The ability for a host to recognize infection is critical for virus clearance and often begins with induction of inflammation. The PB1-F2 of pathogenic influenza A viruses (IAV) contributes to the pathophysiology of infection, although the mechanism for this is unclear. The NLRP3-inflammasome has been implicated in IAV pathogenesis, but whether IAV virulence proteins can be activators of the complex is unknown. We investigated whether PB1-F2-mediated activation of the NLRP3-inflammasome is a mechanism contributing to overt inflammatory responses to IAV infection. We show PB1-F2 induces secretion of pyrogenic cytokine IL-1β by activating the NLRP3-inflammasome, contributing to inflammation triggered by pathogenic IAV. Compared to infection with wild-type virus, mice infected with reverse engineered PB1-F2-deficient IAV resulted in decreased IL-1β secretion and cellular recruitment to the airways. Moreover, mice exposed to PB1-F2 peptide derived from pathogenic IAV had enhanced IL-1β secretion compared to mice exposed to peptide derived from seasonal IAV. Implicating the NLRP3-inflammasome complex specifically, we show PB1-F2 derived from pathogenic IAV induced IL-1β secretion was Caspase-1-dependent in human PBMCs and NLRP3-dependent in mice. Importantly, we demonstrate PB1-F2 is incorporated into the phagolysosomal compartment, and upon acidification, induces ASC speck formation. We also show that high molecular weight aggregated PB1-F2, rather than soluble PB1-F2, induces IL-1β secretion. Furthermore, NLRP3-deficient mice exposed to PB1-F2 peptide or infected with PB1-F2 expressing IAV were unable to efficiently induce the robust inflammatory response as observed in wild-type mice. In addition to viral pore forming toxins, ion channel proteins and RNA, we demonstrate inducers of NLRP3-inflammasome activation may include disordered viral proteins, as exemplified by PB1-F2, acting as host pathogen ‘danger’ signals. Elucidating immunostimulatory PB1-F2 mediation of NLRP3-inflammasome activation is a major step forward in our understanding of the aetiology of disease attributable to exuberant inflammatory responses to IAV infection.

Influenza A, B and C viruses (IAV, IBV, ICV) circulate globally and infect humans, with IAV/IBV causing most severe disease. While CD8 + T-cells confer cross-protection against different IAV strains, CD8 + T-cell responses to IBV/ICV are understudied. We dissected the CD8 + T-cell cross-reactome against influenza viruses and provided the first evidence of CD8 + T-cell cross-reactivity across IAV, IBV and ICV. Using immunopeptidomics, we identified immunodominant CD8 + T-cell epitopes from IBV, protective in mice, and found prominent memory CD8 + T-cells towards both universal and influenza type-specific epitopes in blood and lungs of healthy humans, with lung-derived CD8 + T-cells displaying a tissue-resident phenotype. Importantly, effector CD38 + Ki67 + CD8 + T-cells against novel epitopes were readily detected in IAV-and IBV-infected pediatric and adult patients. Our study introduces a new paradigm, whereby CD8 + T-cells confer unprecedented cross-reactivity across all influenza viruses, a key finding for designing universal vaccines.

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In this study, we describe the synthesis of various lipopeptides based on the sequence of luteinizing hormone-releasing hormone (LHRH) and report on their abilities to induce Abs against this “self” hormone when inoculated into mice in the absence of additional adjuvant. The peptides consisted of a colinear CD4+ T helper cell epitope from the L chain of influenza virus hemagglutinin and LHRH, which has B cell epitopes but no T cell epitopes present in its sequence. Lipids were attached either at the N terminus or between the T cell epitope and LHRH, in the approximate center of the peptide. The lipopeptide constructs displayed different solubilities and immunological properties that depended not only on the lipid content but also on the position of attachment of the lipids. Some of these constructs were highly immunogenic, inducing high titers of Ab, which were capable of efficiently sterilizing female mice when administered in saline by s.c. or intranasal routes. The most effective vaccines were highly soluble, contained the dipalmitoyl-S-glyceryl cysteine moiety, and had this lipid attached at the center of the molecule. The relative ability of the lipopeptides to induce an Ab response in the absence of external adjuvant was reflected by their ability to up-regulate the surface expression of MHC class II molecules on immature dendritic cells. These results demonstrate that the composition and position within peptide vaccines of self-adjuvanting lipid groups can influence the ability to induce the maturation of dendritic cells and, in turn, the magnitude of the resulting Ab response.

