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1.  Heterovariant Cross-Reactive B-Cell Responses Induced by the 2009 Pandemic Influenza Virus A Subtype H1N1 Vaccine 
The Journal of Infectious Diseases  2012;207(2):288-296.
Background. The generation of heterovariant immunity is a highly desirable feature of influenza vaccines. The goal of this study was to compare the heterovariant B-cell response induced by the monovalent inactivated 2009 pandemic influenza A virus subtype H1N1 (A[H1N1]pdm09) vaccine with that induced by the 2009 seasonal trivalent influenza vaccine (sTIV) containing a seasonal influenza A virus subtype H1N1 (A[H1N1]) component in young and elderly adults.
Methods. Plasmablast-derived polyclonal antibodies (PPAb) from young and elderly recipients of A(H1N1)pdm09 vaccine or sTIV were tested for binding activity to various influenza antigens.
Results. In A(H1N1)pdm09 recipients, the PPAb titers against homotypic A(H1N1)pdm09 vaccine were similar to those against the heterovariant seasonal A(H1N1) vaccine and were similar between young and elderly subjects. The PPAb avidity was higher among elderly individuals, compared with young individuals. In contrast, the young sTIV recipients had 10-fold lower heterovariant PPAb titers against the A(H1N1)pdm09 vaccine than against the homotypic seasonal A(H1N1) vaccine. In binding assays with recombinant head and stalk domains of hemagglutinin, PPAb from the A(H1N1)pdm09 recipients but not PPAb from the sTIV recipients bound to the conserved stalk domain.
Conclusion. The A(H1N1)pdm09 vaccine induced production of PPAb with heterovariant reactivity, including antibodies targeting the conserved hemagglutinin stalk domain.
doi:10.1093/infdis/jis664
PMCID: PMC3532823  PMID: 23107783
influenza; vaccine; antibody; cross-reactivity
2.  Lineage Structure of the Human Antibody Repertoire in Response to Influenza Vaccination 
Science translational medicine  2013;5(171):171ra19.
The human antibody repertoire is one of the most important defenses against infectious disease, and the development of vaccines has enabled the conferral of targeted protection to specific pathogens. However, there are many challenges to measuring and analyzing the immunoglobulin sequence repertoire, such as the fact that each B cell contains a distinct antibody sequence encoded in its genome, that the antibody repertoire is not constant but changes over time, and the high similarity between antibody sequences. We have addressed this challenge by using high-throughput long read sequencing to perform immunogenomic characterization of expressed human antibody repertoires in the context of influenza vaccination. Informatic analysis of 5 million antibody heavy chain sequences from healthy individuals allowed us to perform global characterizations of isotype distributions, determine the lineage structure of the repertoire and measure age and antigen related mutational activity. Our analysis of the clonal structure and mutational distribution of individuals’ repertoires shows that elderly subjects have a decreased number of lineages but an increased pre-vaccination mutation load in their repertoire and that some of these subjects have an oligoclonal character to their repertoire in which the diversity of the lineages is greatly reduced relative to younger subjects. We have thus shown that global analysis of the immune system’s clonal structure provides direct insight into the effects of vaccination and provides a detailed molecular portrait of age-related effects.
doi:10.1126/scitranslmed.3004794
PMCID: PMC3699344  PMID: 23390249
3.  Plasmablast-derived polyclonal antibody response after influenza vaccination 
Journal of immunological methods  2010;365(1-2):67-75.
Conventional measurement of antibody responses to vaccines largely relies on serum antibodies, which are primarily produced by bone marrow plasma cells and may not represent the entire vaccine-induced B cell repertoire, including important functional components such as those targeted to mucosal sites. After immunization or infection, activated B cells differentiate into plasmablasts in local lymphoid organs, then traffic through circulation to the target sites where they further develop into plasma cells. On day 7 after influenza vaccination, a burst of plasmablasts, highly enriched for vaccine-specific antibody secreting cells, appears in the peripheral blood. This provides a unique window to the overall B cell response to the vaccine, without interference of pre-existing cross-reactive serum antibody. In this study we isolated B cells from volunteers on day 7 after immunization with the inactivated influenza vaccine and cultured them ex vivo to collect plasmablast-derived polyclonal antibodies (PPAb). The PPAb contained secreted IgG and IgA, which was approximately 0.2 ng per antibody secreting cell. Influenza-specific IgG and IgA binding activity was detected in PPAb at dilutions up to 105 by ELISA. The ratio of the titers of influenza-specific IgA to IgG by ELISA was 4-fold higher in PPAb than in day 28 post-vaccination sera, suggesting that vaccine-induced IgA is enriched in PPAb compared to sera. Functional activity was also detected in PPAb as determined by microneutralization and hemagglutination inhibition assays. In addition to bulk B cell cultures, we also cultured plasmablast subsets sorted by cell surface markers to generate PPAb. These results suggest that PPAb better reflects the mucosal IgA response than serum samples. Since PPAb are exclusively produced by recently activated B cells, it allows assessing vaccine-induced antibody response without interference from pre-existing cross-reactive serum antibodies and permits an assessment of antibody avidity based on antigen specific binding and antibody quantity. Therefore this assay is particularly useful for studying vaccine/infection-induced antibodies against antigens that might have previously circulated, such as antibody responses to rotavirus, dengue or influenza viruses in which cross-reactive antibodies against different virus serotypes/subtypes play a critical role in immunity and/or pathogenesis.
