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1.
Cell ; 173(2): 417-429.e10, 2018 04 05.
Article in English | MEDLINE | ID: mdl-29625056

ABSTRACT

Antibodies to the hemagglutinin (HA) and neuraminidase (NA) glycoproteins are the major mediators of protection against influenza virus infection. Here, we report that current influenza vaccines poorly display key NA epitopes and rarely induce NA-reactive B cells. Conversely, influenza virus infection induces NA-reactive B cells at a frequency that approaches (H1N1) or exceeds (H3N2) that of HA-reactive B cells. NA-reactive antibodies display broad binding activity spanning the entire history of influenza A virus circulation in humans, including the original pandemic strains of both H1N1 and H3N2 subtypes. The antibodies robustly inhibit the enzymatic activity of NA, including oseltamivir-resistant variants, and provide robust prophylactic protection, including against avian H5N1 viruses, in vivo. When used therapeutically, NA-reactive antibodies protected mice from lethal influenza virus challenge even 48 hr post infection. These findings strongly suggest that influenza vaccines should be optimized to improve targeting of NA for durable and broad protection against divergent influenza strains.


Subject(s)
Antibodies, Monoclonal/immunology , Influenza, Human/pathology , Neuraminidase/immunology , Viral Proteins/immunology , Animals , Birds , Cross Reactions , Epitopes/immunology , Female , HEK293 Cells , Humans , Immunoglobulin G/blood , Immunoglobulin G/immunology , Influenza A Virus, H1N1 Subtype/enzymology , Influenza A Virus, H3N2 Subtype/enzymology , Influenza A Virus, H5N1 Subtype/immunology , Influenza A Virus, H5N1 Subtype/pathogenicity , Influenza, Human/immunology , Mice , Mice, Inbred BALB C , Orthomyxoviridae Infections/pathology , Orthomyxoviridae Infections/prevention & control
2.
Nat Immunol ; 21(9): 1046-1057, 2020 09.
Article in English | MEDLINE | ID: mdl-32747818

ABSTRACT

Early recruitment of neutrophils from the blood to sites of tissue infection is a hallmark of innate immune responses. However, little is known about the mechanisms by which apoptotic neutrophils are cleared in infected tissues during resolution and the immunological consequences of in situ efferocytosis. Using intravital multiphoton microscopy, we show previously unrecognized motility patterns of interactions between neutrophils and tissue-resident phagocytes within the influenza-infected mouse airway. Newly infiltrated inflammatory monocytes become a chief pool of phagocytes and play a key role in the clearance of highly motile apoptotic neutrophils during the resolution phase. Apoptotic neutrophils further release epidermal growth factor and promote the differentiation of monocytes into tissue-resident antigen-presenting cells for activation of antiviral T cell effector functions. Collectively, these results suggest that the presence of in situ neutrophil resolution at the infected tissue is critical for optimal CD8+ T cell-mediated immune protection.


Subject(s)
CD8-Positive T-Lymphocytes/immunology , Influenza A virus/physiology , Influenza, Human/immunology , Neutrophils/immunology , Orthomyxoviridae Infections/immunology , Phagocytes/immunology , Receptors, CCR2/metabolism , Animals , Antigen Presentation , Apoptosis , Cell Movement , Cells, Cultured , Humans , Immunity, Innate , Intravital Microscopy , Lymphocyte Activation , Mice , Mice, Inbred C57BL , Mice, Transgenic , Microscopy, Fluorescence, Multiphoton , Receptors, CCR2/genetics
3.
Immunity ; 51(2): 298-309.e6, 2019 08 20.
Article in English | MEDLINE | ID: mdl-31399281

ABSTRACT

T-helper (Th) cell differentiation drives specialized gene programs that dictate effector T cell function at sites of infection. Here, we have shown Th cell differentiation also imposes discrete motility gene programs that shape Th1 and Th2 cell navigation of the inflamed dermis. Th1 cells scanned a smaller tissue area in a G protein-coupled receptor (GPCR) and chemokine-dependent fashion, while Th2 cells scanned a larger tissue area independent of GPCR signals. Differential chemokine reliance for interstitial migration was linked to STAT6 transcription-factor-dependent programming of integrin αVß3 expression: Th2 cell differentiation led to high αVß3 expression relative to Th1 cells. Th1 and Th2 cell modes of motility could be switched simply by manipulating the amount of αVß3 on the cell surface. Deviating motility modes from those established during differentiation impaired effector function. Thus, programmed expression of αVß3 tunes effector T cell reliance on environmental cues for optimal exploration of inflamed tissues.


