and oncogenesis in distinct ways relative to proteins. Here, we show that the tumor suppressor microRNA miR-34a is a cell-fate determinant in early-stage dividing colon cancer stem cells (CCSCs). In pair-cell assays, miR-34a distributes at high levels in differentiating progeny, whereas low levels of miR-34a demarcate self-renewing CCSCs. Moreover, miR-34a loss of function and gain of function alter the balance between self-renewal versus differentiation both in vitro and in vivo. Mechanistically, miR-34a sequesters Notch1 mRNA to generate a sharp threshold response where a bimodal Notch signal specifies the choice between self-renewal and differentiation. In contrast, the canonical cell-fate determinant Numb regulates Notch levels in a continuously graded manner. Altogether, our findings highlight a unique microRNA-regulated mechanism that converts noisy input into a toggle switch for robust cell-fate decisions in CCSCs.

A distal enhancer controls cytokine-dependent human cPLA2α gene expression.

Specific control of group IVA cytosolic phospholipase A2 (cPLA2α or PLA2G4A) expression modulates arachidonic acid production, thus tightly regulating the downstream effects of pro- and anti-inflammatory eicosanoids. The significance of this pathway in human disease is apparent in a range of pathologies from inflammation to tumorigenesis. While much of the regulation of cPLA2α has focused on posttranslational phosphorylation of the protein, studies on transcriptional regulation of this gene have focused only on proximal promoter regions. We have identified a DNase I hypersensitive site encompassing a 5' distal enhancer element containing a highly conserved consensus AP-1 site involved in transcriptional activation of cPLA2α by interleukin (IL)-1ß. Chromatin immunoprecipitation (ChIP), knockdown, knockout, and overexpression analyses have shown that c-Jun acts both in a negative and positive regulatory role. Transcriptional activation of cPLA2α occurs through the phosphorylation of c-Jun in conjunction with increased association of C/EBPß with the distal novel enhancer. The association of C/EBPß with the transcriptional activation complex does not require an obvious DNA binding site. These data provide new and important contributions to the understanding of cPLA2α regulation at the transcriptional level, with implications for eicosanoid metabolism, cellular signaling, and disease pathogenesis.

Highly optimized DNA vaccine targeting human telomerase reverse transcriptase stimulates potent antitumor immunity.

High levels of human telomerase reverse transcriptase (hTERT) are detected in more than 85% of human cancers. Immunologic analysis supports that hTERT is a widely applicable target recognized by T cells and can be potentially studied as a broad cancer immunotherapeutic, or a unique line of defense against tumor recurrence. There remains an urgent need to develop more potent hTERT vaccines. Here, a synthetic highly optimized full-length hTERT DNA vaccine (phTERT) was designed and the induced immunity was examined in mice and non-human primates (NHP). When delivered by electroporation, phTERT elicited strong, broad hTERT-specific CD8 T-cell responses including induction of T cells expressing CD107a, IFN-γ, and TNF-α in mice. The ability of phTERT to overcome tolerance was evaluated in an NHP model, whose TERT is 96% homologous to that of hTERT. Immunized monkeys exhibited robust [average 1,834 spot forming unit (SFU)/10(6) peripheral blood mononuclear cells (PBMC)], diverse (multiple immunodominant epitopes) IFN-γ responses and antigen-specific perforin release (average 332 SFU/10(6) PBMCs), suggesting that phTERT breaks tolerance and induces potent cytotoxic responses in this human-relevant model. Moreover, in an HPV16-associated tumor model, vaccination of phTERT slows tumor growth and improves survival rate in both prophylactic and therapeutic studies. Finally, in vivo cytotoxicity assay confirmed that phTERT-induced CD8 T cells exhibited specific cytotoxic T lymphocyte (CTL) activity, capable of eliminating hTERT-pulsed target cells. These findings support that this synthetic electroporation-delivered DNA phTERT may have a role as a broad therapeutic cancer vaccine candidate.

Regulation of human microsomal prostaglandin E synthase-1 by IL-1ß requires a distal enhancer element with a unique role for C/EBPß.

The studies of PGE2 (prostaglandin E2) biosynthesis have focused primarily on the role of cyclo-oxygenases. Efforts have shifted towards the specific PGE2 terminal synthases, particularly mPGES-1 (microsomal PGE synthase 1), which has emerged as the crucial inducible synthase with roles in pain, cancer and inflammation. mPGES-1 is induced by pro-inflammatory cytokines with studies focusing on the proximal promoter, mediated specifically through Egr-1 (early growth-response factor 1). Numerous studies demonstrate that the mPGES-1 promoter (PTGES) alone cannot account for the level of IL-1ß (interleukin 1ß) induction. We identified two DNase I-hypersensitive sites within the proximal promoter near the Egr-1 element and a novel distal site near -8.6 kb. Functional analysis of the distal site revealed two elements that co-operate with basal promoter expression and a stimulus-dependent enhancer. A specific binding site for C/EBPß (CCAAT/enhancer-binding protein ß) in the enhancer was directly responsible for inducible enhancer activity. ChIP (chromatin immunoprecipitation) analysis demonstrated constitutive Egr-1 binding to the promoter and induced RNA polymerase II and C/EBPß binding to the promoter and enhancer respectively. Knockout/knockdown studies established a functional role for C/EBPß in mPGES-1 gene regulation and the documented interaction between Egr-1 and C/EBPß highlights the proximal promoter co-operation with a novel distal enhancer element in regulating inducible mPGES-1 expression.

Induction of group IVC phospholipase A2 in allergic asthma: transcriptional regulation by TNFα in bronchoepithelial cells.

