Induction of H7N9-Cross-Reactive Antibody-Dependent Cellular Cytotoxicity Antibodies by Human Seasonal Influenza A Viruses that are Directed Toward the Nucleoprotein.

Antibodies that mediate antibody-dependent cellular cytotoxicity (ADCC) against avian influenza virus subtypes, including H7N9 and H5N1, have been detected in human sera. Using NK cell activation and NK cytotoxicity assays, we compared ADCC-mediating antibodies (ADCC-Abs) in sera collected from healthy infants, children and adults against H7N9 virus-infected cells and recombinant hemagglutinin (HA), neuraminidase (NA), and nucleoprotein (NP) proteins. High titers of ADCC-Abs against H7N9 virus-infected cells were detected in sera from adults and children but not infants. ADCC-Abs titers directed against H7N9 HA or NA proteins. Further analysis showed that ADCC-Abs titers were significantly higher toward H7N9 NP, as compared with H7N9 HA or NA proteins, and correlated strongly with ADCC-Abs titers against H7N9 virus-infected cells. Indeed, ADCC-Abs to NPs of seasonal H1N1 and H3N2 viruses correlated strongly with ADCC-Abs to H7N9 NP, suggesting that seasonal influenza infections and vaccinations may induce these cross-reactive antibodies. Targeting ADCC-Abs to internal proteins may be a potential mechanism of universal vaccine design.

High Antibody-Dependent Cellular Cytotoxicity Antibody Titers to H5N1 and H7N9 Avian Influenza A Viruses in Healthy US Adults and Older Children.

Human influenza is a highly contagious acute respiratory illness that is responsible for significant morbidity and excess mortality worldwide. In addition to neutralizing antibodies, there are antibodies that bind to influenza virus-infected cells and mediate lysis of the infected cells by natural killer (NK) cells (antibody-dependent cellular cytotoxicity [ADCC]) or complement (complement-dependent lysis [CDL]). We analyzed sera obtained from 16 healthy adults (18-63 years of age), 52 children (2-17 years of age), and 10 infants (0.75-1 year of age) in the United States, who were unlikely to have been exposed to the avian H7N9 subtype of influenza A virus, by ADCC and CDL assays. As expected, none of these sera had detectable levels of hemagglutination-inhibiting antibodies against the H7N9 virus, but we unexpectedly found high titers of ADCC antibodies to the H7N9 subtype virus in all sera from adults and children aged ≥8 years.

Cross-reactive human B cell and T cell epitopes between influenza A and B viruses.

Influenza A and B viruses form different genera, which were originally distinguished by antigenic differences in their nucleoproteins and matrix 1 proteins. Cross-protection between these two genera has not been observed in animal experiments, which is consistent with the low homology in viral proteins common to both viruses except for one of three polymerase proteins, polymerase basic 1 (PB1). Recently, however, antibody and CD4+ T cell epitopes conserved between the two genera were identified in humans. A protective antibody epitope was located in the stalk region of the surface glycoprotein, hemagglutinin, and a CD4+ T cell epitope was located in the fusion peptide of the hemagglutinin. The fusion peptide was also found to contain antibody epitopes in humans and animals. A short stretch of well-conserved peptide was also identified in the other surface glycoprotein, neuraminidase, and antibodies binding to this peptide were generated by peptide immunization in rabbits. Although PB1, the only protein which has relatively high overall sequence homology between influenza A and B viruses, is not considered an immunodominant protein in the T cell responses to influenza A virus infection, amino acid sequence comparisons show that a considerable number of previously identified T cell epitopes in the PB1 of influenza A viruses are conserved in the PB1 of influenza B viruses. These data indicate that B and T cell cross-reactivity exists between influenza A and B viruses, which may have modulatory effects on the disease process and recovery. Although the antibody titers and the specific T cell frequencies induced by natural infection or standard vaccination may not be high enough to provide cross protection in humans, it might be possible to develop immunization strategies to induce these cross-reactive responses more efficiently.

Telomere length dynamics in human memory T cells specific for viruses causing acute or latent infections.