Subject(s)
Inflammation/immunology , Th1 Cells/immunology , Th2 Cells/immunology , Adoptive Transfer , Animals , Cell Differentiation , Cell Movement , Cells, Cultured , Cellular Reprogramming Techniques , Chemokines/metabolism , Humans , Integrin alphaVbeta3/metabolism , Lymphocyte Activation , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Knockout , STAT6 Transcription Factor/metabolism
4.
Nat Immunol ; 14(9): 949-58, 2013 Sep.
Article in English | MEDLINE | ID: mdl-23933892

ABSTRACT

Leukocytes must traverse inflamed tissues to effectively control local infection. Although motility in dense tissues seems to be integrin independent and based on actomyosin-mediated protrusion and contraction, during inflammation, changes to the extracellular matrix (ECM) may necessitate distinct motility requirements. Indeed, we found that the interstitial motility of T cells was critically dependent on Arg-Gly-Asp (RGD)-binding integrins in the inflamed dermis. Inflammation-induced deposition of fibronectin was functionally linked to higher expression of integrin αV on effector CD4⁺ T cells. By intravital multiphoton imaging, we found that the motility of CD4⁺ T cells was dependent on αV expression. Selective blockade or knockdown of αV arrested T helper type 1 (TH1) cells in the inflamed tissue and attenuated local effector function. Our data demonstrate context-dependent specificity of lymphocyte movement in inflamed tissues that is essential for protective immunity.


Subject(s)
CD4-Positive T-Lymphocytes/immunology , CD4-Positive T-Lymphocytes/metabolism , Cell Movement/immunology , Inflammation/immunology , Inflammation/metabolism , Integrin alphaV/metabolism , Animals , Dermis/immunology , Dermis/metabolism , Extracellular Matrix/metabolism , Gene Expression Regulation , Inflammation/genetics , Integrin alphaV/genetics , Lymph Nodes/immunology , Mice , Oligopeptides/metabolism , Protein Binding , Th1 Cells/immunology , Th1 Cells/metabolism
5.
J Infect Dis ; 227(3): 381-390, 2023 02 01.
Article in English | MEDLINE | ID: mdl-35199825

ABSTRACT

The most effective measure to induce protection from influenza is vaccination. Thus, yearly vaccination is recommended, which, together with infections, establishes diverse repertoires of B cells, antibodies, and T cells. We examined the impact of this accumulated immunity on human responses in adults to split, subunit, and recombinant protein-based influenza vaccines. Enzyme-linked immunosorbent assay (ELISA) assays, to quantify serum antibodies, and peptide-stimulated CD4 T-cell cytokine ELISpots revealed that preexisting levels of hemagglutinin (HA)-specific antibodies were negatively associated with gains in antibody postvaccination, while preexisting levels of CD4 T cells were negatively correlated with vaccine-induced expansion of CD4 T cells. These patterns were seen independently of the vaccine formulation administered and the subjects' influenza vaccine history. Thus, although memory CD4 T cells and serum antibodies consist of components that can enhance vaccine responses, on balance, the accumulated immunity specific for influenza A H1 and H3 proteins is associated with diminished future responses.


Subject(s)
Influenza Vaccines , Influenza, Human , Adult , Humans , Influenza, Human/prevention & control , Antibodies , CD4-Positive T-Lymphocytes , Vaccination , Antibodies, Viral , Hemagglutinin Glycoproteins, Influenza Virus
6.
Proc Natl Acad Sci U S A ; 117(22): 12306-12314, 2020 06 02.
Article in English | MEDLINE | ID: mdl-32439709

ABSTRACT

Tissue-resident memory CD8 T (TRM) cells are a unique immune memory subset that develops and remains in peripheral tissues at the site of infection, providing future host resistance upon reexposure to that pathogen. In the pulmonary system, TRM are identified through S1P antagonist CD69 and expression of integrins CD103/ß7 and CD49a/CD29(ß1). Contrary to the established role of CD69 on CD8 T cells, the functions of CD103 and CD49a on this population are not well defined. This study examines the expression patterns and functions of CD103 and CD49a with a specific focus on their impact on T cell motility during influenza virus infection. We show that the TRM cell surface phenotype develops by 2 wk postinfection, with the majority of the population expressing CD49a and a subset that is also positive for CD103. Despite a previously established role in retaining TRM in peripheral tissues, CD49a facilitates locomotion of virus-specific CD8 T cells, both in vitro and in vivo. These results demonstrate that CD49a may contribute to local surveillance mechanisms of the TRM population.


Subject(s)
Antigens, CD/immunology , Influenza A Virus, H3N2 Subtype/physiology , Influenza, Human/immunology , Integrin alpha Chains/immunology , Integrin alpha1/metabolism , Animals , Antigens, CD/genetics , CD8-Positive T-Lymphocytes/cytology , CD8-Positive T-Lymphocytes/immunology , Cell Adhesion , Cell Movement , Humans , Immunologic Memory , Influenza A Virus, H3N2 Subtype/genetics , Influenza, Human/genetics , Influenza, Human/physiopathology , Influenza, Human/virology , Integrin alpha Chains/genetics , Integrin alpha1/genetics , Mice, Inbred C57BL
7.
PLoS Pathog ; 16(7): e1008506, 2020 07.
Article in English | MEDLINE | ID: mdl-32645119

