Publications by authors named "Shivaprakash Gangappa"

53 Publications

Immunogenicity of standard, high-dose, MF59-adjuvanted, and recombinant-HA seasonal influenza vaccination in older adults.

NPJ Vaccines 2021 Feb 16;6(1):25. Epub 2021 Feb 16.

HKU-Pasteur Research Pole, School of Public Health, The University of Hong Kong, Hong Kong SAR, China.

The vaccine efficacy of standard-dose seasonal inactivated influenza vaccines (S-IIV) can be improved by the use of vaccines with higher antigen content or adjuvants. We conducted a randomized controlled trial in older adults to compare cellular and antibody responses of S-IIV versus enhanced vaccines (eIIV): MF59-adjuvanted (A-eIIV), high-dose (H-eIIV), and recombinant-hemagglutinin (HA) (R-eIIV). All vaccines induced comparable H3-HA-specific IgG and elevated antibody-dependent cellular cytotoxicity (ADCC) activity at day 30 post vaccination. H3-HA-specific ADCC responses were greatest following H-eIIV. Only A-eIIV increased H3-HA-IgG avidity, HA-stalk IgG and ADCC activity. eIIVs also increased polyfunctional CD4+ and CD8+ T cell responses, while cellular immune responses were skewed toward single-cytokine-producing T cells among S-IIV subjects. Our study provides further immunological evidence for the preferential use of eIIVs in older adults as each vaccine platform had an advantage over the standard-dose vaccine in terms of NK cell activation, HA-stalk antibodies, and T cell responses.
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http://dx.doi.org/10.1038/s41541-021-00289-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7886864PMC
February 2021

Prevalent, protective, and convergent IgG recognition of SARS-CoV-2 non-RBD spike epitopes in COVID-19 convalescent plasma.

bioRxiv 2020 Dec 21. Epub 2020 Dec 21.

Although humoral immunity is essential for control of SARS-CoV-2, the molecular composition, binding epitopes and effector functions of the immunoglobulin G (IgG) antibodies that circulate in blood plasma following infection are unknown. Proteomic deconvolution of the circulating IgG repertoire (Ig-Seq ) to the spike ectodomain (S-ECD ) in four convalescent study subjects revealed that the plasma response is oligoclonal and directed predominantly (>80%) to S-ECD epitopes that lie outside the receptor binding domain (RBD). When comparing antibodies directed to either the RBD, the N-terminal domain (NTD) or the S2 subunit (S2) in one subject, just four IgG lineages (1 anti-S2, 2 anti-NTD and 1 anti-RBD) accounted for 93.5% of the repertoire. Although the anti-RBD and one of the anti-NTD antibodies were equally potently neutralizing , we nonetheless found that the anti-NTD antibody was sufficient for protection to lethal viral challenge, either alone or in combination as a cocktail where it dominated the effect of the other plasma antibodies. We identified protective plasma anti-NTD antibodies in 3/4 subjects analyzed and discovered a shared class of antibodies targeting the NTD that utilize unmutated or near-germline IGHV1-24, the most electronegative IGHV gene in the human genome. Structural analysis revealed that binding to NTD is dominated by interactions with the heavy chain, accounting for 89% of the entire interfacial area, with germline residues uniquely encoded by IGHV1-24 contributing 20% (149 Å ). Together with recent reports of germline IGHV1-24 antibodies isolated by B-cell cloning our data reveal a class of shared IgG antibodies that are readily observed in convalescent plasma and underscore the role of NTD-directed antibodies in protection against SARS-CoV-2 infection.
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http://dx.doi.org/10.1101/2020.12.20.423708DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7781304PMC
December 2020

A Dual-Functioning 5'-PPP-NS1shRNA that Activates a RIG-I Antiviral Pathway and Suppresses Influenza NS1.

Mol Ther Nucleic Acids 2020 Mar 31;19:1413-1422. Epub 2020 Jan 31.

Influenza Division, National Center for Immunization and Respiratory Diseases, Centers for Disease Control and Prevention, 1600 Clifton Road, Atlanta, GA 30329, USA. Electronic address:

Retinoic acid-inducible gene-I (RIG-I) is a cytosolic pathogen sensor that is crucial against a number of viral infections. Many viruses have evolved to inhibit pathogen sensors to suppress host innate immune responses. In the case of influenza, nonstructural protein 1 (NS1) suppresses RIG-I function, leading to viral replication, morbidity, and mortality. We show that silencing NS1 with in-vitro-transcribed 5'-triphosphate containing NS1 short hairpin RNA (shRNA) (5'-PPP-NS1shRNA), designed using the conserved region of a number of influenza viruses, not only prevented NS1 expression but also induced RIG-I activation and type I interferon (IFN) expression, resulting in an antiviral state leading to inhibition of influenza virus replication in vitro. In addition, administration of 5'-PPP-NS1shRNA in prophylactic and therapeutic settings resulted in significant inhibition of viral replication following viral challenge in vivo in mice with corresponding increases of RIG-I, IFN-β, and IFN-λ, as well as a decrease in NS1 expression.
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http://dx.doi.org/10.1016/j.omtn.2020.01.025DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7049568PMC
March 2020

Influenza virus NS1- C/EBPβ gene regulatory complex inhibits RIG-I transcription.

