Denise A. Kaminski

A Novel Role for Non-Neutralizing Antibodies against Nucleoprotein in Facilitating Resistance to Influenza Virus

Current influenza vaccines elicit Abs to the hemagglutinin and neuraminidase envelope proteins. Due to antigenic drift, these vaccines must be reformulated annually to include the envelope proteins predicted to dominate in the following season. By contrast, vaccination with the conserved nucleoprotein (NP) elicits immunity against multiple serotypes (heterosubtypic immunity). NP vaccination is generally thought to convey protection primarily via CD8 effector mechanisms. However, significant titers of anti-NP Abs are also induced, yet the involvement of Abs in protection has largely been disregarded. To investigate how Ab responses might contribute to heterosubtypic immunity, we vaccinated C57BL/6 mice with soluble rNP. This approach induced high titers of NP-specific serum Ab, but only poorly detectable NP-specific T cell responses. Nevertheless, rNP immunization significantly reduced morbidity and viral titers after influenza challenge. Importantly, Ab-deficient mice were not protected by this vaccination strategy. Furthermore, rNP-immune serum could transfer protection to naive hosts in an Ab-dependent manner. Therefore, Ab to conserved, internal viral proteins, such as NP, provides an unexpected, yet important mechanism of protection against influenza. These results suggest that vaccines designed to elicit optimal heterosubtypic immunity to influenza should promote both Ab and T cell responses to conserved internal proteins.

Contributions of Antinucleoprotein IgG to Heterosubtypic Immunity against Influenza Virus

Influenza A virus causes recurring seasonal epidemics and occasional influenza pandemics. Because of changes in envelope glycoprotein Ags, neutralizing Abs induced by inactivated vaccines provide limited cross-protection against new viral serotypes. However, prior influenza infection induces heterosubtypic immunity that accelerates viral clearance of a second strain, even if the external proteins are distinct. In mice, cross-protection can also be elicited by systemic immunization with the highly conserved internal nucleoprotein (NP). Both T lymphocytes and Ab contribute to such cross-protection. In this paper, we demonstrate that anti-NP IgG specifically promoted influenza virus clearance in mice by using a mechanism involving both FcRs and CD8+ cells. Furthermore, anti-NP IgG rescued poor heterosubtypic immunity in B cell-deficient mice, correlating with enhanced NP-specific CD8 T cell responses. Thus, Ab against this conserved Ag has potent antiviral activity both in naive and in influenza-immune subjects. Such antiviral activity was not seen when mice were vaccinated with another internal influenza protein, nonstructural 1. The high conservation of NP Ag and the known longevity of Ab responses suggest that anti-NP IgG may provide a critically needed component of a universal influenza vaccine.

Advances in human B cell phenotypic profiling.

To advance our understanding and treatment of disease, research immunologists have been called-upon to place more centralized emphasis on impactful human studies. Such endeavors will inevitably require large-scale study execution and data management regulation (“Big Biology”), necessitating standardized and reliable metrics of immune status and function. A well-known example setting this large-scale effort in-motion is identifying correlations between eventual disease outcome and T lymphocyte phenotype in large HIV-patient cohorts using multiparameter flow cytometry. However, infection, immunodeficiency, and autoimmunity are also characterized by correlative and functional contributions of B lymphocytes, which to-date have received much less attention in the human Big Biology enterprise. Here, we review progress in human B cell phenotyping, analysis, and bioinformatics tools that constitute valuable resources for the B cell research community to effectively join in this effort.

