Standard-Dose Intradermal Influenza Vaccine Elicits Cellular Immune Responses Similar to Those of Intramuscular Vaccine in Men With and Those Without HIV Infection.

BACKGROUND: Human immunodeficiency virus (HIV)-infected persons are at a higher risk of severe influenza. Although we have shown that a standard-dose intradermal influenza vaccine versus a standard-dose intramuscular influenza vaccine does not result in differences in hemagglutination-inhibition titers in this population, a comprehensive examination of cell-mediated immune responses remains lacking. METHODS: Serological, antigen-specific B-cell, and interleukin 2-, interferon γ-, and tumor necrosis factor α-secreting T-cell responses were assessed in 79 HIV-infected men and 79 HIV-uninfected men. RESULTS: The route of vaccination did not affect the immunoglobulin A and immunoglobulin G (IgG) plasmablast or memory B-cell response, although these were severely impaired in the group with a CD4+ T-cell count of <200 cells/µL. The frequencies of IgG memory B cells measured on day 28 after vaccination were highest in the HIV-uninfected group, followed by the group with a CD4+ T-cell count of ≥200 cells/µL and the group with a CD4+ T-cell count of <200 cells/µL. The route of vaccination did not affect the CD4+ or CD8+ T-cell responses measured at various times after vaccination. CONCLUSIONS: The route of vaccination had no effect on antibody responses, antibody avidity, T-cell responses, or B-cell responses in HIV-infected or HIV-uninfected subjects. With the serological and cellular immune responses to influenza vaccination being impaired in HIV-infected individuals with a CD4+ T-cell count of <200 cells/µL, passive immunization strategies need to be explored to protect this population. CLINICAL TRIALS REGISTRATION: NCT01538940.

Impact of automated response systems on in-class cell phone use.

Cell phones have long been known as a potential distraction from attention intensive activities such as studying and driving. Many, however, are developing the cell phone as a powerful tool to augment some of these same activities. Audience response systems (ARSs) are a type of teaching tool that allows educators to poll audience members in real time. Increasingly, cell phones are being integrated into ARSs to make them more versatile and affordable. As cell phones and other personal electronic devices (tablets, laptops) are becoming more common classroom learning tools, we sought to explore how student cell phone use is impacted by this change. Additionally, we studied how a student's seat location and how the time during a term impacts students' cell phone use. To measure student cell phone use in lecture, we observed introductory biology classes at the University of Washington and recorded when students' cell phones were visible. We found that students sitting in the back of the room showed an increased likelihood of having a cell phone out. However contrary to our expectations, students using personal device (cell phone) based polling technology were no more likely to be using cell phones during lecture than their peers using traditional ARSs. Our results suggest that the downsides to using cell phones as teaching tools may be limited. © 2019 The International Union of Biochemistry and Molecular Biology, 47(5):538-546, 2019.

Kinetics of antibody response to influenza vaccination in renal transplant recipients.

Annual vaccination is routinely used in organ transplant recipients for immunization against seasonal influenza. However, detailed analysis of the kinetics of vaccine-induced immune responses in this population is lacking. In this study, we investigated the kinetics of vaccine strains-specific antibody responses to trivalent influenza vaccine in a group of renal transplant recipients and a control group. First, we found that the geometric mean hemagglutination inhibition titer against all 3 vaccine strains in the transplant cohort was significantly low when compared to control subjects. Next, whereas the control group sera showed significantly higher HA-specific IgG and isotype IgG1 antibodies at all four time points, a similar increase in the transplant group was delayed until day 28. Interestingly, within the transplant group, subjects receiving belatacept/MMF/prednisone-based regimen had significantly lower levels of total IgG and HA-specific IgG when compared to tacrolimus/MMF/prednisone-based regimen. Even though IgG-ASC response in both cohorts peaked at day 7 post-vaccination, the frequency of IgG-ASC was significantly low in the transplant group. Taken together, our studies show delayed kinetics and lower levels of influenza vaccine-specific antibody responses in renal transplant recipients and, more importantly, indicate the need to probe and improve current vaccination strategies in renal transplant recipients.