doi:10.1016/j.jim.2010.12.008
PMCID: PMC3039424  PMID: 21182843
Influenza virus; vaccines; antibodies; plasmablasts
4.  Limited efficacy of inactivated influenza vaccine in elderly individuals is associated with decreased production of vaccine-specific antibodies 
The Journal of Clinical Investigation  2011;121(8):3109-3119.
During seasonal influenza epidemics, disease burden is shouldered predominantly by the very young and the elderly. Elderly individuals are particularly affected, in part because vaccine efficacy wanes with age. This has been linked to a reduced ability to induce a robust serum antibody response. Here, we show that this is due to reduced quantities of vaccine-specific antibodies, rather than a lack of antibody avidity or affinity. We measured levels of vaccine-specific plasmablasts by ELISPOT 1 week after immunization of young and elderly adults with inactivated seasonal influenza vaccine. Plasmablast-derived polyclonal antibodies (PPAbs) were generated from bulk-cultured B cells, while recombinant monoclonal antibodies (re-mAbs) were produced from single plasmablasts. The frequency of vaccine-specific plasmablasts and the concentration of PPAbs were lower in the elderly than in young adults, whereas the yields of secreted IgG per plasmablast were not different. Differences were not detected in the overall vaccine-specific avidity or affinity of PPAbs and re-mAbs between the 2 age groups. In contrast, reactivity of the antibodies induced by the inactivated seasonal influenza vaccine toward the 2009 pandemic H1N1 virus, which was not present in the vaccine, was higher in the elderly than in the young. These results indicate that the inferior antibody response to influenza vaccination in the elderly is primarily due to reduced quantities of vaccine-specific antibodies. They also suggest that exposure history affects the cross-reactivity of vaccination-induced antibodies.
doi:10.1172/JCI57834
PMCID: PMC3148747  PMID: 21785218
5.  Influence of Prior Influenza Vaccination on Antibody and B-Cell Responses 
PLoS ONE  2008;3(8):e2975.
Currently two vaccines, trivalent inactivated influenza vaccine (TIV) and live attenuated influenza vaccine (LAIV), are licensed in the USA. Despite previous studies on immune responses induced by these two vaccines, a comparative study of the influence of prior influenza vaccination on serum antibody and B-cell responses to new LAIV or TIV vaccination has not been reported. During the 2005/6 influenza season, we quantified the serum antibody and B-cell responses to LAIV or TIV in adults with differing influenza vaccination histories in the prior year: LAIV, TIV, or neither. Blood samples were collected on days 0, 7–9 and 21–35 after immunization and used for serum HAI assay and B-cell assays. Total and influenza-specific circulating IgG and IgA antibody secreting cells (ASC) in PBMC were detected by direct ELISPOT assay. Memory B cells were also tested by ELISPOT after polyclonal stimulation of PBMC in vitro. Serum antibody, effector, and memory B-cell responses were greater in TIV recipients than LAIV recipients. Prior year TIV recipients had significantly higher baseline HAI titers, but lower HAI response after vaccination with either TIV or LAIV, and lower IgA ASC response after vaccination with TIV than prior year LAIV or no vaccination recipients. Lower levels of baseline HAI titer were associated with a greater fold-increase of HAI titer and ASC number after vaccination, which also differed by type of vaccine. Our findings suggest that the type of vaccine received in the prior year affects the serum antibody and the B-cell responses to subsequent vaccination. In particular, prior year TIV vaccination is associated with sustained higher HAI titer one year later but lower antibody response to new LAIV or TIV vaccination, and a lower effector B-cell response to new TIV but not LAIV vaccination.
doi:10.1371/journal.pone.0002975
PMCID: PMC2500171  PMID: 18714352
6.  Baseline Levels of Influenza-Specific CD4 Memory T-Cells Affect T-Cell Responses to Influenza Vaccines 
PLoS ONE  2008;3(7):e2574.