Antiviral Res 2020 04 21;176:104747. Epub 2020 Feb 21.

Virology Group, International Centre for Genetic Engineering & Biotechnology, New Delhi, 110067, India; School of Science, Tropical Medicine and Biology Multidisciplinary Plateform, Monash University Malaysia, 47500, Bandar Sunway, Selangor DE, Malaysia. Electronic address:

Influenza virus non-structural protein 1 (NS1) counteracts host antiviral innate immune responses by inhibiting Retinoic acid inducible gene-I (RIG-I) activation. However, whether NS1 also specifically regulates RIG-I transcription is unknown. Here, we identify a CCAAT/Enhancer Binding Protein beta (C/EBPβ) binding site in the RIG-I promoter as a repressor element, and show that NS1 promotes C/EBPβ phosphorylation and its recruitment to the RIG-I promoter as a C/EBPβ/NS1 complex. C/EBPβ overexpression and siRNA knockdown in human lung epithelial cells resulted in suppression and activation of RIG-I expression respectively, implying a negative regulatory role of C/EBPβ. Further, C/EBPβ phosphorylation, its interaction with NS1 and occupancy at the RIG-I promoter was associated with RIG-I transcriptional inhibition. These findings provide an important insight into the molecular mechanism by which influenza NS1 commandeers RIG-I transcriptional regulation and suppresses host antiviral responses.
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http://dx.doi.org/10.1016/j.antiviral.2020.104747DOI Listing
April 2020

Comparative Immunogenicity of Several Enhanced Influenza Vaccine Options for Older Adults: A Randomized, Controlled Trial.

Clin Infect Dis 2020 Oct;71(7):1704-1714

Influenza Division, Centers for Disease Control and Prevention, Atlanta, Georgia, USA.

Background: Enhanced influenza vaccines may improve protection for older adults, but comparative immunogenicity data are limited. Our objective was to examine immune responses to enhanced influenza vaccines, compared to standard-dose vaccines, in community-dwelling older adults.

Methods: Community-dwelling older adults aged 65-82 years in Hong Kong were randomly allocated (October 2017-January 2018) to receive 2017-2018 Northern hemisphere formulations of a standard-dose quadrivalent vaccine, MF59-adjuvanted trivalent vaccine, high-dose trivalent vaccine, or recombinant-hemagglutinin (rHA) quadrivalent vaccine. Sera collected from 200 recipients of each vaccine before and at 30-days postvaccination were assessed for antibodies to egg-propagated vaccine strains by hemagglutination inhibition (HAI) and to cell-propagated A/Hong Kong/4801/2014(H3N2) virus by microneutralization (MN). Influenza-specific CD4+ and CD8+ T cell responses were assessed in 20 participants per group.

Results: Mean fold rises (MFR) in HAI titers to egg-propagated A(H1N1) and A(H3N2) and the MFR in MN to cell-propagated A(H3N2) were statistically significantly higher in the enhanced vaccine groups, compared to the standard-dose vaccine. The MFR in MN to cell-propagated A(H3N2) was highest among rHA recipients (4.7), followed by high-dose (3.4) and MF59-adjuvanted (2.9) recipients, compared to standard-dose recipients (2.3). Similarly, the ratio of postvaccination MN titers among rHA recipients to cell-propagated A(H3N2) recipients was 2.57-fold higher than the standard-dose vaccine, which was statistically higher than the high-dose (1.33-fold) and MF59-adjuvanted (1.43-fold) recipient ratios. Enhanced vaccines also resulted in the boosting of T-cell responses.

Conclusions: In this head-to-head comparison, older adults receiving enhanced vaccines showed improved humoral and cell-mediated immune responses, compared to standard-dose vaccine recipients.

Clinical Trials Registration: NCT03330132.
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http://dx.doi.org/10.1093/cid/ciz1034DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7289658PMC
October 2020

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

J Infect Dis 2019 07;220(5):743-751

Influenza Division, National Center for Immunization and Respiratory Diseases, Atlanta, Georgia.

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.
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http://dx.doi.org/10.1093/infdis/jiz205DOI Listing
July 2019

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

Transpl Immunol 2019 04 18;53:51-60. Epub 2019 Jan 18.

Emory Transplant Center, Emory University School of Medicine, Atlanta, GA 30322, United States.

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.
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http://dx.doi.org/10.1016/j.trim.2019.01.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6471676PMC
April 2019

Longevity of adenovirus vector immunity in mice and its implications for vaccine efficacy.

Vaccine 2018 10 25;36(45):6744-6751. Epub 2018 Sep 25.