BACKGROUND: Declining telomere length (TL) is associated with T cell senescence. While TL in naïve and memory T cells declines with increasing age, there is limited data on TL dynamics in virus-specific memory CD4+ T cells in healthy adults. We combined BrdU-labeling of virus-stimulated T cells followed with flow cytometry-fluorescent in situ hybridization for TL determination. We analyzed TL in T cells specific for several virus infections: non-recurring acute (vaccinia virus, VACV), recurring-acute (influenza A virus, IAV), and reactivating viruses (varicella-zoster virus, VZV, and cytomegalovirus, CMV) in 10 healthy subjects. Additionally, five subjects provided multiple blood samples separated by up to 10 years. RESULTS: VACV- and CMV-specific T cells had longer average TL than IAV-specific CD4+ T cells. Although most virus-specific cells were CD45RA-, we observed a minor population of BrdU+ CD45RA+ T cells characterized by long telomeres. Longitudinal analysis demonstrated a slow decline in average TL in virus-specific T cells. However, in one subject, VZV reactivation led to an increase in average TL in VZV-specific memory T cells, suggesting a conversion of longer TL cells from the naïve T cell repertoire. CONCLUSIONS: TLs in memory CD4+ T cells in otherwise healthy adults are heterogeneous and follow distinct virus-specific kinetics. These findings suggests that the distribution of TL and the creation and maintenance of long TL memory T cells could be important for the persistence of long-lived T cell memory.

Complement-dependent lysis of influenza a virus-infected cells by broadly cross-reactive human monoclonal antibodies.

We characterized human monoclonal antibodies (MAbs) cloned from influenza virus-infected patients and from influenza vaccine recipients by complement-dependent lysis (CDL) assay. Most MAbs active in CDL were neutralizing, but not all neutralizing MAbs can mediate CDL. Two of the three stalk-specific neutralizing MAbs tested were able to mediate CDL and were more cross-reactive to temporally distant H1N1 strains than the conventional hemagglutination-inhibiting and neutralizing MAbs. One of the stalk-specific MAbs was subtype cross-reactive to H1 and H2 hemagglutinins, suggesting a role for stalk-specific antibodies in protection against influenza illness, especially by a novel viral subtype which can cause pandemics.

Broadly recognized, cross-reactive SARS-CoV-2 CD4 T cell epitopes are highly conserved across human coronaviruses and presented by common HLA alleles.

Sequence homology between SARS-CoV-2 and common-cold human coronaviruses (HCoVs) raises the possibility that memory responses to prior HCoV infection can affect T cell response in COVID-19. We studied T cell responses to SARS-CoV-2 and HCoVs in convalescent COVID-19 donors and identified a highly conserved SARS-CoV-2 sequence, S811-831, with overlapping epitopes presented by common MHC class II proteins HLA-DQ5 and HLA-DP4. These epitopes are recognized by low-abundance CD4 T cells from convalescent COVID-19 donors, mRNA vaccine recipients, and uninfected donors. TCR sequencing revealed a diverse repertoire with public TCRs. T cell cross-reactivity is driven by the high conservation across human and animal coronaviruses of T cell contact residues in both HLA-DQ5 and HLA-DP4 binding frames, with distinct patterns of HCoV cross-reactivity explained by MHC class II binding preferences and substitutions at secondary TCR contact sites. These data highlight S811-831 as a highly conserved CD4 T cell epitope broadly recognized across human populations.

Pandemic influenza: implications for preparation and delivery of critical care services.

In a 5-week span during the 1918 influenza A pandemic, more than 2000 patients were admitted to Cook County Hospital in Chicago, with a diagnosis of either influenza or pneumonia; 642 patients, approximately 31% of those admitted, died, with deaths occurring predominantly in patients of age 25 to 30 years. This review summarizes basic information on the biology, epidemiology, control, treatment and prevention of influenza overall, and then addresses the potential impact of pandemic influenza in an intensive care unit setting. Issues that require consideration include workforce staffing and safety, resource management, alternate sites of care surge of patients, altered standards of care, and crisis communication.

Non-neutralizing antibody responses following A(H1N1)pdm09 influenza vaccination with or without AS03 adjuvant system.