Background
Factors affecting immune responses to influenza vaccines have not been studied systematically. We hypothesized that T-cell and antibody responses to the vaccines are functions of pre-existing host immunity against influenza antigens.
Methodology/Principal Findings
During the 2004 and 2005 influenza seasons, we have collected data on cellular and humoral immune reactivity to influenza virus in blood samples collected before and after immunization with inactivated or live attenuated influenza vaccines in healthy children and adults. We first used cross-validated lasso regression on the 2004 dataset to identify a group of candidate baseline correlates with T-cell and antibody responses to vaccines, defined as fold-increase in influenza-specific T-cells and serum HAI titer after vaccination. The following baseline parameters were examined: percentages of influenza-reactive IFN-γ+ cells in T and NK cell subsets, percentages of influenza-specific memory B-cells, HAI titer, age, and type of vaccine. The candidate baseline correlates were then tested with the independent 2005 dataset. Baseline percentage of influenza-specific IFN-γ+ CD4 T-cells was identified as a significant correlate of CD4 and CD8 T-cell responses, with lower baseline levels associated with larger T-cell responses. Baseline HAI titer and vaccine type were identified as significant correlates for HAI response, with lower baseline levels and the inactivated vaccine associated with larger HAI responses. Previously we reported that baseline levels of CD56dim NK reactivity against influenza virus inversely correlated with the immediate T-cell response to vaccination, and that NK reactivity induced by influenza virus depended on IL-2 produced by influenza-specific memory T-cells. Taken together these results suggest a novel mechanism for the homeostasis of virus-specific T-cells, which involves interaction between memory helper T-cells, CD56dim NK and DC.
Significance
These results demonstrate that assessment of baseline biomarkers may predict immunologic outcome of influenza vaccination and may reveal some of the mechanisms responsible for variable immune responses following vaccination and natural infection.
doi:10.1371/journal.pone.0002574
PMCID: PMC2440350  PMID: 18596908
7.  Comparison of the Influenza Virus-Specific Effector and Memory B-Cell Responses to Immunization of Children and Adults with Live Attenuated or Inactivated Influenza Virus Vaccines▿  
Journal of Virology  2006;81(1):215-228.
Cellular immune responses to influenza virus infection and influenza virus vaccination have not been rigorously characterized. We quantified the effector and memory B-cell responses in children and adults after administration of either live attenuated (LAIV) or inactivated (TIV) influenza virus vaccines and compared these to antibody responses. Peripheral blood mononuclear cells were collected at days 0, 7 to 12, and 27 to 42 after immunization of younger children (6 months to 4 years old), older children (5 to 9 years old), and adults. Influenza virus-specific effector immunoglobulin A (IgA) and IgG circulating antibody-secreting cells (ASC) and stimulated memory B cells were detected using an enzyme-linked immunospot assay. Circulating influenza virus-specific IgG and IgA ASC were detected 7 to 12 days after TIV and after LAIV immunization. Seventy-nine percent or more of adults and older children had demonstrable IgG ASC responses, while IgA ASC responses were detected in 29 to 53% of the subjects. The IgG ASC response rate to LAIV immunization in adults was significantly higher than the response rate measured by standard serum antibody assays (26.3% and 15.8% by neutralization and hemagglutination inhibition assays, respectively). IgG ASC and serum antibody responses were relatively low in the younger children compared to older children and adults. TIV, but not LAIV, significantly increased the percentage of circulating influenza virus-specific memory B cells detected at 27 to 42 days after immunization in children and adults. In conclusion, although both influenza vaccines are effective, we found significant differences in the B-cell and antibody responses elicited after LAIV or TIV immunization in adults and older children and between young children and older age groups.
doi:10.1128/JVI.01957-06
PMCID: PMC1797237  PMID: 17050593
8.  Interference between Host Resistance to Listeria monocytogenes Infection and Ovalbumin-Induced Allergic Responses in Mice 
Infection and Immunity  2001;69(3):1883-1888.
Listeria monocytogenes promotes the induction of the T-helper 1 (Th1) cell response, while ovalbumin (OVA) induces a Th2 cell response and allergic reactions, such as airway hyperreactivity and immunoglobulin E (IgE) production. When mice were immunized with OVA on day 7 after L. monocytogenes infection, eosinophilia in bronchoalveolar lavage and the production of total IgE, OVA-specific IgE, interleukin-4 (IL-4), and IL-5 in the circulation were markedly suppressed. Cytokine responses, including IL-4, IL-5, IL-10, IL-13, and gamma interferon, to OVA were decreased in the spleen cell cultures obtained from OVA-immunized mice that had been infected with L. monocytogenes. Conversely, when OVA-immunized mice were infected with L. monocytogenes, conversion from the nonlethal infection to the lethal infection occurred. Host resistance to L. monocytogenes infection in OVA-immunized mice was enhanced by the administration of anti–IL-10 monoclonal antibody. The present study indicates that striking interference is observed between Th1-inducing L. monocytogenes infection and Th2-driven OVA-induced airway hyperreactivity.
doi:10.1128/IAI.69.3.1883-1888.2001
PMCID: PMC98097  PMID: 11179368
9.  Host Resistance to Listeria monocytogenes Infection Is Enhanced but Resistance to Staphylococcus aureus Infection Is Reduced in Acute Graft-versus-Host Disease in Mice 
Infection and Immunity  2000;68(7):4340-4343.