Department of Comparative Pathobiology, Purdue Institute for Immunology, Inflammation and Infectious Disease, and Purdue University Center for Cancer Research, College of Veterinary Medicine, Purdue University, West Lafayette, IN, USA. Electronic address:

There is a high incidence of adenovirus (AdV) infection in humans due to the presence of more than 60 types of human adenoviruses (HAdVs). The majority of individuals are exposed to one or more HAdV types early in their lives, leading to the development of AdV type-specific neutralizing antibodies. Similarly, immunization or gene therapy with AdV vectors leads to immune responses to the AdV vector. This 'vector immunity' is a concern for AdV vector-based applications for vaccines or gene therapy, especially when the repeated administration of a vector is required. The objective of this investigation was to establish whether AdV neutralizing antibody titers decline sufficiently in a year to permit annual vaccination with the same AdV vector. Naïve or human adenoviral vector group C, type 5 (HAdV-C5)-primed mice were mock-inoculated (with PBS) or inoculated i.m. with 10 PFU of either HAd-GFP [HAdV-C5 vector expressing the green fluorescent protein (GFP)] to mimic the conditions for the first inoculation with an AdV vector-based vaccine. At 1, 3, 6, and 10 months post-HAd-GFP inoculation, naïve- or HAdV-primed animals were vaccinated i.m. with 10 PFU of HAd-H5HA [HAdV-C5 vector expressing hemagglutinin (HA) of H5N1 influenza virus]. There was a significant continual decrease in vector immunity titers with time, thereby leading to significant continual increases in the levels of HA-specific humoral and cell-mediated immune responses. In addition, significant improvement in protection efficacy against challenge with an antigenically heterologous H5N1 virus was observed in HAdV-primed animals at 6 months and onwards. These results indicate that the annual immunization with the same AdV vector may be effective due to a significant decline in vector immunity.
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http://dx.doi.org/10.1016/j.vaccine.2018.09.031DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6200586PMC
October 2018

A Bovine Adenoviral Vector-Based H5N1 Influenza -Vaccine Provides Enhanced Immunogenicity and Protection at a Significantly Low Dose.

Mol Ther Methods Clin Dev 2018 Sep 17;10:210-222. Epub 2018 Jul 17.

Department of Comparative Pathobiology, Purdue Institute for Inflammation, Immunology, and Infectious Disease, College of Veterinary Medicine, Purdue University, West Lafayette, IN, USA.

Several human and nonhuman adenovirus (AdV) vectors including bovine AdV type 3 (BAdV-3) were developed as gene delivery vectors to supplement and/or elude human AdV (HAdV)-specific neutralizing antibodies (vector immunity). Here we evaluated the vaccine immunogenicity and efficacy of BAdV-3 vector (BAd-H5HA) expressing hemagglutinin (HA) of a H5N1 influenza virus in a dose escalation study in mice with the intranasal (IN) or intramuscular (IM) route of inoculation in comparison with the HAdV type C5 (HAdV-C5) vector (HAd-H5HA) expressing HA of a H5N1 influenza virus. Dose-related increases in the immune responses were clearly noticeable. A single IM inoculation with BAd-H5HA resulted in enhanced cellular immune responses compared with that of HAd-H5HA and conferred complete protection following challenge with a heterologous H5N1 virus at the dose of 3 × 10 plaque-forming units (PFUs), whereas a significant amount of influenza virus was detected in the lungs of mice immunized with 1 × 10 PFUs of HAd-H5HA. Similarly, compared with that of HAd-H5HA, a single IN inoculation with BAd-H5HA produced significantly enhanced humoral (HA-specific immunoglobulin [IgG] and its subclasses, as well as HA-specific IgA) and cellular immune responses, and conferred complete protection following challenge with a heterologous H5N1 virus. Complete protection with BAd-H5HA was observed with the lowest vaccine dose (1 × 10 PFUs), but similar protection with HAd-H5HA was observed at the highest vaccine dose (1 × 10 PFUs). These results suggest that at least 30-fold dose sparing can be achieved with BAd-H5HA vector compared with HAd-H5HA vaccine vector.
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http://dx.doi.org/10.1016/j.omtm.2018.07.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6082999PMC
September 2018

Nasal delivery of H5N1 avian influenza vaccine formulated with GenJet™ or in vivo-jetPEI induces enhanced serological, cellular and protective immune responses.

Drug Deliv 2018 Nov;25(1):773-779

a Immunology and Pathogenesis Branch , National Center for Immunization and Respiratory Diseases , Atlanta , GA , USA.

Avian influenza virus infection is a serious public health threat and preventive vaccination is the most cost-effective public health intervention strategy. Unfortunately, currently available unadjuvanted avian influenza vaccines are poorly immunogenic and alternative vaccine formulations and delivery strategies are in urgent need to reduce the high risk of avian influenza pandemics. Cationic polymers have been widely used as vectors for gene delivery in vitro and in vivo. In this study, we formulated H5N1 influenza vaccines with GenJet™ or in vivo-jetPEI, and showed that these formulations significantly enhanced the immunogenicity of H5N1 vaccines and conferred protective immunity in a mouse model. Detailed analyses of adaptive immune responses revealed that both formulations induced mixed T1/T2 antigen-specific CD4 T-cell responses, antigen-specific cytotoxic CD8 T-cell and memory B-cell responses. Our findings suggest that cationic polymers merit future development as potential adjuvants for mucosal delivery of poorly immunogenic vaccines.
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http://dx.doi.org/10.1080/10717544.2018.1450909DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6058713PMC
November 2018

Adenovirus vector-based multi-epitope vaccine provides partial protection against H5, H7, and H9 avian influenza viruses.

PLoS One 2017 12;12(10):e0186244. Epub 2017 Oct 12.