ABSTRACT

Circulating androgens can modulate immune cell activity, but the impact of androgens on viral pathogenesis remains unclear. Previous data demonstrate that testosterone reduces the severity of influenza A virus (IAV) infection in male mice by mitigating pulmonary inflammation rather than by affecting viral replication. To examine the immune responses mediated by testosterone to mitigate IAV-induced inflammation, adult male mice remained gonadally intact or were gonadectomized and treated with either placebo or androgen-filled (i.e., testosterone or dihydrotestosterone) capsules prior to sublethal IAV infection. Like intact males, treatment of gonadectomized males with androgens improved the outcome of IAV infection, which was not mediated by changes in the control of virus replication or pulmonary cytokine activity. Instead, androgens accelerated pulmonary leukocyte contraction to limit inflammation. To identify which immune cells were contracting in response to androgens, the composition of pulmonary cellular infiltrates was analyzed and revealed that androgens specifically accelerated the contraction of total pulmonary inflammatory monocytes during peak disease, as well as CD8+ T cells, IAV-specific CD8+ T numbers, cytokine production and degranulation by IAV-specific CD8+ T cells, and the influx of eosinophils into the lungs following clearance of IAV. Neither depletion of eosinophils nor adoptive transfer of CD8+ T cells could reverse the ability of testosterone to protect males against IAV suggesting these were secondary immunologic effects. The effects of testosterone on the contraction of immune cell numbers and activity were blocked by co-administration of the androgen receptor antagonist flutamide and mimicked by treatment with dihydrotestosterone, which was also able to reduce the severity of IAV in female mice. These data suggest that androgen receptor signaling creates a local pulmonary environment that promotes downregulation of detrimental inflammatory immune responses to protect against prolonged influenza disease.


Subject(s)
Influenza A virus/immunology , Lung/drug effects , Orthomyxoviridae Infections/immunology , Receptors, Androgen/metabolism , Testosterone/pharmacology , Animals , Female , Inflammation/immunology , Inflammation/virology , Lung/immunology , Male , Mice, Inbred C57BL , Rats , Receptors, Androgen/drug effects , Signal Transduction/drug effects , Signal Transduction/immunology
8.
PLoS Pathog ; 16(5): e1008393, 2020 05.
Article in English | MEDLINE | ID: mdl-32433711

ABSTRACT

Infection with (SAg)-producing bacteria may precede or follow infection with or vaccination against influenza A viruses (IAVs). However, how SAgs alter the breadth of IAV-specific CD8+ T cell (TCD8) responses is unknown. Moreover, whether recall responses mediating heterosubtypic immunity to IAVs are manipulated by SAgs remains unexplored. We employed wild-type (WT) and mutant bacterial SAgs, SAg-sufficient/deficient Staphylococcus aureus strains, and WT, mouse-adapted and reassortant IAV strains in multiple in vivo settings to address the above questions. Contrary to the popular view that SAgs delete or anergize T cells, systemic administration of staphylococcal enterotoxin B (SEB) or Mycoplasma arthritidis mitogen before intraperitoneal IAV immunization enlarged the clonal size of 'select' IAV-specific TCD8 and reshuffled the hierarchical pattern of primary TCD8 responses. This was mechanistically linked to the TCR Vß makeup of the impacted clones rather than their immunodominance status. Importantly, SAg-expanded TCD8 retained their IFN-γ production and cognate cytolytic capacities. The enhancing effect of SEB on immunodominant TCD8 was also evident in primary responses to vaccination with heat-inactivated and live attenuated IAV strains administered intramuscularly and intranasally, respectively. Interestingly, in prime-boost immunization settings, the outcome of SEB administration depended strictly upon the time point at which this SAg was introduced. Accordingly, SEB injection before priming raised CD127highKLRG1low memory precursor frequencies and augmented the anamnestic responses of SEB-binding TCD8. By comparison, introducing SEB before boosting diminished recall responses to IAV-derived epitopes drastically and indiscriminately. This was accompanied by lower Ki67 and higher Fas, LAG-3 and PD-1 levels consistent with a pro-apoptotic and/or exhausted phenotype. Therefore, SAgs can have contrasting impacts on anti-IAV immunity depending on the naïve/memory status and the TCR composition of exposed TCD8. Finally, local administration of SEB or infection with SEB-producing S. aureus enhanced pulmonary TCD8 responses to IAV. Our findings have clear implications for superinfections and prophylactic vaccination.


Subject(s)
Immunologic Memory/immunology , Influenza A virus/immunology , Superantigens/immunology , Animals , CD8-Positive T-Lymphocytes/immunology , Epitopes/immunology , Female , Humans , Immunologic Memory/physiology , Influenza A virus/metabolism , Influenza, Human/immunology , Influenza, Human/metabolism , Lymphocyte Activation/immunology , Mice , Mice, Inbred BALB C , Staphylococcus aureus/immunology , Superantigens/physiology , Superinfection/immunology , Vaccination
9.
PLoS Pathog ; 16(4): e1008409, 2020 04.
Article in English | MEDLINE | ID: mdl-32287326