BACKGROUND: Non-neutralizing antibodies inducing complement-dependent lysis (CDL) and antibody-dependent cell-mediated cytotoxicity (ADCC) activity may contribute to protection against influenza infection. We investigated CDL and ADCC responses in healthy adults randomized to receive either non-adjuvanted or AS03-adjuvanted monovalent A(H1N1)pdm09 vaccine (containing 15 µg/3.75 µg of hemagglutinin, respectively) on a 2-dose schedule 21 days apart. METHODS: We conducted an exploratory analysis of a subset of 106 subjects having no prior history of A(H1N1)pdm09 infection or seasonal influenza vaccination enrolled in a previously reported study (NCT00985673). Antibody responses against the homologous A/California/7/2009 (H1N1) vaccine strain and a related A/Brisbane/59/2007 (H1N1) seasonal influenza strain were analyzed up to Day 42. RESULTS: Baseline seropositivity determined with hemagglutination inhibition (HI), CDL and ADCC antibody titers against viral strains was high; A/California/7/2009 (HI [40.4-48.1%]; CDL [34.6-36.0%]; ADCC [92.1-92.3%]); A/Brisbane/59/2007 (HI [73.1-88.9%]; CDL [38.0-42.0%]; ADCC [86.8-97.0%]). CDL seropositivity increased following vaccination with both adjuvanted and non-adjuvanted formulations (A/California/7/2009 [95.9-100%]; A/Brisbane/59/2007 [75.5-79.6%]). At Day 21, increases in CDL and ADCC antibody geometric mean titers against both strains were observed for both formulations. After 2 doses of AS03-adjuvanted vaccine, vaccine responses of 95.8% (≥9-fold increase from baseline in CDL titers) and 34.3% (≥16-fold increase from baseline in ADCC titers) were seen against A/California/7/2009; and 22.4% and 42.9%, respectively, against A/Brisbane/59/2007. Vaccine responses after 2 doses of the non-adjuvanted vaccine were broadly similar. CONCLUSIONS: Broadly comparable non-neutralizing immune responses were observed following vaccination with non-adjuvanted and AS03-adjuvanted A(H1N1)pdm09 formulations; including activity against a related vaccine strain.

Dynamics of the CD8 T-cell response following yellow fever virus 17D immunization.

Management of yellow fever is focused on the prevention of illness by the use of the yellow fever virus (YFV) 17D vaccine. The role of neutralizing antibodies in protection is generally accepted with YFV-specific T cells likely contributing to the control of viral replication. We studied CD8(+) T-cell responses to four defined human leucocyte antigen-B35-restricted epitopes in YFV vaccine recipients as a model of the kinetics of cytotoxic T-lymphocyte responses to an acute human viral infection. Multiple features of these epitope-specific responses were analysed after vaccination including magnitude, cytokine production, phenotype and T-cell receptor repertoire. Peak peptide-specific interferon-gamma (IFN-gamma) responses of almost 1% of CD8(+) T cells were seen as early as 2 weeks post-vaccination; however, dominant responses varied between donors. Peptide-specific responses were still detectable at 54 months post-vaccination. Tetramer-positive cells, at high frequencies, were detected as early as 7-9 days, before detectable IFN-gamma-producing cells, suggesting a defect in the functional capacity of some antigen-specific cells early post-vaccination. The predominant memory phenotype of the tetramer-positive population was a differentiated effector (CD45RA(+) CCR7(-) CD62L(-)) phenotype. The T-cell receptor Vbeta analysis revealed a diverse oligoclonal repertoire in tetramer-positive T-cell populations in two individuals. These characteristics of the YFV-specific T-cell response could contribute to vaccine effectiveness.

Influenza A virus matrix protein 1-specific human CD8+ T-cell response induced in trivalent inactivated vaccine recipients.

Among 17 HLA-A2-positive healthy adults, CD8+ T-cell responses against an HLA-A2-restricted matrix protein 1 (M1) epitope increased after immunization with trivalent inactivated influenza vaccine (TIV) in two individuals. The presence of M1 in TIV was confirmed by Western blotting. T-cell cytotoxicity assays showed that TIV is processed and the epitope is presented by antigen-presenting cells to an M1 epitope-specific CD8+ T-cell line for specific lysis. These data show that TIV, which is formulated to contain surface glycoproteins to induce serotype-specific antibody responses, also contains M1, capable of inducing subtype cross-reactive CD8+ T-cell responses in some vaccinees.

CpG Improves Influenza Vaccine Efficacy in Young Adult but Not Aged Mice.

Several studies have shown a reduced efficacy of influenza vaccines in the elderly compared to young adults. In this study, we evaluated the immunogenicity and protective efficacy of a commercially available inactivated influenza vaccine (Fluzone®) in young adult and aged mice. C57/BL6 mice were administered a single or double immunization of Fluzone® with or without CpG and challenged intranasally with H1N1 A/California/09 virus. A double immunization of Fluzone® adjuvanted with CpG elicited the highest level of protection in young adult mice which was associated with increases in influenza specific IgG, elevated HAI titres, reduced viral titres and lung inflammation. In contrast, the vaccine schedule which provided fully protective immunity in young adult mice conferred limited protection in aged mice. Antigen presenting cells from aged mice were found to be less responsive to in vitro stimulation by Fluzone and CpG which may partially explain this result. Our data are supportive of studies that have shown limited effectiveness of influenza vaccines in the elderly and provide important information relevant to the design of more immunogenic vaccines in this age group.

