The genome of the Lymantria dispar multinucleocapsid nucleopolyhedrovirus (LdMNPV) was sequenced and analyzed. It is composed of 161,046 bases with a G + C content of 57.5% and contains 163 putative open reading frames (ORFs) of >/=150 nucleotides. Homologs were found to 95 of the 155 genes predicted for the Autographa californica MNPV (AcMNPV) genome. More than 9% of the LdMNPV genome was occupied by 16 repeated genes related to AcMNPV ORF2. Readily identifiable homologs of several genes that have been reported to play important roles in the AcMNPV life cycle are not present; these include ie-2, a transcriptional transactivator, and gp64, a major envelope glycoprotein of the nonoccluded form of the virus. A number of genes lacking in AcMNPV but present in other baculoviruses were identified; these include two viral enhancing factor homologs, a second copy of a conotoxin-like gene, and a dutpase homolog. Although a single gene predicted to encode a large subunit of ribonucleotide reductase was found, two different copies of the small subunit gene were present. In addition, homologs of genes not previously reported for baculoviruses were identified, including a predicted protein with homology to DNA ligases and another that has motifs most closely related to a yeast mitochondrial helicase. Thirteen homologous regions (hrs) containing 54 repeated sequences that include 30-bp imperfect palindromes were identified. The imperfect palindromes are related to those from other baculoviruses.

The nucleotide sequence of the Orgyia pseudotsugata multinucleocapsid nuclear polyhedrosis virus (OpMNPV) genome was completed and analyzed. It is composed of 131,990 bases with a G + C content of 55% and contains 152 putative genes of 150 nucleotides or greater. Major differences in gene content and arrangement between OpMNPV and the Autographa californica MNPV were found. These include the presence in OpMNPV of three complete iap gene homologs, two conotoxin gene homologs, two protein tyrosine phosphatase homologs, and genes encoding homologs of dUTPase and the large and small subunits of ribonucleotide reductase. Seven major intergenic repeated regions were identified. Five of these are homologous regions that are related to similar regions from other baculoviruses.

Toll-like receptor (TLR) agonists are being developed for use as vaccine adjuvants and as stand-alone immunomodulators because of their ability to stimulate innate and adaptive immune responses. Among the most thoroughly studied TLR agonists are the lipid A molecules that target the TLR4 complex. One promising candidate, monophosphoryl lipid A, which is a derivative of lipid A from Salmonella minnesota, has proven to be safe and effective as a vaccine adjuvant in > 120,000 human doses. A new class of synthetic lipid A mimetics, the aminoalkyl glucosaminide 4-phosphates (AGPs), have been engineered specifically to target human TLR4 and are showing promise as vaccine adjuvants and as monotherapeutic agents capable of eliciting nonspecific protection against a wide range of infectious pathogens. In this review, the authors provide an update of the preclinical and clinical experiences with the TLR4 agonists, MPL (Corixa Corporation) adjuvant and the AGPs.

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MPL (Corixa) adjuvant is a chemically modified derivative of lipopolysaccharide that displays greatly reduced toxicity while maintaining most of the immunostimulatory activity of lipopolysaccharide. MPL adjuvant has been used extensively in clinical trials as a component in prophylactic and therapeutic vaccines targeting infectious disease, cancer and allergies. With over 33,000 doses administered to date, MPL adjuvant has emerged as a safe and effective vaccine adjuvant. Recently, scientists at Corixa Corporation have developed a library of synthetic lipid A mimetics (aminoalkyl glucosaminide 4-phosphates) with demonstrated immunostimulatory properties. Similar to MPL adjuvant, these synthetic compounds signal through Toll-like receptor 4 to stimulate the innate immune system. One of these compounds, Ribi.529 (RC-529), has emerged as a leading adjuvant with a similar efficacy and safety profile to MPL adjuvant in both preclinical and clinical studies.