Department of Comparative Pathobiology and Purdue Institute for Inflammation, Immunology, and Infectious Disease, Purdue University, West Lafayette, IN, United States of America.

The emergence of H5, H7, and H9 avian influenza virus subtypes in humans reveals their pandemic potential. Although human-to-human transmission has been limited, the genetic reassortment of the avian and human/porcine influenza viruses or mutations in some of the genes resulting in virus replication in the upper respiratory tract of humans could generate novel pandemic influenza viruses. Current vaccines do not provide cross protection against antigenically distinct strains of the H5, H7, and H9 influenza viruses. Therefore, newer vaccine approaches are needed to overcome these potential threats. We developed an egg-independent, adenovirus vector-based, multi-epitope (ME) vaccine approach using the relatively conserved immunogenic domains of the H5N1 influenza virus [M2 ectodomain (M2e), hemagglutinin (HA) fusion domain (HFD), T-cell epitope of nucleoprotein (TNP). and HA α-helix domain (HαD)]. Our ME vaccine induced humoral and cell-mediated immune responses and caused a significant reduction in the viral loads in the lungs of vaccinated mice that were challenged with antigenically distinct H5, H7, or H9 avian influenza viruses. These results suggest that our ME vaccine approach provided broad protection against the avian influenza viruses. Further improvement of this vaccine will lead to a pre-pandemic vaccine that may lower morbidity, hinder transmission, and prevent mortality in a pandemic situation before a strain-matched vaccine becomes available.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0186244PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5638338PMC
November 2017

An Adjuvanted A(H5N1) Subvirion Vaccine Elicits Virus-Specific Antibody Response and Improves Protection Against Lethal Influenza Viral Challenge in Mouse Model of Protein Energy Malnutrition.

J Infect Dis 2017 09;216(suppl_4):S560-S565

Influenza Division, National Center for Immunization and Respiratory Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia.

Background: Protein energy malnutrition (PEM) increases susceptibility to infectious diseases, including influenza infection, but no studies have addressed the potential influences of PEM on the immunogenicity and protective efficacy of avian influenza A(H5N1) vaccine.

Methods: We investigated the role of PEM on vaccine-mediated protection after a lethal challenge with recombinant A(H5N1) virus using isocaloric diets providing either adequate protein (AP; 18% protein) or very low protein (VLP; 2% protein) in an established murine model of influenza vaccination.

Results: We demonstrated that mice maintained on a VLP diet succumb to lethal challenge at greater rates than mice maintained on an AP diet, despite comparable immunization regimens. Importantly, there was no virus-induced mortality in both VLP and AP groups of mice when either group was immunized with adjuvanted low-dose A(H5N1) subvirion vaccine.

Conclusions: Our results suggest that adjuvanted vaccination in populations where PEM is endemic may be one strategy to boost vaccination-promoted immunity and improve outcomes associated with highly pathogenic A(H5N1).
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http://dx.doi.org/10.1093/infdis/jiw585DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5737928PMC
September 2017

Nasal delivery of Protollin-adjuvanted H5N1 vaccine induces enhanced systemic as well as mucosal immunity in mice.

Vaccine 2017 06 9;35(25):3318-3325. Epub 2017 May 9.

Immunology and Pathogenesis Branch, Influenza Division, National Center for Immunization and Respiratory Diseases, Centers for Disease Control and Prevention, Atlanta, GA 30329, USA. Electronic address:

Sporadic, yet frequent human infections with avian H5N1 influenza A viruses continue to pose a potential pandemic threat. Poor immunogenicity of unadjuvanted H5N1 vaccines warrants developing novel adjuvants and formulations as well as alternate delivery systems to improve their immunogenicity and efficacy. Here, we show that Protollin, a nasal adjuvant composed of Neisseria meningitides outer membrane proteins non-covalently linked to Shigella flexneri 2a lipopolysaccharide, is a potent nasal adjuvant for an inactivated split virion H5N1 clade 1 A/Viet Nam1203/2004 (A/VN/1203/04) vaccine in a mouse model. Protollin-adjuvanted vaccines elicited enhanced serum protective hemagglutination inhibition titers, mucosal IgA responses, and H5N1-specific cell-mediated immunity that resulted in complete protection against a lethal challenge with a homologous virus as well as a heterologous clade 2 virus A/Indonesia/05/2005 (A/IN/05/05). Detailed analysis of adaptive immunity revealed that Protollin increased the frequency of lymphoid- as well as local tissue-resident antibody-secreting cells, local germinal center reaction of B cells, broad-spectrum of CD4 T cell response. Our findings suggest that nasal delivery of H5N1 vaccine with Protollin adjuvant can overcome the poor immunogenicity of H5N1 vaccines, induce both cellular and humoral immune responses, enhance protection against challenge with clade 1 and clade 2 H5N1 viruses and achieve significant antigen dose-sparing.
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http://dx.doi.org/10.1016/j.vaccine.2017.05.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7115484PMC
June 2017

Influenza virus exploits tunneling nanotubes for cell-to-cell spread.

Sci Rep 2017 01 6;7:40360. Epub 2017 Jan 6.

Immunology and Pathogenesis Branch, Influenza Division, Centers for Disease Control and Prevention, 1600 Clifton Road, Atlanta, GA 30329-4027, USA.