ABSTRACT

The continual emergence of novel influenza A strains from non-human hosts requires constant vigilance and the need for ongoing research to identify strains that may pose a human public health risk. Since 1999, canine H3 influenza A viruses (CIVs) have caused many thousands or millions of respiratory infections in dogs in the United States. While no human infections with CIVs have been reported to date, these viruses could pose a zoonotic risk. In these studies, the National Institutes of Allergy and Infectious Diseases (NIAID) Centers of Excellence for Influenza Research and Surveillance (CEIRS) network collaboratively demonstrated that CIVs replicated in some primary human cells and transmitted effectively in mammalian models. While people born after 1970 had little or no pre-existing humoral immunity against CIVs, the viruses were sensitive to existing antivirals and we identified a panel of H3 cross-reactive human monoclonal antibodies (hmAbs) that could have prophylactic and/or therapeutic value. Our data predict these CIVs posed a low risk to humans. Importantly, we showed that the CEIRS network could work together to provide basic research information important for characterizing emerging influenza viruses, although there were valuable lessons learned.


Subject(s)
Communicable Diseases, Emerging/veterinary , Dog Diseases/virology , Influenza A Virus, H3N2 Subtype/isolation & purification , Influenza A Virus, H3N8 Subtype/isolation & purification , Influenza A virus/isolation & purification , Zoonoses/virology , Animals , Communicable Diseases, Emerging/transmission , Communicable Diseases, Emerging/virology , Dog Diseases/transmission , Dogs , Ferrets , Guinea Pigs , Humans , Influenza A Virus, H3N2 Subtype/classification , Influenza A Virus, H3N2 Subtype/genetics , Influenza A Virus, H3N8 Subtype/classification , Influenza A Virus, H3N8 Subtype/genetics , Influenza A virus/classification , Influenza A virus/genetics , Influenza, Human/transmission , Influenza, Human/virology , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Inbred DBA , United States , Zoonoses/transmission
10.
PLoS Comput Biol ; 17(12): e1009617, 2021 12.
Article in English | MEDLINE | ID: mdl-34962914

ABSTRACT

Respiratory syncytial virus (RSV) infection results in millions of hospitalizations and thousands of deaths each year. Variations in the adaptive and innate immune response appear to be associated with RSV severity. To investigate the host response to RSV infection in infants, we performed a systems-level study of RSV pathophysiology, incorporating high-throughput measurements of the peripheral innate and adaptive immune systems and the airway epithelium and microbiota. We implemented a novel multi-omic data integration method based on multilayered principal component analysis, penalized regression, and feature weight back-propagation, which enabled us to identify cellular pathways associated with RSV severity. In both airway and immune cells, we found an association between RSV severity and activation of pathways controlling Th17 and acute phase response signaling, as well as inhibition of B cell receptor signaling. Dysregulation of both the humoral and mucosal response to RSV may play a critical role in determining illness severity.


Subject(s)
Genomics/methods , Respiratory Syncytial Virus Infections , Humans , Immunity, Innate/genetics , Immunity, Innate/immunology , Infant , Machine Learning , Microbiota/immunology , Nasal Cavity/cytology , Nasal Cavity/immunology , Nasal Cavity/metabolism , RNA-Seq , Respiratory Syncytial Virus Infections/genetics , Respiratory Syncytial Virus Infections/immunology , Respiratory Syncytial Virus Infections/metabolism , Respiratory Syncytial Virus Infections/physiopathology , Severity of Illness Index
11.
J Infect Dis ; 223(9): 1650-1658, 2021 05 20.
Article in English | MEDLINE | ID: mdl-32926147

ABSTRACT

BACKGROUND: Respiratory syncytial virus (RSV) is a leading cause of infant respiratory disease. Infant airway microbiota has been associated with respiratory disease risk and severity. The extent to which interactions between RSV and microbiota occur in the airway, and their impact on respiratory disease susceptibility and severity, are unknown. METHODS: We carried out 16S rRNA microbiota profiling of infants in the first year of life from (1) a cross-sectional cohort of 89 RSV-infected infants sampled during illness and 102 matched healthy controls, and (2) a matched longitudinal cohort of 12 infants who developed RSV infection and 12 who did not, sampled before, during, and after infection. RESULTS: We identified 12 taxa significantly associated with RSV infection. All 12 taxa were differentially abundant during infection, with 8 associated with disease severity. Nasal microbiota composition was more discriminative of healthy vs infected than of disease severity. CONCLUSIONS: Our findings elucidate the chronology of nasal microbiota dysbiosis and suggest an altered developmental trajectory associated with RSV infection. Microbial temporal dynamics reveal indicators of disease risk, correlates of illness and severity, and impact of RSV infection on microbiota composition.