CD4 Depletion or CD40L Blockade Results in Antigen-Specific Tolerance in a Red Blood Cell Alloimmunization Model.

Approximately 3-10% of human red blood cell (RBC) transfusion recipients form alloantibodies to non-self, non-ABO blood group antigens expressed on donor RBCs, with these alloantibodies having the potential to be clinically significant in transfusion and pregnancy settings. However, the majority of transfused individuals never form detectable alloantibodies. Expanding upon observations that children initially transfused with RBCs at a young age are less likely to form alloantibodies throughout their lives, we hypothesized that "non-responders" may not only be ignorant of antigens on RBCs but instead tolerized. We investigated this question in a reductionist murine model, in which transgenic donors express the human glycophorin A (hGPA) antigen in an RBC-specific manner. Although wild-type mice treated with poly IC and transfused with hGPA RBCs generated robust anti-hGPA IgG alloantibodies that led to rapid clearance of incompatible RBCs, those transfused in the absence of an adjuvant failed to become alloimmunized. Animals depleted of CD4+ cells or treated with CD40L blockade prior to initial hGPA RBC exposure, in the presence of poly IC, failed to generate detectable anti-hGPA IgG alloantibodies. These non-responders to a primary transfusion remained unable to generate anti-hGPA IgG alloantibodies upon secondary hGPA exposure and did not prematurely clear transfused hGPA RBCs even after their CD4 cells had returned or their CD40L blockade had resolved. This observed tolerance was antigen (hGPA) specific, as robust IgG responses to transfused RBCs expressing a third-party antigen occurred in all studied groups. Experiments completed in an RBC alloimmunization model that allowed evaluation of antigen-specific CD4+ T-cells (HOD (hen egg lysozyme, ovalbumin, and human duffyb)) demonstrated that CD40L blockade prevented the expansion of ovalbumin 323-339 specific T-cells after HOD RBC transfusion and also prevented germinal center formation. Taken together, our data suggest that recipients may indeed become tolerized to antigens expressed on RBCs, with the recipient's immune status upon initial RBC exposure dictating future responses. Although questions surrounding mechanism(s) and sustainability of tolerance remain, these data lay the groundwork for future work investigating RBC immunity versus tolerance in reductionist models and in humans.

Making evolution stick: using sticky notes to teach the mechanisms of evolutionary change.

Evolution and its mechanisms of action are concepts that unite all aspects of biology, but remain some of the most difficult for students to understand. To address this challenge, we designed a hands-on activity that introduces fundamental mechanisms of evolutionary change: natural selection, genetic drift, and gene flow. In small groups, students use a population of sticky notes to reveal the consequences of each mechanism on phenotype frequency. In a followup homework assignment, students then explore how changes in phenotype frequency reflect changes in allele frequency in the population. This activity is suitable for anyone learning the basics of evolution, from high-school through the undergraduate level. We have provided detailed instructions, in-class worksheets, follow-up homework, and extensions that allow the activity to be simplified or made more complex as needed. In our own classrooms, we have observed that the concrete and collaborative nature of this activity enables students to deepen their understanding of the mechanisms through which evolution occurs. We have designed this study such that, in completing this activity, we hope to offer students the opportunity to confront potential misconceptions about evolution and gain a solid foundation for future explorations in the discipline.

Rapamycin Does Not Impede Survival or Induction of Antibody Responses to Primary and Heterosubtypic Influenza Infections in Mice.

Impairment of immune defenses can contribute to severe influenza infections. Rapamycin is an immunosuppressive drug often used to prevent transplant rejection and is currently undergoing clinical trials for treating cancers and autoimmune diseases. We investigated whether rapamycin has deleterious effects during lethal influenza viral infections. We treated mice with two concentrations of rapamycin and infected them with A/Puerto Rico/8/1934 (A/PR8), followed by a heterosubtypic A/Hong Kong/1/68 (A/HK68) challenge. Our data show similar morbidity, mortality, and lung viral titer with both rapamycin treatment doses compared to untreated controls, with a delay in morbidity onset in rapamycin high dose recipients during primary infection. Rapamycin treatment at high dose also led to increase in percent cytokine producing T cells in the spleen. However, all infected animals had similar serum antibody responses against A/PR8. Post-A/HK68 challenge, rapamycin had no impeding effect on morbidity or mortality and had similar serum antibody levels against A/PR8 and A/HK68. We conclude that rapamycin treatment does not adversely affect morbidity, mortality, or antibody production during lethal influenza infections.