Predictors of erythropoietin use in patients with cardiorenal anaemia syndrome.

OBJECTIVES: Chronic kidney disease (CKD) and anemia are common in patients with heart failure (HF) - these 3 conditions have been coined the Cardiorenal Anemia Sydrome (CRAS). The National Kidney Foundation Kidney Disease Outcomes Quality Initiative (NKF-K/DOQI) guidelines do not specifically address patients with CRAS, creating uncertainty in erythropoietin (EPO) prescribing. We sought to determine predictors of EPO use in patients with CRAS. METHODS: We conducted a retrospective cohort study at the Veteran's Affairs Greater Los Angeles Healthcare System (VAGLAHS), a 300+ bed facility that provides primary and tertiary inpatient, and ambulatory care services, between January 1, 2003 to December 31, 2006. A multiple logistic regression model was constructed to identify predictors of EPO use among CRAS patients. KEY FINDINGS: Of 2058 patients with CRAS, 213 (10.3%) were prescribed EPO. There were significant differences in baseline characteristics between the EPO and non-EPO groups. The following predictors were found to be associated with EPO prescription: iron supplementation (odds ratio [OR] 52.70, 95% confidence interval [CI] 11.70-237.46), renal clinic appointment (OR 2.60, 95% CI 1.79-3.76), malignancy (OR 1.52, 95% CI 1.07-2.16) and use of hydralazine/nitrates (OR 1.41, 95% CI 1.03-1.92). There was an inverse association found between EPO prescription and baseline hemoglobin (OR 0.61, 95% CI 0.53-0.70) and eGFR (OR 0.96, 95% CI 0.94-0.97). CONCLUSION: A small proportion of patients eligible for EPO therapy according to guidelines at the time of the study were prescribed the indicated therapy. Markers of declining renal function or those suggesting need for anemia therapy were identified as EPO predictors.

Pre-eclampsia has an adverse impact on maternal and fetal health.

Pre-eclampsia (preE) is a multifaceted complication found uniquely in the pregnant patient and one that has puzzled scientists for years. PreE is not a single disorder, but a complex syndrome that is produced by various pathophysiological triggers and mechanisms affecting about 5% of obstetrical patients. PreE is a major cause of premature delivery and maternal and fetal morbidity and mortality. PreE is characterized by de novo development of hypertension and proteinuria after 20 weeks of gestation and affects nearly every organ system, with the most severe consequences being eclampsia, pulmonary edema, intrauterine growth restriction, and thrombocytopenia. PreE alters the intrauterine environment by modulating the pattern of hormonal signals and activating the detrimental cellular signaling that has been transported to the fetus. The fetus has to adapt to this intrauterine environment with detrimental signals. The adaptive changes increase the risk of disease later in life. This review defines the predisposition and causes of preE and the cellular signaling detrimental to maternal health during preE. Moreover, the risk factors for diseases that are transmitted to the offspring have been addressed in this review. The detrimental signaling molecules that have been overexpressed in preE patients raises the possibility that those signals could be therapeutically blocked one day.

Serological documentation of maternal influenza exposure and bipolar disorder in adult offspring.

OBJECTIVE: The authors examined whether serologically confirmed maternal exposure to influenza was associated with an increased risk of bipolar disorder in the offspring and with subtypes of bipolar disorder, with and without psychotic features. METHOD: The study used a nested case-control design in the Child Health and Development Study birth cohort. In all, 85 individuals with bipolar disorder were identified following extensive ascertainment and diagnostic assessment and matched to 170 comparison subjects in the analysis. Serological documentation of maternal exposure to influenza was determined using the hemagglutination inhibition assay. RESULTS: No association was observed between serologically documented maternal exposure to influenza and bipolar disorder in offspring. However, maternal serological influenza exposure was related to a significant fivefold greater risk of bipolar disorder with psychotic features. CONCLUSIONS: The results suggest that maternal influenza exposure may increase the risk for offspring to develop bipolar disorder with psychotic features. Taken together with earlier associations between prenatal influenza exposure and schizophrenia, these results may suggest that prenatal influenza is a risk factor for psychosis rather than for a specific psychotic disorder diagnosis.

Relationship of preexisting influenza hemagglutination inhibition, complement-dependent lytic, and antibody-dependent cellular cytotoxicity antibodies to the development of clinical illness in a prospective study of A(H1N1)pdm09 Influenza in children.