Persistent infection with the high-risk subset of genitotropic human papillomavirus (HPV) genotypes is a necessary cause of cervical cancer. Given the global burden of cervical cancer, a low-cost, broadly protective vaccine is needed. RG-1 is a crossneutralizing and protective monoclonal antibody that recognizes residues 17-36 of HPV16 minor capsid protein L2. Because this epitope is highly conserved in divergent HPV types, we determined whether vaccination with HPV16 L2 17-36 peptide is broadly protective. The peptide was administered to BALB/c mice three times at monthly intervals, either alone or in the context of a synthetic lipopeptide vaccine candidate (P25-P2C-HPV) produced by linkage of the HPV peptide with a broadly recognized T helper epitope (P25) and the Toll-like receptor-2 (TLR2) ligand dipalmitoyl-S-glyceryl cysteine (P2C). In contrast to vaccination with the L2 17-36 peptide or P25-P2C alone, a potent L2-specific antibody response was generated to the P25-P2C-HPV lipopeptide when delivered either s.c. or intranasally. Sera from mice vaccinated with the P25-P2C-HPV lipopeptide neutralized not only HPV16 pseudovirions but also other evolutionarily divergent oncogenic genital (HPV18, HPV45) and cutaneous (HPV5, BPV1) types. The L2-specific antibody response depended on MHC class II, CD40, and MyD88 signaling. Additionally, vaccination with the P25-P2C-HPV lipopeptide protected mice from homologous challenge with HPV16 pseudovirions at cutaneous and genital sites and heterologous challenge with HPV45 pseudovirions. If provided in the appropriate context, therefore, HPV16 L2 17-36 might be used in a totally synthetic cross-protective HPV vaccine. G enitotropic human papillomavirus (HPV) infections are considered the most common sexually transmitted infection in the United States (1). The major manifestations of anogenital HPV include genital warts (condyloma acuminatum) and anogenital intraepithelial neoplasia. If left untreated, a small fraction of persistent high-risk HPV infections progresses to cancer. The presence of HPV DNA has been reported in 99.7% of cervical carcinomas worldwide, indicating that HPV infection is a necessary cause of this cancer and that this disease can be prevented by prophylactic HPV vaccination (2).Approximately 35 of the Ͼ100 subtypes of HPV are specific for the anogenital epithelium and have varying potentials for malignant transformation (3). Of the 15 oncogenic genital HPV types, HPV16 is the most common, followed by HPV18 and HPV45 (contributing Ϸ50%, 20%, and 10%, respectively, of cervical cancer cases worldwide). Public health efforts have successfully reduced the incidence and mortality of cervical cancer with the implementation of cervical cytology screening programs. Women who do not undergo regular screening account for most of the patients with invasive cancers (4), and cervical cancer remains the second most common cause of cancer death in women worldwide and the most prevalent cancer in women of sub-Saharan Africa, Central America, south-central Asia, and Me...

Infection with group A streptococci can result in acute and post-infectious pathology, including rheumatic fever and rheumatic heart disease. These diseases are associated with poverty and are increasing in incidence, particularly in developing countries and amongst indigenous populations, such as Australia's Aboriginal population, who suffer the highest incidence worldwide. Immunity to group A streptococci is mediated by antibodies against the M protein, a coiled-coil alpha helical surface protein of the bacterium. Vaccine development faces two substantial obstacles. Although opsonic antibodies directed against the N terminus of the protein are mostly responsible for serotypic immunity, more than 100 serotypes exist. Furthermore, whereas the pathogenesis of rheumatic fever is not well understood, increasing evidence indicates an autoimmune process. To develop a suitable vaccine candidate, we first identified a minimum, helical, non-host-cross-reactive peptide from the conserved C-terminal half of the protein and displayed this within a non-M-protein peptide sequence designed to maintain helical folding and antigenicity, J14 (refs. 8,9). As this region of the M protein is identical in only 70% of group A streptococci isolates, the optimal candidate might consist of the conserved determinant with common N-terminal sequences found in communities with endemic group A streptococci. We linked seven serotypic peptides with J14 using a new chemistry technique that enables the immunogen to display all the individual peptides pendant from an alkane backbone. This construct demonstrated excellent immunogenicity and protection in mice.

Addressing the transmission enigma of the neglected disease Buruli ulcer (BU) is a World Health Organization priority. In Australia, we have observed an association between mosquitoes harboring the causative agent, Mycobacterium ulcerans, and BU. Here we tested a contaminated skin model of BU transmission by dipping the tails from healthy mice in cultures of the causative agent, Mycobacterium ulcerans. Tails were exposed to mosquito (Aedes notoscriptus and Aedes aegypti) blood feeding or punctured with sterile needles. Two of 12 of mice with M. ulcerans contaminated tails exposed to feeding A. notoscriptus mosquitoes developed BU. There were no mice exposed to A. aegypti that developed BU. Eighty-eight percent of mice (21/24) subjected to contaminated tail needle puncture developed BU. Mouse tails coated only in bacteria did not develop disease. A median incubation time of 12 weeks, consistent with data from human infections, was noted. We then specifically tested the M. ulcerans infectious dose-50 (ID50) in this contaminated skin surface infection model with needle puncture and observed an ID50 of 2.6 colony-forming units. We have uncovered a biologically plausible mechanical transmission mode of BU via natural or anthropogenic skin punctures.

The protective role played by the innate immune system during early stages of infection suggests that compounds which stimulate innate responses could be used as antimicrobial or antiviral agents. In this study, we demonstrate that the Toll-like receptor-2 agonist Pam2Cys, when administered intranasally, triggers a cascade of inflammatory and innate immune signals, acting as an immunostimulant by attracting neutrophils and macrophages and inducing secretion of IL-2, IL-6, IL-10, IFN-γ, MCP-1 and TNF-α. These changes provide increased resistance against influenza A virus challenge and also reduce the potential for transmission of infection. Pam2Cys treatment also reduced weight loss and lethality associated with virulent influenza virus infection in a Toll-like receptor-2-dependent manner. Treatment did not affect the animals' ability to generate an adaptive immune response, measured by the induction of functional influenza A virus-specific CD8 + T cells following exposure to virus. Because this compound demonstrates efficacy against distinct strains of influenza, it could be a candidate for development as an agent against influenza and possibly other respiratory pathogens.