Lorena E. Brown

Biological activity of monoclonal antibodies to operationally defined antigenic regions on the hemagglutinin molecule of A/Seal/Massachusetts/1 /80 (H7N7) influenza virus

Abstract Monoclonal antibodies to the hemagglutinin (HA) molecule of A/Seal/Mass/1/80 (H7N7) have been prepared and used to establish an operational antigenic map. Four nonoverlapping antigenic areas on the HA of seal influenza viruses were defined. Monoclonal antibodies belonging to two of the groups (III and IV) failed to inhibit hemagglutination of intact virus yet effectively neutralized viral infectivity. These findings could not be explained by differences in affinities and were interpreted in terms of functional differences between the assays. Antibodies that failed to inhibit hemagglutination may bind nearer to the hydrophobic end of the HA molecule and might not block the receptor binding site for erythrocytes. Antibodies to these areas may inhibit infectivity by interference with cell fusion or viral replication. The monoclonal antibodies that failed to inhibit hemagglutination of intact virus nevertheless did inhibit HA activity of purified isolated rosettes of hemagglutinin. The mechanism of inhibition is not resolved but may involve the access of a larger number of antibody molecules to the HA when it is in the form of rosettes compared to when it is located on the viral membrane. The reactivity of seal influenza virus and other H7 viruses from birds with the monoclonal antibodies show that some avian strains possess HA molecules that are closely related to those of seal virus and may therefore be related to viruses that were important in the evolution of this strain. Since HI tests do not necessarily detect all antibodies that neutralize viral infectivity, the question is raised as to whether HI assays alone can be used for determining the efficacy of all influenza virus vaccines.

Highly Immunogenic and Totally Synthetic Lipopeptides as Self-Adjuvanting Immunocontraceptive Vaccines

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.

Antiviral Activity of the Long Chain Pentraxin PTX3 against Influenza Viruses

Proteins of the innate immune system can act as natural inhibitors of influenza virus, limiting growth and spread of the virus in the early stages of infection before the induction of adaptive immune responses. In this study, we identify the long pentraxin PTX3 as a potent innate inhibitor of influenza viruses both in vitro and in vivo. Human and murine PTX3 bound to influenza virus and mediated a range of antiviral activities, including inhibition of hemagglutination, neutralization of virus infectivity and inhibition of viral neuraminidase. Antiviral activity was associated with binding of the viral hemagglutinin glycoprotein to sialylated ligands present on PTX3. Using a mouse model we found PTX3 to be rapidly induced following influenza infection and that PTX3−/− mice were more susceptible than wild-type mice to infection by PTX3-sensitive virus strains. Therapeutic treatment of mice with human PTX3 promoted survival and reduced viral load in the lungs following infection with PTX3-sensitive, but not PTX3-resistant, influenza viruses. Together, these studies describe a novel antiviral role for PTX3 in early host defense against influenza infections both in vitro and in vivo and describe the therapeutic potential of PTX3 in ameliorating disease during influenza infection.

ISCOMTM-based vaccines: The second decade

The immunostimulating complex or ‘iscom’ was first described 20 years ago as an antigen delivery system with powerful immunostimulating activity. Iscoms are cage‐like structures, typically 40 nm in diameter, that are comprised of antigen, cholesterol, phospholipid and saponin. ISCOMTM‐based vaccines have been shown to promote both antibody and cellular immune responses in a variety of experimental animal models. This review focuses on the evaluation of ISCOMTM‐based vaccines in animals over the past 10 years, as well as examining the progress that has been achieved in the development of human vaccines based on ISCOMTM adjuvant technology.

Influenza A virus facilitates Streptococcus pneumoniae transmission and disease

Streptococcus pneumoniae (the pneumococcus) kills ~1.6 million people annually. Pneumococcal infections predominantly manifest as pneumonia, sepsis, meningitis, and otitis media. S. pneumoniae is also a member of the normal nasopharyngeal flora, colonizing up to 80% of children. Infection with influenza A virus (IAV) has been associated with both pneumococcal disease and transmission. However, to date no animal model has been available to investigate the role of IAV in the spread of S. pneumoniae. Here we investigate pneumococcal‐influenza synergism with a particular focus on the role of IAV on pneumococcal transmission. Infant mice were colonized with S. pneumoniae and subsequently infected with IAV 3 d later. Using this novel model we show increased pneumococcal colonization and disease in the presence of IAV. Notably, in vivo imaging showed that IAV was essential for the transmission of S. pneumoniae from colonized (“index”) mice to their naive cohoused littermates (“contacts”). Transmission occurred only when all mice were infected with IAV and was prevented when an IAV‐neutralizing antibody was used to inhibit IAV replication in either index mice or contact mice. Together, these data provide novel insights into pneumococcal‐influenza synergism and may indicate a previously unappreciated role of IAV in the spread of S. pneumoniae. —Diavatopoulos, D. A, Short, K. R., Price, J. T., Wilksch, J. J., Brown, L. E., Briles, D. E., Strugnell, R. A, Wijburg, O. L. Influenza A virus facilitates Streptococcus pneumoniae transmission and disease. FASEB J. 24, 1789–1798 (2010). www.fasebj.org

Single-cell perforin and granzyme expression reveals the anatomical localization of effector CD8+ T cells in influenza virus-infected mice