Subject(s)
Dysbiosis , Microbiota , Nose/microbiology , Respiratory Syncytial Virus Infections , Cross-Sectional Studies , Dysbiosis/etiology , Humans , Infant , RNA, Ribosomal, 16S/genetics , Respiratory Syncytial Virus Infections/complications , Respiratory Syncytial Virus, Human , Severity of Illness Index
12.
J Infect Dis ; 223(9): 1639-1649, 2021 05 20.
Article in English | MEDLINE | ID: mdl-32926149

ABSTRACT

BACKGROUND: Respiratory syncytial virus (RSV) is the leading cause of severe respiratory disease in infants. The causes and correlates of severe illness in the majority of infants are poorly defined. METHODS: We recruited a cohort of RSV-infected infants and simultaneously assayed the molecular status of their airways and the presence of airway microbiota. We used rigorous statistical approaches to identify gene expression patterns associated with disease severity and microbiota composition, separately and in combination. RESULTS: We measured comprehensive airway gene expression patterns in 106 infants with primary RSV infection. We identified an airway gene expression signature of severe illness dominated by excessive chemokine expression. We also found an association between Haemophilus influenzae, disease severity, and airway lymphocyte accumulation. Exploring the time of onset of clinical symptoms revealed acute activation of interferon signaling following RSV infection in infants with mild or moderate illness, which was absent in subjects with severe illness. CONCLUSIONS: Our data reveal that airway gene expression patterns distinguish mild/moderate from severe illness. Furthermore, our data identify biomarkers that may be therapeutic targets or useful for measuring efficacy of intervention responses.


Subject(s)
Microbiota , Respiratory Syncytial Virus Infections , Respiratory System/metabolism , Transcriptome , Humans , Infant , Respiratory Syncytial Virus Infections/genetics , Respiratory Syncytial Virus, Human , Respiratory System/virology , Severity of Illness Index
13.
J Virol ; 94(4)2020 01 31.
Article in English | MEDLINE | ID: mdl-31748399

ABSTRACT

Influenza A virus (IAV) causes significant morbidity and mortality, despite the availability of viral vaccines. The efficacy of live attenuated influenza vaccines (LAIVs) has been especially poor in recent years. One potential reason is that the master donor virus (MDV), on which all LAIVs are based, contains either the internal genes of the 1960 A/Ann Arbor/6/60 or the 1957 A/Leningrad/17/57 H2N2 viruses (i.e., they diverge considerably from currently circulating strains). We previously showed that introduction of the temperature-sensitive (ts) residue signature of the AA/60 MDV into a 2009 pandemic A/California/04/09 H1N1 virus (Cal/09) results in only 10-fold in vivo attenuation in mice. We have previously shown that the ts residue signature of the Russian A/Leningrad/17/57 H2N2 LAIV (Len LAIV) more robustly attenuates the prototypical A/Puerto Rico/8/1934 (PR8) H1N1 virus. In this work, we therefore introduced the ts signature from Len LAIV into Cal/09. This new Cal/09 LAIV is ts in vitro, highly attenuated (att) in mice, and protects from a lethal homologous challenge. In addition, when our Cal/09 LAIV with PR8 hemagglutinin and neuraminidase was used to vaccinate mice, it provided enhanced protection against a wild-type Cal/09 challenge relative to a PR8 LAIV with the same attenuating mutations. These findings suggest it may be possible to improve the efficacy of LAIVs by better matching the sequence of the MDV to currently circulating strains.IMPORTANCE Seasonal influenza infection remains a major cause of disease and death, underscoring the need for improved vaccines. Among current influenza vaccines, the live attenuated influenza vaccine (LAIV) is unique in its ability to elicit T-cell immunity to the conserved internal proteins of the virus. Despite this, LAIV has shown limited efficacy in recent years. One possible reason is that the conserved, internal genes of all current LAIVs derive from virus strains that were isolated between 1957 and 1960 and that, as a result, do not resemble currently circulating influenza viruses. We have therefore developed and tested a new LAIV, based on a currently circulating pandemic strain of influenza. Our results show that this new LAIV elicits improved protective immunity compared to a more conventional LAIV.


Subject(s)
Influenza A virus/genetics , Influenza Vaccines/genetics , Influenza, Human/genetics , Animals , Antibodies, Neutralizing , Antibodies, Viral/immunology , Dogs , Female , HEK293 Cells , Humans , Immunogenicity, Vaccine/immunology , Influenza A Virus, H1N1 Subtype/genetics , Influenza A Virus, H1N1 Subtype/immunology , Influenza A Virus, H2N2 Subtype/genetics , Influenza A Virus, H2N2 Subtype/immunology , Influenza A virus/immunology , Influenza Vaccines/immunology , Influenza, Human/immunology , Influenza, Human/virology , Madin Darby Canine Kidney Cells , Mice , Mice, Inbred C57BL , Vaccines, Attenuated/immunology
14.
BMC Bioinformatics ; 21(1): 256, 2020 Jun 19.
Article in English | MEDLINE | ID: mdl-32560624