Tunneling nanotubes (TNTs) represent a novel route of intercellular communication. While previous work has shown that TNTs facilitate the exchange of viral or prion proteins from infected to naïve cells, it is not clear whether the viral genome is also transferred via this mechanism and further, whether transfer via this route can result in productive replication of the infectious agents in the recipient cell. Here we present evidence that lung epithelial cells are connected by TNTs, and in spite of the presence of neutralizing antibodies and an antiviral agent, Oseltamivir, influenza virus can exploit these networks to transfer viral proteins and genome from the infected to naïve cell, resulting in productive viral replication in the naïve cells. These observations indicate that influenza viruses can spread using these intercellular networks that connect epithelial cells, evading immune and antiviral defenses and provide an explanation for the incidence of influenza infections even in influenza-immune individuals and vaccine failures.
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http://dx.doi.org/10.1038/srep40360DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5216422PMC
January 2017

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

Viral Immunol 2016 10 22;29(8):487-493. Epub 2016 Jul 22.

1 Immunology and Pathogenesis Branch, Influenza Division, National Center for Immunization and Respiratory Diseases, Centers for Disease Control and Prevention , Atlanta, Georgia .

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.
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http://dx.doi.org/10.1089/vim.2016.0056DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5749914PMC
October 2016

An oil-in-water nanoemulsion enhances immunogenicity of H5N1 vaccine in mice.

Nanomedicine 2016 10 23;12(7):1909-1917. Epub 2016 Apr 23.

Immunology and Pathogenesis Branch, Influenza Division, National Center for Immunization and Respiratory Diseases, Centers for Disease Control and Prevention, Atlanta, GA, USA.

To enhance the immunogenicity of the Influenza H5N1 vaccine, we developed an oil-in-water nanoemulsion (NE) adjuvant. NE displayed good temperature stability and maintained particle size. More importantly, it significantly enhanced IL-6 and MCP-1 production to recruit innate cells, including neutrophils, monocytes/macrophages and dendritic cells to the local environment. Furthermore, NE enhanced dendritic cell function to induce robust antigen-specific T and B cell immune responses. NE-adjuvanted H5N1 vaccine not only elicited significantly higher and long-lasting antibody responses, but also conferred enhanced protection against homologous clade 1 as well as heterologous clade 2 H5N1 virus challenge in young as well as in aged mice. The pre-existing immunity to seasonal influenza did not affect the immunogenicity of NE-adjuvanted H5N1 vaccine.
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http://dx.doi.org/10.1016/j.nano.2016.04.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5240149PMC
October 2016

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

Cell Immunol 2016 Jun-Jul;304-305:55-8. Epub 2016 Apr 9.

Immunology and Pathogenesis Branch, Influenza Division, National Center for Immunization and Respiratory Diseases, Centers for Disease Control and Prevention, 1600 Clifton Road, Atlanta, GA, USA.

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.
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http://dx.doi.org/10.1016/j.cellimm.2016.04.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5240151PMC
February 2017

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

PLoS One 2016 24;11(2):e0149864. Epub 2016 Feb 24.

Influenza Division, Centers for Disease Control and Prevention, Atlanta, Georgia, United States of America.

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.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0149864PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4766197PMC
July 2016

A highly immunogenic vaccine against A/H7N9 influenza virus.

Vaccine 2016 Feb 4;34(6):744-9. Epub 2016 Jan 4.

Immunology and Pathogenesis Branch, Influenza Division, National Center for Immunization and Respiratory Diseases, Centers for Disease Control and Prevention, 1600 Clifton Road, Atlanta, GA, USA. Electronic address:

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.
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http://dx.doi.org/10.1016/j.vaccine.2015.12.062DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4735641PMC
February 2016

Increased Dietary Salt Intake Does Not Influence Influenza A Virus-Induced Disease Severity in Mice.

Viral Immunol 2015 Nov 18;28(9):532-7. Epub 2015 Aug 18.

1 Immunology and Pathogenesis Branch, Influenza Division, National Center for Immunization and Respiratory Diseases, Centers for Disease Control and Prevention , Atlanta, Georgia .

Influenza viruses are pathogens of significant public health importance. The influence of nutritional status on severity of disease has become increasingly recognized. In particular, high dietary salt intake has been linked to cardiovascular disease, but the effects on infectious diseases have not been studied. This study investigated the impact on influenza-induced morbidity and mortality in mice fed isocaloric diets containing 10-fold increments of sodium by altering the salt levels. Following infection, despite higher levels of IFN-gamma cytokine in the lung as well as virus-neutralizing antibody in the serum of mice fed the lowest salt level, the amounts of dietary salt intake had no substantial impact on the disease severity or the ability to respond immunologically to the infection.
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http://dx.doi.org/10.1089/vim.2015.0037DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5737935PMC
November 2015

A Newly Emerged Swine-Origin Influenza A(H3N2) Variant Dampens Host Antiviral Immunity but Induces Potent Inflammasome Activation.

J Infect Dis 2015 Dec 11;212(12):1923-9. Epub 2015 Jun 11.

Immunology and Pathogenesis Branch.