The hemagglutination inhibition (HAI) antibody titer is considered the primary immune correlate of protection for influenza. However, recent studies have highlighted the limitations on the use of the HAI titer as a correlate in at-risk populations such as children and older adults. In addition to the neutralization of cell-free virus by antibodies to hemagglutinin and interference of virus release from infected cells by antibodies to neuraminidase, influenza virus-specific antibodies specifically can bind to infected cells and lyse virus-infected cells through the activation of complement or natural killer (NK) cells, via antibody-dependent cellular cytotoxicity (ADCC) or complement-dependent lysis (CDL). We evaluated preexisting HAI, CDL, and ADCC antibodies in young children enrolled in a prospective cohort study of dengue during the epidemic with influenza A(H1N1)pdm09 virus to determine associations between preexisting antibodies and the occurrence of clinical or subclinical influenza virus infection. Though both preexisting HAI and CDL antibodies were associated with protection against clinical influenza, our data suggested that CDL was not a better correlate than HAI. We found that ADCC antibodies behaved differently from HAI and CDL antibodies. Unlike HAI and CDL antibodies, preexisting ADCC antibodies did not correlate with protection against clinical influenza. In fact, ADCC antibodies were detected more frequently in the clinical influenza group than the subclinical group. In addition, in contrast to HAI and CDL antibodies, HAI and the ADCC antibodies titers did not correlate. We also found that ADCC, but not CDL or HAI antibodies, positively correlated with the ages of the children.

Comparison of complement dependent lytic, hemagglutination inhibition and microneutralization antibody responses in influenza vaccinated individuals.

Virus specific, non-neutralizing antibodies such as complement dependent lytic (CDL) antibodies may reduce morbidity following infection through the clearance of infectious virus particles and infected cells. We examined hemagglutination inhibition (HAI), microneutralization (MN) and CDL antibody titers to influenza A H1 and H3 virus strains in 23 healthy young adults who received the 2005-2006 trivalent inactivated influenza vaccine. Post vaccination, we detected statistically significant increases in MN and CDL antibodies but not in HAI antibodies. Statistically significantly higher fold increases in CDL antibodies post vaccination were seen compared with MN and HAI antibodies post vaccination. However, the overall fold increases were modest, likely related to the fact that most of the subjects had received influenza vaccination previously. This study showed that influenza vaccination is not only capable of increasing the level of antibodies that neutralize virus but also antibodies that can cause lysis of infected cells. The biological significance of these CDL antibodies merits further investigation in clinical studies.

Genome-wide screening of human T-cell epitopes in influenza A virus reveals a broad spectrum of CD4(+) T-cell responses to internal proteins, hemagglutinins, and neuraminidases.

We performed a genome-wide screening for T-cell epitopes using synthetic peptides that encompass all of the influenza A viral proteins, including subtype variants for hemagglutinin (HA; H1, H3, and H5) and neuraminidase (NA; human and avian N1 and N2) proteins, based on the sequence information of recently circulating strains. We identified a total of 83 peptides, 54 of them novel, to which specific T cells were detectable in interferon-gamma (IFN-gamma) enzyme-linked immunosorbent spot assays using peripheral blood mononuclear cells from four healthy adult donors. The surface glycoproteins, HA and NA, major components of vaccines, expressed many T-cell epitopes. HA and matrix protein 1 expressed more T-cell epitopes than other viral proteins, most of which were recognized by CD4(+) T cells. We established several cytotoxic CD4(+) T-cell lines from these donors. We also analyzed H1 and H3 HA-specific T-cell responses using the peripheral blood mononuclear cells of 30 hospital workers. Fifty-three percent of donors gave a positive response to H3 HA peptides, whereas 17% gave a positive response to H1 HA peptides. Our genome-wide screening is useful in identifying T-cell epitopes and is complementary to the approach based on the predicted binding peptides to well-studied HLA-A, -B, and -DR alleles.

In vitro evidence that commercial influenza vaccines are not similar in their ability to activate human T cell responses.

We evaluated three commercial trivalent inactivated vaccines (TIVs) from the 2007-2008 season in terms of their ability to elicit in vitro T cell responses. T cell-mediated immunity may offer a more cross-reactive vaccine approach for the prevention of pandemic or epidemic influenza. Human cytotoxic T cell lines demonstrated differences in matrix protein 1 and nucleocapsid protein recognition of autologous target cells. Peripheral blood mononuclear cells stimulated with each of the TIVs showed statistically significant differences between the vaccines in the numbers of IFNgamma producing cells activated. These data suggest that TIV vaccines are not similar in their ability to activate human T cell responses.