RIG-I ligand enhances the immunogenicity of recombinant H7HA protein.

Avian H7N9 influenza virus infection with fatal outcomes continues to pose a pandemic threat and highly immunogenic vaccines are urgently needed. In this report we show that baculovirus-derived recombinant H7 hemagglutinin protein, when delivered with RIG-I ligand, induced enhanced antibody and T cell responses and conferred protection against lethal challenge with a homologous H7N9 virus. These findings indicate the potential utility of RIG-I ligands as vaccine adjuvants to increase the immunogenicity of recombinant H7 hemagglutinin.

NADPH Oxidase 1 Is Associated with Altered Host Survival and T Cell Phenotypes after Influenza A Virus Infection in Mice.

The role of the reactive oxygen species-producing NADPH oxidase family of enzymes in the pathology of influenza A virus infection remains enigmatic. Previous reports implicated NADPH oxidase 2 in influenza A virus-induced inflammation. In contrast, NADPH oxidase 1 (Nox1) was reported to decrease inflammation in mice within 7 days post-influenza A virus infection. However, the effect of NADPH oxidase 1 on lethality and adaptive immunity after influenza A virus challenge has not been explored. Here we report improved survival and decreased morbidity in mice with catalytically inactive NADPH oxidase 1 (Nox1*/Y) compared with controls after challenge with A/PR/8/34 influenza A virus. While changes in lung inflammation were not obvious between Nox1*/Y and control mice, we observed alterations in the T cell response to influenza A virus by day 15 post-infection, including increased interleukin-7 receptor-expressing virus-specific CD8+ T cells in lungs and draining lymph nodes of Nox1*/Y, and increased cytokine-producing T cells in lungs and spleen. Furthermore, a greater percentage of conventional and interstitial dendritic cells from Nox1*/Y draining lymph nodes expressed the co-stimulatory ligand CD40 within 6 days post-infection. Results indicate that NADPH oxidase 1 modulates the innate and adaptive cellular immune response to influenza virus infection, while also playing a role in host survival. Results suggest that NADPH oxidase 1 inhibitors may be beneficial as adjunct therapeutics during acute influenza infection.

A highly immunogenic vaccine against A/H7N9 influenza virus.

Since the first case of human infection in March 2013, continued reports of H7N9 cases highlight a potential pandemic threat. Highly immunogenic vaccines to this virus are urgently needed to protect vulnerable populations who lack protective immunity. In this study, an egg- and adjuvant-independent adenoviral vector-based, hemagglutinin H7 subtype influenza vaccine (HAd-H7HA) demonstrated enhanced cell-mediated immunity as well as serum antibody responses in a mouse model. Most importantly, this vaccine provided complete protection against homologous A/H7N9 viral challenge suggesting its potential utility as a pandemic vaccine.

Antigen modulation confers protection to red blood cells from antibody through Fcγ receptor ligation.

Autoantibodies and alloantibodies can damage self-tissue or transplanted tissues through either fixation of complement or ligation of FcγRs. Several pathways have been described that imbue self-tissues with resistance to damage from complement fixation, as a protective measure against damage from these Abs. However, it has been unclear whether parallel pathways exist to provide protection from FcγR ligation by bound Abs. In this article, we describe a novel pathway by which cell surface Ag is specifically decreased as a result of Ab binding (Ag modulation) to the extent of conferring protection to recognized cells from Fcγ-dependent clearance. Moreover, the Ag modulation in this system requires FcγR ligation. Together, these findings provide unique evidence of self-protective pathways for FcγR-mediated Ab damage.

Resistance of a subset of red blood cells to clearance by antibodies in a mouse model of incompatible transfusion.