We compared the innate immune response to a newly emerged swine-origin influenza A(H3N2) variant containing the M gene from 2009 pandemic influenza A(H1N1), termed "A(H3N2)vpM," to the immune responses to the 2010 swine-origin influenza A(H3N2) variant and seasonal influenza A(H3N2). Our results demonstrated that A(H3N2)vpM-induced myeloid dendritic cells secreted significantly lower levels of type I interferon (IFN) but produced significantly higher levels of proinflammatory cytokines and induced potent inflammasome activation. The reduction in antiviral immunity with increased inflammatory responses upon A(H3N2)vpM infection suggest that these viruses have the potential for increased disease severity in susceptible hosts.
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http://dx.doi.org/10.1093/infdis/jiv330DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5240150PMC
December 2015

NLRC5 interacts with RIG-I to induce a robust antiviral response against influenza virus infection.

Eur J Immunol 2015 Mar 23;45(3):758-72. Epub 2014 Dec 23.

Influenza Division, National Center for Immunization and Respiratory Diseases, Centers for Disease Control and Prevention, Atlanta, GA, USA.

The NLR protein, NLRC5 is an important regulator of MHC class I gene expression, however, the role of NLRC5 in other innate immune responses is less well defined. In the present study, we report that NLRC5 binds RIG-I and that this interaction is critical for robust antiviral responses against influenza virus. Overexpression of NLRC5 in the human lung epithelial cell line, A549, and normal human bronchial epithelial cells resulted in impaired replication of influenza virus A/Puerto Rico/8/34 virus (PR8) and enhanced IFN-β expression. Influenza virus leads to induction of IFN-β that drives RIG-I and NLRC5 expression in host cells. Our results suggest that NLRC5 extends and stabilizes influenza virus induced RIG-I expression and delays expression of the viral inhibitor protein NS1. We show that NS1 binds to NLRC5 to suppress its function. Interaction domain mapping revealed that NLRC5 interacts with RIG-I via its N-terminal death domain and that NLRC5 enhanced antiviral activity in an leucine-rich repeat domain independent manner. Taken together, our findings identify a novel role for NLRC5 in RIG-I-mediated antiviral host responses against influenza virus infection, distinguished from the role of NLRC5 in MHC class I gene regulation.
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http://dx.doi.org/10.1002/eji.201344412DOI Listing
March 2015

Activation of the RIG-I pathway during influenza vaccination enhances the germinal center reaction, promotes T follicular helper cell induction, and provides a dose-sparing effect and protective immunity.

J Virol 2014 Dec 24;88(24):13990-4001. Epub 2014 Sep 24.

Department of Pediatrics and Emory Vaccine Center, Emory University School of Medicine, Atlanta, Georgia, USA ICGEB-Emory Vaccine Center, International Center for Genetic Engineering and Biotechnology, New Delhi, India

Unlabelled: Pattern recognition receptors (PRR) sense certain molecular patterns uniquely expressed by pathogens. Retinoic-acid-inducible gene I (RIG-I) is a cytosolic PRR that senses viral nucleic acids and induces innate immune activation and secretion of type I interferons (IFNs). Here, using influenza vaccine antigens, we investigated the consequences of activating the RIG-I pathway for antigen-specific adaptive immune responses. We found that mice immunized with influenza vaccine antigens coadministered with 5'ppp-double-stranded RNA (dsRNA), a RIG-I ligand, developed robust levels of hemagglutination-inhibiting antibodies, enhanced germinal center reaction, and T follicular helper cell responses. In addition, RIG-I activation enhanced antibody affinity maturation and plasma cell responses in the draining lymph nodes, spleen, and bone marrow and conferred protective immunity against virus challenge. Importantly, activation of the RIG-I pathway was able to reduce the antigen requirement by 10- to 100-fold in inducing optimal influenza-specific cellular and humoral responses, including protective immunity. The effects induced by 5'ppp-dsRNA were significantly dependent on type I IFN and IPS-1 (an adapter protein downstream of the RIG-I pathway) signaling but were independent of the MyD88- and TLR3-mediated pathways. Our results show that activation of the RIG-I-like receptor pathway programs the innate immunity to achieve qualitatively and quantitatively enhanced protective cellular adaptive immune responses even at low antigen doses, and this indicates the potential utility of RIG-I ligands as molecular adjuvants for viral vaccines.

Importance: The recently discovered RNA helicase family of RIG-I-like receptors (RLRs) is a critical component of host defense mechanisms responsible for detecting viruses and triggering innate antiviral cytokines that help control viral replication and dissemination. In this study, we show that the RLR pathway can be effectively exploited to enhance adaptive immunity and protective immune memory against viral infection. Our results show that activation of the RIG-I pathway along with influenza vaccination programs the innate immunity to induce qualitatively and quantitatively superior protective adaptive immunity against pandemic influenza viruses. More importantly, RIG-I activation at the time of vaccination allows induction of robust adaptive responses even at low vaccine antigen doses. These results highlight the potential utility of exploiting the RIG-I pathway to enhance viral-vaccine-specific immunity and have broader implications for designing better vaccines in general.
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http://dx.doi.org/10.1128/JVI.02273-14DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4249139PMC
December 2014

Protein energy malnutrition decreases immunity and increases susceptibility to influenza infection in mice.

J Infect Dis 2013 Feb 4;207(3):501-10. Epub 2012 Sep 4.