Acute graft-versus-host disease (GVHD) is characterized by the production of high levels of T helper 1 (Th1)-type cytokines. Bone marrow transplantation from allogeneic C57BL/6 cells to CBF1 mice produced acute GVHD. Host resistance to Th1-driven Listeria monocytogenes was enhanced, whereas host resistance to Th2-driven Staphylococcus aureus was reduced during acute GVHD. These results suggest that opposite host responses are observed between Th1-driven and Th2-driven bacterial infections in acute GVHD.
PMCID: PMC101764  PMID: 10858256
10.  Interleukin-4 and Interleukin-10 Are Involved in Host Resistance to Staphylococcus aureus Infection through Regulation of Gamma Interferon 
Infection and Immunity  2000;68(5):2424-2430.
Our previous study showed that gamma interferon (IFN-γ), a T-helper 1 (Th1)-type cytokine, plays a detrimental role in Staphylococcus aureus infection in mice. In this study, the role of Th2-type cytokines such as interleukin-4 (IL-4) and IL-10 in S. aureus infection was investigated. IL-10 mRNA was induced in parallel with IFN-γ in the spleens and kidneys of mice during S. aureus infection, whereas IL-4 mRNA was induced in the spleens but not in the kidneys of these animals. Spleen cells obtained from S. aureus-infected mice produced lower titers of IFN-γ and higher titers of IL-4 and IL-10 in response to heat-killed S. aureus than did those from uninfected mice. Administration of anti-IL-4 monoclonal antibody (MAb) or anti-IL-10 MAb inhibited the elimination of S. aureus cells from the kidneys of mice. IFN-γ mRNA expression was enhanced in the spleens of anti-IL-4 MAb- or anti-IL-10 MAb-treated mice and also in the kidneys of anti-IL-4 MAb-treated animals. Next, we evaluated the role of IFN-γ in S. aureus infection in IFN-γ−/− mice. An increase in survival rates, a decrease in bacterial numbers in the kidneys, and an amelioration of histologic abnormalities in these organs were observed in IFN-γ−/− mice compared with those in IFN-γ+/+ mice. Administration of MAb against IL-4 or IL-10 failed to affect bacterial growth in the spleens and kidneys of IFN-γ−/− mice irrespective of the expression of Th2 response. These results suggest that S. aureus infection induced a Th2 response and that IL-4 and IL-10 might play a protective role through the regulation of IFN-γ in S. aureus infection.
PMCID: PMC97441  PMID: 10768926
11.  Protective Role of Nitric Oxide in Staphylococcus aureus Infection in Mice 
Infection and Immunity  1998;66(3):1017-1022.
This study was carried out to determine the role of nitric oxide (NO) in Staphylococcus aureus infection in mice. NO production in spleen cell cultures was induced by heat-killed S. aureus. Expression of mRNA of the inducible isoform of NO synthase (iNOS) was induced in the spleens and kidneys of S. aureus-infected mice. When mice were treated with monoclonal antibodies (MAbs) against tumor necrosis factor alpha (TNF-α) or gamma interferon (IFN-γ) before S. aureus infection, the induction of iNOS mRNA expression in the kidneys was inhibited. These MAbs also inhibited NO production in spleen cell cultures stimulated with heat-killed S. aureus. NO production in the spleen cell cultures and levels of urinary nitrate plus nitrite were suppressed by treatment with aminoguanidine (AG), a selective inhibitor of iNOS. The survival rates of AG-treated mice were significantly decreased by either lethal or sublethal S. aureus infections. However, an effect of AG administration on bacterial growth was not observed in the spleens and kidneys of mice during either type of infection. Production of TNF-α and IFN-γ was not affected by AG treatment in vitro and in vivo. These results suggest that NO plays an important role in protection from lethality by the infection, but the protective role of NO in host resistance against S. aureus infection was not proved. Moreover, our results show that TNF-α and IFN-γ regulate NO production while NO may not be involved in the regulation of the production of these cytokines during S. aureus infection.
PMCID: PMC108010  PMID: 9488390

Results 1-11 (11)