ABSTRACT

BACKGROUND: In 2009, a novel influenza vaccine was distributed worldwide to combat the H1N1 influenza "swine flu" pandemic. However, antibodies induced by the vaccine display differences in their specificity and cross-reactivity dependent on pre-existing immunity. Here, we present a computational model that can capture the effect of pre-existing immunity on influenza vaccine responses. The model predicts the region of the virus hemagglutinin (HA) protein targeted by antibodies after vaccination as well as the level of cross-reactivity induced by the vaccine. We tested our model by simulating a scenario similar to the 2009 pandemic vaccine and compared the results to antibody binding data obtained from human subjects vaccinated with the monovalent 2009 H1N1 influenza vaccine. RESULTS: We found that both specificity and cross-reactivity of the antibodies induced by the 2009 H1N1 influenza HA protein were affected by the viral strain the individual was originally exposed. Specifically, the level of antigenic relatedness between the original exposure HA antigen and the 2009 HA protein affected antigenic-site immunodominance. Moreover, antibody cross-reactivity was increased when the individual's pre-existing immunity was specific to an HA protein antigenically distinct from the 2009 pandemic strain. Comparison of simulation data with antibody binding data from human serum samples demonstrated qualitative and quantitative similarities between the model and real-life immune responses to the 2009 vaccine. CONCLUSION: We provide a novel method to evaluate expected outcomes in antibody specificity and cross-reactivity after influenza vaccination in individuals with different influenza HA antigen exposure histories. The model produced similar outcomes as what has been previously reported in humans after receiving the 2009 influenza pandemic vaccine. Our results suggest that differences in cross-reactivity after influenza vaccination should be expected in individuals with different exposure histories.


Subject(s)
Antibodies, Viral/immunology , Hemagglutinin Glycoproteins, Influenza Virus/immunology , Influenza A Virus, H1N1 Subtype/immunology , Influenza Vaccines/immunology , Models, Immunological , Amino Acid Sequence , Antibodies, Viral/blood , Antigens, Viral/chemistry , Antigens, Viral/immunology , Computer Simulation , Cross Reactions , Hemagglutinin Glycoproteins, Influenza Virus/chemistry , Humans
15.
Clin Infect Dis ; 71(6): 1447-1453, 2020 09 12.
Article in English | MEDLINE | ID: mdl-31598646

ABSTRACT

BACKGROUND: The H3N2 component of egg-based 2017-2018 influenza vaccines possessed an adaptive substitution that alters antigenicity. Several influenza vaccines include antigens that are produced through alternative systems, but a systematic comparison of different vaccines used during the 2017-2018 season has not been completed. METHODS: We compared antibody responses in humans vaccinated with Fluzone (egg-based, n = 23), Fluzone High-Dose (egg-based, n = 16), Flublok (recombinant protein-based, n = 23), or Flucelvax (cell-based, n = 23) during the 2017-2018 season. We completed neutralization assays using an egg-adapted H3N2 virus, a cell-based H3N2 virus, wild-type 3c2.A and 3c2.A2 H3N2 viruses, and the H1N1 vaccine strain. We also performed enzyme-linked immunosorbent assays using a recombinant wild-type 3c2.A hemagglutinin. Antibody responses were compared in adjusted analysis. RESULTS: Postvaccination neutralizing antibody titers to 3c2.A and 3c2.A2 were higher in Flublok recipients compared with Flucelvax or Fluzone recipients (P < .01). Postvaccination titers to 3c2.A and 3c2.A2 were similar in Flublok and Fluzone High-Dose recipients, though seroconversion rates trended higher in Flublok recipients. Postvaccination titers in Flucelvax recipients were low to all H3N2 viruses tested, including the cell-based H3N2 strain. Postvaccination neutralizing antibody titers to H1N1 were similar among the different vaccine groups. CONCLUSIONS: These data suggest that influenza vaccine antigen match and dose are both important for eliciting optimal H3N2 antibody responses in humans. Future studies should be designed to determine if our findings directly impact vaccine effectiveness. CLINICAL TRIALS REGISTRATION: NCT03068949.


Subject(s)
Influenza A Virus, H1N1 Subtype , Influenza Vaccines , Influenza, Human , Antibodies, Viral , Antibody Formation , Antigens, Viral , Hemagglutination Inhibition Tests , Hemagglutinin Glycoproteins, Influenza Virus , Humans , Influenza A Virus, H3N2 Subtype , Influenza, Human/prevention & control , Recombinant Proteins , Seasons
16.
Anal Chem ; 92(18): 12322-12329, 2020 09 15.
Article in English | MEDLINE | ID: mdl-32829631

ABSTRACT

Solid-phase extraction (SPE) is a general preconcentration method for sample preparation that can be performed on a variety of specimens. The miniaturization of SPE within a 3D printed microfluidic device further allows for fast and simple extraction of analytes while also enabling integration of SPE with other sample preparation and separation methods. Here, we present the development and application of a reversed-phase lauryl methacrylate-based monolith, formed in 3D printed microfluidic devices, which can selectively retain peptides and proteins. The effectiveness of these SPE monoliths and 3D printed microfluidic devices was tested using a panel of nine preterm birth biomarkers of varying hydrophobicities and ranging in mass from 2 to 470 kDa. The biomarkers were selectively retained, fluorescently labeled, and eluted separately from the excess fluorescent label in 3D printed microfluidic systems. These are the first results demonstrating microfluidic analysis processes on a complete panel of preterm birth biomarkers, an important step toward developing a miniaturized, fully integrated analysis system.