Airway inflammation in allergen-induced asthma is associated with eicosanoid release. These bioactive lipids exhibit anti- and pro-inflammatory activities with relevance to pulmonary pathophysiology. We hypothesized that sensitization/challenge using an extract from the ubiquitous fungus Aspergillus fumigatus in a mouse model of allergic asthma would result in altered phospholipase gene expression, thus modulating the downstream eicosanoid pathway. We observed the most significant induction in the group IVC PLA2 (phospholipase A2) [also known as cPLA2γ (cytosolic PLA2γ) or PLA2G4C]. Our results infer that A. fumigatus extract can induce cPLA2γ levels directly in eosinophils, whereas induction in lung epithelial cells is most likely to be a consequence of TNFα (tumour necrosis factor α) secretion by A. fumigatus-activated macrophages. The mechanism of TNFα-dependent induction of cPLA2γ gene expression was elucidated through a combination of promoter deletions, ChIP (chromatin immunoprecipitation) and overexpression studies in human bronchoepithelial cells, leading to the identification of functionally relevant CRE (cAMP-response element), NF-κB (nuclear factor κB) and E-box promoter elements. ChIP analysis demonstrated that RNA polymerase II, ATF-2 (activating transcription factor 2)-c-Jun, p65-p65 and USF (upstream stimulating factor) 1-USF2 complexes are recruited to the cPLA2γ enhancer/promoter in response to TNFα, with overexpression and dominant-negative studies implying a strong level of co-operation and interplay between these factors. Overall, our results link cytokine-mediated alterations in cPLA2γ gene expression with allergic asthma and outline a complex regulatory mechanism.

cPLA(2)α gene activation by IL-1ß is dependent on an upstream kinase pathway, enzymatic activation and downstream 15-lipoxygenase activity: a positive feedback loop.

Cytosolic phospholipase A(2)α (cPLA(2)α) is the most widely studied member of the Group IV PLA(2) family. The enzyme is Ca(2+)-dependent with specificity for phospholipid substrates containing arachidonic acid. As the pinnacle of the arachidonic acid pathway, cPLA(2)α has a primary role in the biosynthesis of a diverse family of eicosanoid metabolites, with potent physiological, inflammatory and pathological consequences. cPLA(2)α activity is regulated by pro-inflammatory stimuli through pathways involving increased intracellular Ca(2+) levels, phosphorylation coupled to increased enzymatic activity and de novo gene transcription. This study addresses the signal transduction pathways for protein phosphorylation and gene induction following IL-1ß stimulation in human fetal lung fibroblasts. Our results utilizing both inhibitors and kinase-deficient cells demonstrate that cPLA(2)α is phosphorylated within 10min of IL-1ß treatment, an event requiring p38 MAPK as well as the upstream kinase, MKK3/MKK6. Inhibition of p38 MAPK also blocks the phosphorylation of a downstream, nuclear kinase, MSK-1. Our results further demonstrate that the activities of both cPLA(2)α and a downstream lipoxygenase (15-LOX2) are required for IL-1ß-dependent induction of cPLA(2)α mRNA expression. Overall, these data support an MKK3/MKK6→p38 MAPK→MSK-1→cPLA(2)α→15-LOX2-dependent, positive feedback loop where a protein's enzymatic activity is required to regulate its own gene induction by a pro-inflammatory stimulus.

Mutation of single hydrophobic residue I27, L35, F39, L58, L65, L67, or L71 in the N terminus of VP5 abolishes interaction with the scaffold protein and prevents closure of herpes simplex virus type 1 capsid shells.

Protein-protein interactions drive the assembly of the herpes simplex virus type 1 (HSV-1) capsid. A key interaction occurs between the C-terminal tail of the scaffold protein (pre-22a) and the major capsid protein (VP5). Previously (Z. Hong, M. Beaudet-Miller, J. Durkin, R. Zhang, and A. D. Kwong, J. Virol. 70:533-540, 1996) it was shown that the minimal domain in the scaffold protein necessary for this interaction was composed of a hydrophobic amphipathic helix. The goal of this study was to identify the hydrophobic residues in VP5 important for this bimolecular interaction. Results from the genetic analysis of second-site revertant virus mutants identified the importance of the N terminus of VP5 for the interaction with the scaffold protein. This allowed us to focus our efforts on a small region of this large polypeptide. Twenty-four hydrophobic residues, starting at L23 and ending at F84, were mutated to alanine. All the mutants were first screened for interaction with pre-22a in the yeast two-hybrid assay. From this in vitro assay, seven residues, I27, L35, F39, L58, L65, L67, and L71, that eliminated the interaction when mutated were identified. All 24 mutants were introduced into the virus genome with a genetic marker rescue/marker transfer system. For this system, viruses and cell lines that greatly facilitated the introduction of the mutants into the genome were made. The same seven mutants that abolished interaction of VP5 with pre-22a resulted in an absolute requirement for wild-type VP5 for growth of the viruses. The viruses encoding these mutations in VP5 were capable of forming capsid shells comprised of VP5, VP19C, VP23, and VP26, but the closure of these shells into an icosahedral structure was prevented. Mutation at L75 did not affect the ability of this protein to interact with pre-22a, as judged from the in vitro assay, but this mutation specified a lethal effect for virus growth and abolished the formation of any detectable assembled structure. Thus, it appears that the L75 residue is important for another essential interaction of VP5 with the capsid shell proteins. The congruence of the data from the previous and present studies demonstrates the key roles of two regions in the N terminus of this large protein that are crucial for this bimolecular interaction. Thus, residues I27, L35, and F39 comprise the first subdomain and residues L58, L65, L67 and L71 comprise a second subdomain of VP5. These seven hydrophobic residues are important for the interaction of VP5 with the scaffold protein and consequently the formation of an icosahedral shell structure that encloses the viral genome.