Influenza Division, National Center for Immunization and Respiratory Diseases, National Center for Emerging and Zoonotic Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia 30333, USA.

Background: Protein energy malnutrition (PEM), a common cause of secondary immune deficiency in children, is associated with an increased risk of infections. Very few studies have addressed the relevance of PEM as a risk factor for influenza.

Methods: We investigated the influence of PEM on susceptibility to, and immune responses following, influenza virus infection using isocaloric diets providing either adequate protein (AP; 18%) or very low protein (VLP; 2%) in a mouse model.

Results: We found that mice maintained on the VLP diet, when compared to mice fed with the AP diet, exhibited more severe disease following influenza infection based on virus persistence, trafficking of inflammatory cell types to the lung tissue, and virus-induced mortality. Furthermore, groups of mice maintained on the VLP diet showed significantly lower virus-specific antibody response and a reduction in influenza nuclear protein-specific CD8(+) T cells compared with mice fed on the AP diet. Importantly, switching diets for the group maintained on the VLP diet to the AP diet improved virus clearance, as well as protective immunity to viral challenge.

Conclusions: Our results highlight the impact of protein energy on immunity to influenza infection and suggest that balanced protein energy replenishment may be one strategy to boost immunity against influenza viral infections.
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http://dx.doi.org/10.1093/infdis/jis527DOI Listing
February 2013

Rapid differentiation of monocytes into type I IFN-producing myeloid dendritic cells as an antiviral strategy against influenza virus infection.

J Immunol 2012 Sep 1;189(5):2257-65. Epub 2012 Aug 1.

Immunology and Pathogenesis Branch, Influenza Division, National Center for Immunization and Respiratory Diseases, Centers for Disease Control and Prevention, Atlanta, GA 30333, USA.

Myeloid dendritic cells (mDCs) have long been thought to function as classical APCs for T cell responses. However, we demonstrate that influenza viruses induce rapid differentiation of human monocytes into mDCs. Unlike the classic mDCs, the virus-induced mDCs failed to upregulate DC maturation markers and were unable to induce allogeneic lymphoproliferation. Virus-induced mDCs secreted little, if any, proinflammatory cytokines; however, they secreted a substantial amount of chemoattractants for monocytes (MCP-1 and IP-10). Interestingly, the differentiated mDCs secreted type I IFN and upregulated the expression of IFN-stimulated genes (tetherin, IFITM3, and viperin), as well as cytosolic viral RNA sensors (RIG-I and MDA5). Additionally, culture supernatants from virus-induced mDCs suppressed the replication of virus in vitro. Furthermore, depletion of monocytes in a mouse model of influenza infection caused significant reduction of lung mDC numbers, as well as type I IFN production in the lung. Consequently, increased lung virus titer and higher mortality were observed. Taken together, our results demonstrate that the host responds to influenza virus infection by initiating rapid differentiation of circulating monocytes into IFN-producing mDCs, which contribute to innate antiviral immune responses.
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http://dx.doi.org/10.4049/jimmunol.1200168DOI Listing
September 2012

The 3' untranslated regions of influenza genomic sequences are 5'PPP-independent ligands for RIG-I.

PLoS One 2012 15;7(3):e32661. Epub 2012 Mar 15.

Influenza Division, National Center for Immunization and Respiratory Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia, United States of America.

Retinoic acid inducible gene-I (RIG-I) is a key regulator of antiviral immunity. RIG-I is generally thought to be activated by ssRNA species containing a 5'-triphosphate (PPP) group or by unphosphorylated dsRNA up to ~300 bp in length. However, it is not yet clear how changes in the length, nucleotide sequence, secondary structure, and 5' end modification affect the abilities of these ligands to bind and activate RIG-I. To further investigate these parameters in the context of naturally occurring ligands, we examined RNA sequences derived from the 5' and 3' untranslated regions (UTR) of the influenza virus NS1 gene segment. As expected, RIG-I-dependent interferon-β (IFN-β) induction by sequences from the 5' UTR of the influenza cRNA or its complement (26 nt in length) required the presence of a 5'PPP group. In contrast, activation of RIG-I by the 3' UTR cRNA sequence or its complement (172 nt) exhibited only a partial 5'PPP-dependence, as capping the 5' end or treatment with CIP showed a modest reduction in RIG-I activation. Furthermore, induction of IFN-β by a smaller, U/A-rich region within the 3' UTR was completely 5'PPP-independent. Our findings demonstrated that RNA sequence, length, and secondary structure all contributed to whether or not the 5'PPP moiety is needed for interferon induction by RIG-I.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0032661PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3305289PMC
August 2012

Influenza A virus neuraminidase protein enhances cell survival through interaction with carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6) protein.

J Biol Chem 2012 Apr 6;287(18):15109-17. Epub 2012 Mar 6.

Virology Group, International Centre for Genetic Engineering and Biotechnology, Aruna Asaf Ali Road, New Delhi 110067, India.