A compound family of synthetic lipid A mimetics (termed the aminoalkyl glucosaminide phosphates [AGPs]) was evaluated in murine infectious disease models of protection against challenge with Listeria monocytogenes and influenza virus. For the Listeria model, intravenous administration of AGPs was followed by intravenous bacterial challenge 24 h later. Spleens were harvested 2 days postchallenge for the enumeration of CFU. For the influenza virus model, mice were challenged with virus via the intranasal/intrapulmonary route 48 h after intranasal/intrapulmonary administration of AGPs. The severity of disease was assessed daily for 3 weeks following challenge. Several types of AGPs provided strong protection against influenza virus or Listeria challenge in wild-type mice, but they were inactive in the C3H/HeJ mouse, demonstrating the dependence of the AGPs on toll-like receptor 4 (TLR4) signaling for the protective effect. Structure-activity relationship studies showed that the activation of innate immune effectors by AGPs depends primarily on the lengths of the secondary acyl chains within the three acyl-oxy-acyl residues and also on the nature of the functional group attached to the aglycon component. We conclude that the administration of synthetic TLR4 agonists provides rapid pharmacologic induction of innate resistance to infectious challenge by two different pathogen classes, that this effect is mediated via TLR4, and that structural differences between AGPs can have dramatic effects on agonist activity in vivo.The toll-like receptors (TLRs) comprise an evolutionarily conserved receptor family that is capable of detecting and responding to microbial challenge (1). The TLRs recognize a variety of pathogen-specific components, including lipopolysaccharide (LPS), CpG DNA, and microbial membrane and cell wall components (20). Toll-like receptor 4 (TLR4) is critical for the recognition of LPS (30, 31), and considerable progress has recently been made in understanding the interaction of TLR4 with critical accessory molecules implicated in LPS recognition (8,9,17,21,22, 32,40,41). In this regard, it appears that LPS binding protein (LBP) promotes the binding of LPS to CD14, which in turn facilitates the association of the lipid A component of LPS with MD-2 to form a soluble complex that serves as an activating ligand for TLR4 on the cell surface. Binding of the MD-2/LPS complex to TLR4 results in the aggregation of TLR4 into lipid rafts and the activation of several distinct intracellular signaling pathways that results in increased transcription of many genes encoding cytokines, defensins, chemokines, and alpha/beta interferons (28, 38). These effector molecules determine a wide range of biological activities, including further production of cytokines, enhancement of microbicidal activity of phagocytic cells, and migration/maturation of dendritic cells (5,23,36).Before the discovery that LPS interacts with TLR4, evidence demonstrating the importance of innate immune activation in controlling infection with gram-neg...

Lipopolysaccharide (LPS) has long been known to enhance innate and adaptive immune responses; however, its extreme toxicity precludes its use in clinical settings. The combined toxicity and adjuvanticity of LPS have contributed to the view that immunological adjuvants need to be highly inflammatory to be maximally effective. Here, we compared the effects of LPS with its less-toxic derivatives, monophosphoryl lipid A (MPL) and a chemical mimetic, RC529, on CD4+ T cell clonal expansion, long-term survival, and T helper cell type 1 (Th1) differentiation. We found that LPS, MPL, and RC529 had similar effects on CD4+ T cell clonal expansion, cell division, and ex vivo survival. Analysis of the ability of activated CD4+ T cells to produce interferon-gamma following a 21-day immunization and challenge protocol with LPS and MPL resulted in similar Th1 differentiation. In contrast, we found that LPS was more effective in promoting long-term CD4+ T cell responses, as we recovered nearly sixfold more cells following immunization/challenge as compared with treatment with MPL. Our results indicate that low-inflammation adjuvants, such as MPL and RC529, are capable of enhancing short-term CD4+ T cell clonal expansion and Th1 differentiation, but inflammatory signaling aids in the long-term retention of antigen-specific T cells.

Influenza disease is a global health issue that causes significant morbidity and mortality through seasonal epidemics. Currently, inactivated influenza virus vaccines given intramuscularly or live attenuated influenza virus vaccines administered intranasally are the only approved options for vaccination against influenza virus in humans. We evaluated the efficacy of a synthetic toll-like receptor 4 agonist CRX-601 as an adjuvant for enhancing vaccine-induced protection against influenza infection. Intranasal administration of CRX-601 adjuvant combined with detergent split-influenza antigen (A/Uruguay/716/2007 (H3N2)) generated strong local and systemic immunity against co-administered influenza antigens while exhibiting high efficacy against two heterotypic influenza challenges. Intranasal vaccination with CRX-601 adjuvanted vaccines promoted antigen-specific IgG and IgA antibody responses and the generation of polyfunctional antigen-specific Th17 cells (CD4+IL-17A+TNFα+). Following challenge with influenza virus, vaccinated mice transiently exhibited increased weight loss and morbidity during early stages of disease but eventually controlled infection. This disease exacerbation following influenza infection in vaccinated mice was dependent on both the route of vaccination and the addition of the adjuvant. Neutralization of IL-17A confirmed a detrimental role for this cytokine during influenza infection. The expansion of vaccine-primed Th17 cells during influenza infection was also accompanied by an augmented lung neutrophilic response, which was partially responsible for mediating the increased morbidity. This discovery is of significance in the field of vaccinology, as it highlights the importance of both route of vaccination and adjuvant selection in vaccine development