BACKGROUND: Alloimmunization to antigens on transfused red blood cells (RBCs) represents a major barrier to chronic transfusion. In extreme cases of multiple alloimmunization, clinicians may be faced with the decision of transfusing incompatible RBCs or risking death from lack of transfusion. The disastrous results of hemolytic transfusion reactions are well understood, and major pathways of clearance have been described. However, well described but poorly understood is the survival of a subset of incompatible donor RBCs during hemolysis, despite antibody binding. STUDY DESIGN AND METHODS: We utilize a tractable murine model of incompatible transfusion in which RBCs from transgenic donor mice expressing human glycophorin A (hGPA) are transfused into recipients passively immunized with anti-hGPA. RESULTS: As in humans, the majority of RBCs are cleared but a subset of incompatible donor RBCs persist in circulation, despite being bound by antibodies. Data contained herein reject the hypothesis that lack of clearance is due to insufficient antibody or overwhelming of phagocytic machinery; rather, we establish that surviving RBCs represent a distinct population resistant to clearance. CONCLUSIONS: These studies demonstrate that surviving RBCs during incompatible transfusion can represent a population that is resistant to clearance.

Biphasic clearance of incompatible red blood cells through a novel mechanism requiring neither complement nor Fcγ receptors in a murine model.

BACKGROUND: Antibody binding to red blood cells (RBCs) can induce potentially fatal outcomes, including hemolytic transfusion reactions (HTRs), hemolytic disease of the fetus and newborn, and autoimmune hemolytic anemia. The mechanism(s) of RBC destruction following antibody binding is typically thought to require complement activation and/or the involvement of Fcγ receptors (FcγRs). In the current report, we analyzed mechanisms of HTRs during incompatible transfusions of murine RBCs expressing human glycophorin A (hGPA) into mice with anti-hGPA. STUDY DESIGN AND METHODS: C3 and Fcγ receptor knockout, splenectomized, Fcγ receptor blocking antibody-treated, and clodronate-treated mice were passively immunized with anti-hGPA (10F7 or 6A7) and transfused with RBCs expressing the hGPA antigen. Posttransfusion blood and serum were collected and analyzed via flow cytometry and confocal microscopy. RESULTS: This HTR model results in both rapid clearance and cytokine storm. Neither complement nor FcγRs were required for RBC clearance; in contrast, FcγRs were required for cytokine storm. Circulating aggregates of hGPA RBCs were visible during the HTR. Splenectomy and phagocyte depletion by clodronate had no effect on acute RBC clearance; however, incompatible RBCs reentered over 24 hours in clodronate-treated mice. CONCLUSION: These data demonstrate a biphasic HTR, the first phase involving sequestration of incompatible hGPA RBCs and the second phase involving phagocytosis of sequestered RBCs. However, the mechanism(s) of phagocytosis in the second phase required neither C3 nor FcγRs. These findings demonstrate novel mechanistic biology of HTRs.

Cytokine storm in a mouse model of IgG-mediated hemolytic transfusion reactions.

Cytokines are hypothesized to play a central role in the pathophysiology of IgG-mediated hemolytic transfusion reactions (HTRs), and deeper understanding is required for improving therapy for these events. After establishing well-defined mouse models of HTRs, we tested whether cytokines were involved. Red blood cells (RBCs) from human glycophorin A transgenic (hGPA-Tg) or wild-type (WT) mice were transfused into non-Tg recipients passively immunized with monoclonal antibodies (Mabs). Only transfusions of incompatible RBCs induced IgG-mediated HTRs, exemplified by rapid clearance and hemoglobinuria. Very high plasma levels of monocyte chemoattractant protein-1 (MCP-1) and interleukin-6 (IL-6), and lower levels of tumor necrosis factor-alpha (TNF-alpha), were induced after incompatible transfusion. No significant changes in IL-10, IL-12, or interferon-gamma (IFN-gamma) levels were observed. The proinflammatory cytokines elaborated in this in vivo mouse model are also implicated in the systemic inflammatory response syndrome (SIRS) and confirm the hypothesis that cytokine storm occurs as a result of HTRs.