Regulation of Antinucleoprotein IgG by Systemic Vaccination and Its Effect on Influenza Virus Clearance

ABSTRACT Seasonal influenza epidemics recur due to antigenic drift of envelope glycoprotein antigens and immune evasion of circulating viruses. Additionally, antigenic shift can lead to influenza pandemics. Thus, a universal vaccine that protects against multiple influenza virus strains could alleviate the continuing impact of this virus on human health. In mice, accelerated clearance of a new viral strain (cross-protection) can be elicited by prior infection (heterosubtypic immunity) or by immunization with the highly conserved internal nucleoprotein (NP). Both heterosubtypic immunity and NP-immune protection require antibody production. Here, we show that systemic immunization with NP readily accelerated clearance of a 2009 pandemic H1N1 influenza virus isolate in an antibody-dependent manner. However, human immunization with trivalent inactivated influenza virus vaccine (TIV) only rarely and modestly boosted existing levels of anti-NP IgG. Similar results were observed in mice, although the reaction could be enhanced with adjuvants, by adjusting the stoichiometry among NP and other vaccine components, and by increasing the interval between TIV prime and boost. Importantly, mouse heterosubtypic immunity that had waned over several months could be enhanced by injecting purified anti-NP IgG or by boosting with NP protein, correlating with a long-lived increase in anti-NP antibody titers. Thus, current immunization strategies poorly induce NP-immune antibody that is nonetheless capable of contributing to long-lived cross-protection. The high conservation of NP antigen and the known longevity of antibody responses suggest that the antiviral activity of anti-NP IgG may provide a critically needed component of a universal influenza vaccine.

Adaptive immunity and adipose tissue biology.

Studies of immunity typically focus on understanding how hematopoietic cells interact within conventional secondary lymphoid tissues. However, immune reactions and their regulation occur in various environments within the body. Adipose tissue is one tissue that can influence and be influenced by adjacent and embedded lymphocytes. Despite the abundance and wide distribution of such tissue, and despite a growing obesity epidemic, studies of these interactions have been only marginally appreciated in the past. Here, we review advances in understanding of lymphoid structures within adipose tissue, the relationship between adipose tissue and adaptive immune function, and evidence for how this relationship contributes to obesity-associated diseases.

Deletion of microRNA-155 reduces autoantibody responses and alleviates lupus-like disease in the Fas(lpr) mouse

Significance The host immune system provides diverse defense mechanisms to fight harmful bacteria and viruses. One of these mechanisms is the production of antibodies targeting infectious agents. However, the production of antibodies has to be tightly controlled. Insufficient control of the immune system may result in the development of autoimmune diseases, including lupus. Lupus is characterized by the production of antibodies attacking the kidneys, leading to life-threatening kidney failure if untreated. In this study, we show that microRNA-155, one member of a family of regulatory molecules, promotes the production of antibodies. By deleting microRNA-155, we can prevent the production of harmful antibodies and alleviate lupus-like disease in mice. Our results suggest the possibility of targeting microRNA-155 to treat autoimmune diseases. MicroRNA-155 (miR-155) regulates antibody responses and subsequent B-cell effector functions to exogenous antigens. However, the role of miR-155 in systemic autoimmunity is not known. Using the death receptor deficient (Faslpr) lupus-prone mouse, we show here that ablation of miR-155 reduced autoantibody responses accompanied by a decrease in serum IgG but not IgM anti-dsDNA antibodies and a reduction of kidney inflammation. MiR-155 deletion in Faslpr B cells restored the reduced SH2 domain-containing inositol 5′-phosphatase 1 to normal levels. In addition, coaggregation of the Fc γ receptor IIB with the B-cell receptor in miR-155−/−-Faslpr B cells resulted in decreased ERK activation, proliferation, and production of switched antibodies compared with miR-155 sufficient Faslpr B cells. Thus, by controlling the levels of SH2 domain-containing inositol 5′-phosphatase 1, miR-155 in part maintains an activation threshold that allows B cells to respond to antigens.