The influenza virus neuraminidase (NA) protein primarily aids in the release of progeny virions from infected cells. Here, we demonstrate a novel role for NA in enhancing host cell survival by activating the Src/Akt signaling axis via an interaction with carcinoembryonic antigen-related cell adhesion molecule 6/cluster of differentiation 66c (C6). NA/C6 interaction leads to increased tyrosyl phosphorylation of Src, FAK, Akt, GSK3β, and Bcl-2, which affects cell survival, proliferation, migration, differentiation, and apoptosis. siRNA-mediated suppression of C6 resulted in a down-regulation of activated Src, FAK, and Akt, increased apoptosis, and reduced expression of viral proteins and viral titers in influenza virus-infected human lung adenocarcinoma epithelial and normal human bronchial epithelial cells. These findings indicate that influenza NA not only aids in the release of progeny virions, but also cell survival during viral replication.
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http://dx.doi.org/10.1074/jbc.M111.328070DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3340274PMC
April 2012

PAMPer and tRIGer: ligand-induced activation of RIG-I.

Trends Biochem Sci 2011 Jun 14;36(6):314-9. Epub 2011 Apr 14.

Influenza Division, National Center for Immunization and Respiratory Diseases, Centers for Disease Control and Prevention, Atlanta GA, 30333, United States.

Retinoic-acid-inducible gene-I (RIG-I) is an important component of the innate immune response to many RNA viruses that limits viral replication until adaptive immunity becomes available to clear the infection. Upon binding to the nucleic acid genomes and replication intermediates of these viruses, RIG-I undergoes a complex activation process that involves post-translational modifications and structural rearrangements. Once activated, RIG-I upregulates well-studied signal transduction pathways that lead to the production of type-I interferons (IFNs) and a large variety of antiviral IFN-stimulated genes. Thus, an effective antiviral response is dependent on the interaction between pathogen-derived ligands and RIG-I. Recent work has begun to clarify the required characteristics of RIG-I activators and is setting the stage for the identification of authentic ligands used during viral infection.
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http://dx.doi.org/10.1016/j.tibs.2011.03.003DOI Listing
June 2011

Infection of lung epithelial cells with pandemic 2009 A(H1N1) influenza viruses reveals isolate-specific differences in infectivity and host cellular responses.

Viral Immunol 2011 Apr;24(2):89-99

Immunology and Pathogenesis Branch, Influenza Division, National Center for Immunization and Respiratory Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia 30333, USA.

To better understand the early virus-host interactions of the pandemic 2009 A(H1N1) viruses in humans, we examined early host responses following infection of human epithelial cell cultures with three 2009 A(H1N1) viruses (A/California/08/2009, A/Mexico/4108/2009, and A/Texas/15/2009), or a seasonal H1N1 vaccine strain (A/Solomon Islands/3/2006). We report here that infection with pandemic A/California/08/2009 and A/Mexico/4108/2009 viruses resulted in differences in virus infectivity compared to either pandemic A/Texas/15/2009 or the seasonal H1N1 vaccine strain. In addition, IFN-β levels were decreased in cell cultures infected with either the A/California/08/2009 or the A/Mexico/4108/2009 virus. Furthermore, infection with A/California/08/2009 and A/Mexico/4108/2009 viruses resulted in lower expression of four key proinflammatory markers (IL-6, RANTES, IP-10, and MIP-1β) compared with infection with either A/Texas/15/2009 or A/Solomon Islands/3/2006. Taken together, our results demonstrate that 2009 A(H1N1) viruses isolated during the Spring wave induced varying degrees of early host antiviral and inflammatory responses in human respiratory epithelial cells, highlighting the strain-specific nature of these responses, which play a role in clinical disease.
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http://dx.doi.org/10.1089/vim.2010.0122DOI Listing
April 2011

Pathogenic virus-specific T cells cause disease during treatment with the calcineurin inhibitor FK506: implications for transplantation.

J Exp Med 2010 Oct 4;207(11):2355-67. Epub 2010 Oct 4.

Emory Vaccine Center, Emory University School of Medicine, Atlanta, GA 30322, USA.

Recently, several cases of fatal lymphocytic choriomeningitis virus (LCMV) infection occurred in transplant recipients being treated with the immunosuppressive calcineurin inhibitor FK506. These findings were surprising because LCMV is a noncytolytic virus. To understand how a noncytolytic virus can cause disease under conditions of immunosuppression, we used the mouse LCMV model and found that, similar to the observations in human transplant recipients, LCMV infection of FK506-treated mice resulted in a lethal disease characterized by viremia, lack of seroconversion, and minimal lymphocytic infiltrates in the tissues. However, despite the apparent absence of an antiviral immune response, this disease was orchestrated by virus-specific T cells. FK506 did not prevent the generation and proliferation of LCMV-specific T cells but instead altered their differentiation so that these effector T cells lost the ability to control virus but were still capable of mediating disease. These pathogenic T cells initiated a cytokine storm characterized by high levels of tumor necrosis factor (TNF) and interleukin 6 (IL-6), and depletion of T cells or blockade of these inflammatory cytokines prevented the lethal disease. Our study shows that inhibiting calcineurin can generate pathogenic T cells and indicates that T cell-mediated viral disease can occur even under conditions of immunosuppression. Furthermore, we identify a potential strategy (blockade of TNF and IL-6) for treatment of transplant recipients who have acute complications of viral infection.
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http://dx.doi.org/10.1084/jem.20100124DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2964579PMC
October 2010