Subject(s)
Fluorescence , Lab-On-A-Chip Devices , Premature Birth/diagnosis , Printing, Three-Dimensional , Solid Phase Extraction , Biomarkers/analysis , Fluorescent Dyes/chemistry , Humans
17.
J Virol ; 93(21)2019 11 01.
Article in English | MEDLINE | ID: mdl-31434731

ABSTRACT

We describe a novel function for the interferon (IFN)-induced protein 44-like (IFI44L) gene in negatively modulating innate immune responses induced after virus infections. Furthermore, we show that decreasing IFI44L expression impairs virus production and that IFI44L expression negatively modulates the antiviral state induced by an analog of double-stranded RNA (dsRNA) or by IFN treatment. The mechanism likely involves the interaction of IFI44L with cellular FK506-binding protein 5 (FKBP5), which in turn interacts with kinases essential for type I and III IFN responses, such as inhibitor of nuclear factor kappa B (IκB) kinase alpha (IKKα), IKKß, and IKKε. Consequently, binding of IFI44L to FKBP5 decreased interferon regulatory factor 3 (IRF-3)-mediated and nuclear factor kappa-B (NF-κB) inhibitor (IκBα)-mediated phosphorylation by IKKε and IKKß, respectively. According to these results, IFI44L is a good target for treatment of diseases associated with excessive IFN levels and/or proinflammatory responses and for reduction of viral replication.IMPORTANCE Excessive innate immune responses can be deleterious for the host, and therefore, negative feedback is needed. Here, we describe a completely novel function for IFI44L in negatively modulating innate immune responses induced after virus infections. In addition, we show that decreasing IFI44L expression impairs virus production and that IFI44L expression negatively modulates the antiviral state induced by an analog of dsRNA or by IFN treatment. IFI44L binds to the cellular protein FKBP5, which in turn interacts with kinases essential for type I and III IFN induction and signaling, such as the kinases IKKα, IKKß, and IKKε. IFI44L binding to FKBP5 decreased the phosphorylation of IRF-3 and IκBα mediated by IKKε and IKKß, respectively, providing an explanation for the function of IFI44L in negatively modulating IFN responses. Therefore, IFI44L is a candidate target for reducing virus replication.


Subject(s)
I-kappa B Kinase/metabolism , Immunity, Innate/immunology , Interferons/pharmacology , Tacrolimus Binding Proteins/metabolism , Tumor Suppressor Proteins/metabolism , Virus Replication , Amino Acid Sequence , Antiviral Agents/pharmacology , Feedback, Physiological , Humans , I-kappa B Kinase/genetics , Immunity, Innate/drug effects , Influenza, Human/immunology , Influenza, Human/pathology , Influenza, Human/virology , NF-kappa B , Neoplasms/immunology , Neoplasms/pathology , Neoplasms/virology , Orthomyxoviridae/drug effects , Orthomyxoviridae/immunology , Phosphorylation , Sequence Homology , Signal Transduction , Tacrolimus Binding Proteins/genetics , Tumor Cells, Cultured , Tumor Suppressor Proteins/genetics
18.
J Virol ; 93(21)2019 11 01.
Article in English | MEDLINE | ID: mdl-31434733

ABSTRACT

Vaccination is the best measure of protection against influenza virus infection. Vaccine-induced antibody responses target mainly the hemagglutinin (HA) surface glycoprotein, composed of the head and the stalk domains. Recently two novel vaccine platforms have been developed for seasonal influenza vaccination: a recombinant HA vaccine produced in insect cells (Flublok) and Flucelvax, prepared from virions produced in mammalian cells. In order to compare the fine specificity of the antibodies induced by these two novel vaccine platforms, we characterized 42 Flublok-induced monoclonal antibodies (MAbs) and 38 Flucelvax-induced MAbs for avidity, cross-reactivity, and any selectivity toward the head versus the stalk domain. These studies revealed that Flublok induced a greater proportion of MAbs targeting epitopes near the receptor-binding domain on HA head (hemagglutinin inhibition-positive MAbs) than Flucelvax, while the two vaccines induced similar low frequencies of stalk-reactive MAbs. Finally, mice immunized with Flublok and Flucelvax also induced similar frequencies of stalk-reactive antibody-secreting cells, showing that HA head immunodominance is independent of immune memory bias. Collectively, our results suggest that these vaccine formulations are similarly immunogenic but differ in the preferences of the elicited antibodies toward the receptor-binding domain on the HA head.IMPORTANCE There are ongoing efforts to increase the efficacy of influenza vaccines and to promote production strategies that can rapidly respond to newly emerging viruses. It is important to understand if current alternative seasonal vaccines, such as Flublok and Flucelvax, that use alternate production strategies can induce protective influenza-specific antibodies and to evaluate what type of epitopes are targeted by distinct vaccine formulations.