Influenza virus infection activates cytolytic T lymphocytes (CTL) that contribute to viral clearance by releasing perforin and granzymes from cytoplasmic granules. Virus-specific, perforin-dependent CD8+ CTL were detected in freshly isolated cells from the mouse lung parenchyma but not from the mediastinal lymph nodes (MLN), where they are primed, or from the spleen during primary influenza virus infection. To determine whether this difference was due to the low frequency or incomplete maturation of effector CTL in MLN, we measured expression of perforin, granzymes A, B, and C, and IFN-γ mRNAs in CD8+ populations and single cells immediately after isolation from virus-infected mice. Quantitative PCR revealed significant expression of perforin, granzyme A, granzyme B, and IFN-γ in activated CD8+ cells from MLN, spleen, and lung parenchyma. Granzyme C expression was not detected. Individual activated or nucleoprotein peptide/class I tetramer-binding CD8+ cells from the three tissues expressed diverse combinations of perforin, granzyme, and IFN-γ mRNAs. Although cells from lung expressed granzymes A and B at higher frequency, each of the tissues contained cells that coexpressed perforin with granzymes A and/or B. The main difference between MLN and lung was the elevated frequency of activated CD8+ T cells in the lung, rather than their perforin/granzyme expression profile. The data suggest that some CTL mature into perforin/granzyme-expressing effector cells in MLN but reach detectable frequencies only when they accumulate in the infected lung.

Potent Immunity to Low Doses of Influenza Vaccine by Probabilistic Guided Micro-Targeted Skin Delivery in a Mouse Model

Background Over 14 million people die each year from infectious diseases despite extensive vaccine use [1]. The needle and syringe—first invented in 1853—is still the primary delivery device, injecting liquid vaccine into muscle. Vaccines could be far more effective if they were precisely delivered into the narrow layer just beneath the skin surface that contains a much higher density of potent antigen-presenting cells (APCs) essential to generate a protective immune response. We hypothesized that successful vaccination could be achieved this way with far lower antigen doses than required by the needle and syringe. Methodology/Principal Findings To meet this objective, using a probability-based theoretical analysis for targeting skin APCs, we designed the Nanopatch™, which contains an array of densely packed projections (21025/cm2) invisible to the human eye (110 µm in length, tapering to tips with a sharpness of <1000 nm), that are dry-coated with vaccine and applied to the skin for two minutes. Here we show that the Nanopatches deliver a seasonal influenza vaccine (Fluvax® 2008) to directly contact thousands of APCs, in excellent agreement with theoretical prediction. By physically targeting vaccine directly to these cells we induced protective levels of functional antibody responses in mice and also protection against an influenza virus challenge that are comparable to the vaccine delivered intramuscularly with the needle and syringe—but with less than 1/100th of the delivered antigen. Conclusions/Significance Our results represent a marked improvement—an order of magnitude greater than reported by others—for injected doses administered by other delivery methods, without reliance on an added adjuvant, and with only a single vaccination. This study provides a proven mathematical/engineering delivery device template for extension into human studies—and we speculate that successful translation of these findings into humans could uniquely assist with problems of vaccine shortages and distribution—together with alleviating fear of the needle and the need for trained practitioners to administer vaccine, e.g., during an influenza pandemic.

The Role of Neutrophils during Mild and Severe Influenza Virus Infections of Mice

Neutrophils have been implicated in both protective and pathological responses following influenza virus infections. We have used mAb 1A8 (anti-Ly6G) to specifically deplete LyG6high neutrophils and induce neutropenia in mice infected with virus strains known to differ in virulence. Mice were also treated with mAb RB6-8C5 (anti-Ly6C/G or anti-Gr-1), a mAb widely used to investigate the role of neutrophils in mice that has been shown to bind and deplete additional leukocyte subsets. Using mAb 1A8, we confirm the beneficial role of neutrophils in mice infected with virus strains of intermediate (HKx31; H3N2) or high (PR8; H1N1) virulence whereas treatment of mice infected with an avirulent strain (BJx109; H3N2) did not affect disease or virus replication. Treatment of BJx109-infected mice with mAb RB6-8C5 was, however, associated with significant weight loss and enhanced virus replication indicating that other Gr-1+ cells, not neutrophils, limit disease severity during mild influenza infections.

Activation of the NLRP3 Inflammasome by IAV Virulence Protein PB1-F2 Contributes to Severe Pathophysiology and Disease

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.

Prospects for an influenza vaccine that induces cross-protective cytotoxic T lymphocytes

Our approach to vaccination against influenza is unique. For no other pathogen do we construct and produce a new vaccine every year in the face of uncertainty about the strains that will be circulating when it is used. The huge global cooperative effort that underpins this process reflects our awareness of the need to control this major pathogen. Moreover, the threat of devastation by a pandemic due to a newly emerging viral subtype has triggered an intense effort to improve and accelerate the production of vaccines for use if a pandemic arises. However, type A influenza viruses responsible for seasonal epidemics and those with the potential to cause a pandemic share amino acid sequences that form the targets of cytotoxic T lymphocytes (CTL). CTL activated by currently circulating viruses, therefore, offer a possible means to limit the impact of infection with future variant seasonal strains and even new subtypes. This review examines how cross‐protective CTL can be exploited to improve influenza vaccination and issues that need to be considered when attempting to induce this type of immunity. We discuss the role of CTL responses in viral control and review the current knowledge relating to specificity and longevity of memory CD8+ T cells, how vaccine antigen can be loaded into antigen‐presenting cells to prime these responses and factors influencing the class of response induced. Application of these principles to the next generation of influenza vaccines should lead to much greater control of infection.