Antibodies Against Conserved Antigens Provide Opportunities for Reform in Influenza Vaccine Design

High-performance neutralizing antibody against influenza virus typically recognizes the globular head region of its hemagglutinin (HA) envelope glycoprotein. To-date, approved human vaccination strategies have been designed to induce such antibodies as a sole means of preventing the consequences of this infection. However, frequent amino-acid changes in the HA globular head allow for efficient immune evasion. Consequently, vaccines inducing such neutralizing antibodies need to be annually re-designed and re-administered at a great expense. These vaccines furthermore provide little-to-no immunity against antigenic-shift strains, which arise from complete replacement of HA or of neuraminidase genes, and pose pandemic risks. To address these issues, laboratory research has focused on inducing immunity effective against all strains, regardless of changes in the HA globular head. Despite prior dogma that such cross-protection needs to be induced by cellular immunity alone, several advances in recent years demonstrate that antibodies of other specificities are capable of cross-strain protection in mice. This review discusses the reactivity, induction, efficacy, and mechanisms of antibodies that react with poorly accessible epitopes in the HA stalk, with the matrix 2 membrane ion channel, and even with the internal nucleoprotein. These advances warrant further investigation of the inducibility and efficacy of such revolutionary antibody strategies in humans.

Primary Sjögren's syndrome is characterized by distinct phenotypic and transcriptional profiles of IgD+ unswitched memory B cells.

The significance of distinct B cell abnormalities in primary Sjögrens syndrome (SS) remains to be established. We undertook this study to analyze the phenotype and messenger RNA (mRNA) transcript profiles of B cell subsets in patients with primary SS and to compare them with those in sicca syndrome patients and healthy controls.

Multiparameter Flow Cytometry and Bioanalytics for B Cell Profiling in Systemic Lupus Erythematosus

B lymphocyte involvement in systemic lupus erythematosus has been recognized for several decades, mainly in the context of autoantibody production. Both mouse and human studies reveal that different types of antibody responses, as well as antibody-independent effector functions can be ascribed to distinct subpopulations (subsets) of circulating B cells. Characterizing human B cell subsets can advance the field of autoimmunity even further by establishing B cell signatures associated with disease severity, progression, and response-to-treatment. For this purpose, we have developed specialized B cell reagent panels for multiparameter flow cytometry, and combine their use with advanced bioinformatics strategies that together will likely be advantageous for improving the characterization, prognosis, and for possibly improving treatment regimens of chronic inflammatory diseases such as lupus.

9G4+ antibodies isolated from HIV-infected patients neutralize HIV-1 and have distinct autoreactivity profiles.

Potent HIV-1 specific broadly neutralizing antibodies (BNA) are uncommon in HIV infected individuals, and have proven hard to elicit by vaccination. Several, isolated monoclonal BNA are polyreactive and also recognize self-antigens, suggesting a breach of immune tolerance in persons living with HIV (PLWH). Persons with systemic lupus erythematosus (SLE) often have elevated levels of autoreactive antibodies encoded by the VH4-34 heavy chain immunoglobulin gene whose protein product can be detected by the 9G4 rat monoclonal antibody. We have recently found that levels of these “9G4+” antibodies are also elevated in PLWH. However, the putative autoreactive nature of these antibodies and the relationship of such reactivities with HIV neutralization have not been investigated. We therefore examined the autoreactivity and HIV neutralization potential of 9G4+ antibodies from PLWH. Results show that 9G4+ antibodies from PLWH bound to recombinant HIV-1 envelope (Env) and neutralized viral infectivity in vitro, whereas 9G4+ antibodies from persons with SLE did not bind to Env and failed to neutralize viral infectivity. In addition, while 9G4+ antibodies from PLWH retained the canonical anti-i reactivity that mediates B cell binding, they did not display other autoreactivities common to SLE 9G4+ antibodies, such as binding to cardiolipin and DNA and had much lower reactivity with apoptotic cells. Taken together, these data indicate that the autoreactivity of 9G4+ antibodies from PLWH is distinct from that of SLE patients, and therefore, their expansion is not due to a general breakdown of B cell tolerance but is instead determined in a more disease-specific manner by self-antigens that become immunogenic in the context of, and possibly due to HIV infection. Further studies of 9G4+ B cells may shed light on the regulation of B cell tolerance and interface between the generation of specific autoreactivities and the induction of antiviral immunity in persons living with HIV.