Subject(s)
Antibodies, Monoclonal/immunology , Antibodies, Viral/immunology , Hemagglutinin Glycoproteins, Influenza Virus/immunology , Influenza A virus/immunology , Influenza Vaccines/immunology , Influenza, Human/immunology , Orthomyxoviridae Infections/immunology , Vaccines, Inactivated/immunology , Adolescent , Adult , Amino Acid Sequence , Animals , Antibodies, Neutralizing/immunology , Cohort Studies , Female , Hemagglutination Inhibition Tests , Humans , Influenza Vaccines/administration & dosage , Influenza, Human/prevention & control , Influenza, Human/virology , Male , Mice, Inbred BALB C , Middle Aged , Orthomyxoviridae Infections/prevention & control , Orthomyxoviridae Infections/virology , Sequence Homology , Vaccination , Vaccines, Inactivated/administration & dosage , Young Adult
19.
J Virol ; 93(8)2019 04 15.
Article in English | MEDLINE | ID: mdl-30728266

ABSTRACT

Memory B cells (MBCs) are key determinants of the B cell response to influenza virus infection and vaccination, but the effect of different forms of influenza antigen exposure on MBC populations has received little attention. We analyzed peripheral blood mononuclear cells and plasma collected following human H3N2 influenza infection to investigate the relationship between hemagglutinin-specific antibody production and changes in the size and character of hemagglutinin-reactive MBC populations. Infection produced increased concentrations of plasma IgG reactive to the H3 head of the infecting virus, to the conserved stalk, and to a broad chronological range of H3s consistent with original antigenic sin responses. H3-reactive IgG MBC expansion after infection included reactivity to head and stalk domains. Notably, expansion of H3 head-reactive MBC populations was particularly broad and reflected original antigenic sin patterns of IgG production. Findings also suggest that early-life H3N2 infection "imprints" for strong H3 stalk-specific MBC expansion. Despite the breadth of MBC expansion, the MBC response included an increase in affinity for the H3 head of the infecting virus. Overall, our findings indicate that H3-reactive MBC expansion following H3N2 infection is consistent with maintenance of response patterns established early in life, but nevertheless includes MBC adaptation to the infecting virus.IMPORTANCE Rapid and vigorous virus-specific antibody responses to influenza virus infection and vaccination result from activation of preexisting virus-specific memory B cells (MBCs). Understanding the effects of different forms of influenza virus exposure on MBC populations is therefore an important guide to the development of effective immunization strategies. We demonstrate that exposure to the influenza hemagglutinin via natural infection enhances broad protection through expansion of hemagglutinin-reactive MBC populations that recognize head and stalk regions of the molecule. Notably, we show that hemagglutinin-reactive MBC expansion reflects imprinting by early-life infection and that this might apply to stalk-reactive, as well as to head-reactive, MBCs. Our findings provide experimental support for the role of MBCs in maintaining imprinting effects and suggest a mechanism by which imprinting might confer heterosubtypic protection against avian influenza viruses. It will be important to compare our findings to the situation after influenza vaccination.


Subject(s)
B-Lymphocytes/immunology , Hemagglutinin Glycoproteins, Influenza Virus/immunology , Immunologic Memory , Influenza A Virus, H3N2 Subtype/immunology , Influenza, Human/immunology , Seasons , Antibodies, Viral/immunology , Humans , Immunoglobulin G/immunology , Influenza A Virus, H1N1 Subtype
20.
BMC Bioinformatics ; 20(1): 185, 2019 Apr 15.
Article in English | MEDLINE | ID: mdl-30987598

ABSTRACT

BACKGROUND: For many practical hypothesis testing (H-T) applications, the data are correlated and/or with heterogeneous variance structure. The regression t-test for weighted linear mixed-effects regression (LMER) is a legitimate choice because it accounts for complex covariance structure; however, high computational costs and occasional convergence issues make it impractical for analyzing high-throughput data. In this paper, we propose computationally efficient parametric and semiparametric tests based on a set of specialized matrix techniques dubbed as the PB-transformation. The PB-transformation has two advantages: 1. The PB-transformed data will have a scalar variance-covariance matrix. 2. The original H-T problem will be reduced to an equivalent one-sample H-T problem. The transformed problem can then be approached by either the one-sample Student's t-test or Wilcoxon signed rank test. RESULTS: In simulation studies, the proposed methods outperform commonly used alternative methods under both normal and double exponential distributions. In particular, the PB-transformed t-test produces notably better results than the weighted LMER test, especially in the high correlation case, using only a small fraction of computational cost (3 versus 933 s). We apply these two methods to a set of RNA-seq gene expression data collected in a breast cancer study. Pathway analyses show that the PB-transformed t-test reveals more biologically relevant findings in relation to breast cancer than the weighted LMER test. CONCLUSIONS: As fast and numerically stable replacements for the weighted LMER test, the PB-transformed tests are especially suitable for "messy" high-throughput data that include both independent and matched/repeated samples. By using our method, the practitioners no longer have to choose between using partial data (applying paired tests to only the matched samples) or ignoring the correlation in the data (applying two sample tests to data with some correlated samples). Our method is implemented as an R package 'PBtest' and is available at https://github.com/yunzhang813/PBtest-R-Package .


Subject(s)
Breast Neoplasms/genetics , Computer Simulation , Female , Gene Expression Profiling , Gene Expression Regulation, Neoplastic , Humans , Models, Genetic , ROC Curve , Regression Analysis
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