Lung mucosal immunity to NTHi vaccine antigens: Antibodies in sputum of chronic obstructive pulmonary disease patients

Chronic obstructive pulmonary disease (COPD) is a common chronic respiratory illness in older adults. A major cause of COPD-related morbidity and mortality is acute exacerbation of COPD (AECOPD). Bacteria in the lungs play a role in exacerbation development, and the most common pathogen is non-typeable Haemophilus influenzae (NTHi). A vaccine to prevent AECOPD containing NTHi surface antigens was tested in a clinical trial. This study measured IgG and IgA against NTHi vaccine antigens in sputum. Sputum samples from 40 COPD patients vaccinated with the NTHi vaccine were collected at baseline and 30 days after the second dose. IgG and IgA antibodies against the target antigens and albumin were analyzed in the sputum. We compared antibody signals before and after vaccination, analyzed correlation with disease severity and between sputum and serum samples, and assessed transudation. Antigen-specific IgG were absent before vaccination and present with high titers after vaccination. Antigen-specific IgA before and after vaccination were low but significantly different for two antigens. IgG correlated between sputum and serum, and between sputum and disease severity. Sputum albumin was higher in patients with severe COPD than in those with moderate COPD, suggesting changes in transudation played a role. We demonstrated that immunization with the NTHi vaccine induces antigen-specific antibodies in sputum. The correlation between IgG from sputum and serum and the presence of albumin in the sputum of severe COPD patients suggested transudation of antibodies from the serum to the lungs, although local IgG production could not be excluded. Clinical Trial Registration: NCT02075541 What is the context?Chronic obstructive pulmonary disease (COPD) is the most common chronic respiratory illness in older adults and the third leading cause of death worldwide.One bacterium in the lungs, non-typeable Haemophilus influenzae (NTHi), is responsible for acute exacerbation of the disease, characterized by an increase in airway wall inflammation and symptoms, leading to high morbidity and mortality.A vaccine targeting NTHi was previously developed but did not show efficacy in reducing exacerbations in COPD patients, probably because the vaccine did not elicit an immune response in the lung mucosae, where the bacteria are located. Chronic obstructive pulmonary disease (COPD) is the most common chronic respiratory illness in older adults and the third leading cause of death worldwide. One bacterium in the lungs, non-typeable Haemophilus influenzae (NTHi), is responsible for acute exacerbation of the disease, characterized by an increase in airway wall inflammation and symptoms, leading to high morbidity and mortality. A vaccine targeting NTHi was previously developed but did not show efficacy in reducing exacerbations in COPD patients, probably because the vaccine did not elicit an immune response in the lung mucosae, where the bacteria are located. What is the impact?Parenteral immunization with new vaccines targeting NTHi is able to elicit immune defense at the level of lung mucosae.Now that antibodies can be measured in sputum, new vaccines against COPD exacerbations or other lung infections can be tested for efficacy in the actual target tissue.Also, lung immunity against specific pathogens can now be tested. Parenteral immunization with new vaccines targeting NTHi is able to elicit immune defense at the level of lung mucosae. Now that antibodies can be measured in sputum, new vaccines against COPD exacerbations or other lung infections can be tested for efficacy in the actual target tissue. Also, lung immunity against specific pathogens can now be tested. What is new?We determined that antigen-specific antibodies were present in the lungs after vaccination; these were assessed in sputum after vaccination with NTHi surface antigens.NTHi-specific IgG were present in the lungs and appeared to have arrived there primarily by transudation, a type of leakage from the serum to the lung mucosae.Transudation appeared to be stronger in severe than in moderate COPD patients. We determined that antigen-specific antibodies were present in the lungs after vaccination; these were assessed in sputum after vaccination with NTHi surface antigens. NTHi-specific IgG were present in the lungs and appeared to have arrived there primarily by transudation, a type of leakage from the serum to the lung mucosae. Transudation appeared to be stronger in severe than in moderate COPD patients.</p

H1⁺ IgG⁺ MBCs frequencies measured by flow-cytometry and by ELISPOT correlated linearly.

<p><b>A–B.</b> Specificity of the staining with the rH1 bait from A/Solomon Island/3/06. PBMCs (1.6×10⁸) from anonymous blood donors were stained with Live/Dead, incubated with an H3N2 mono-bulk vaccine subunit (from A/Panama/2007/1999), and then stained with Alexa647-conjugated HSA (6×10⁷), or Alexa647-conjugated rH1 (1×10⁸), and with an antiCD20 mAb. <b>A.</b> Binding pattern of HSA (A647-HSA; left panel) and of rH1 (A647-rH1; right panel) in the CD20⁺ B-cell gates. <b>B.</b> H1⁺ B-cells identified in A were sorted (n = 8215), mixed with autologous CD20^neg cells in the ratio of 1∶50 and activated with CpG and IL-2 for 5 days <i>in vitro</i>. Unsorted PBMC and CD20^neg cells were also cultured in the same manner as controls. After 5 days, equal numbers of cultured cells were harvested and assayed by ELISPOT for numbers of cells secreting IgG and IgG specific for H1N1 (from A/Solomon Island/3/06). <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070620#s2" target="_blank">Results</a> are expressed as number of antibody secreting cells (ASC) normalized to 10⁶ cultured cells assayed by ELISPOT. Nd indicates undetectable ASC. <b>C.</b> Distribution of IgG⁺ B-cells among H1^neg and H1⁺ B cells expressing or not the CD27 B cell memory marker; shown is one representative subject. <b>D.</b> Replicates of frozen PBMCs from 4 anonymous blood donors were assayed by conventional ELISPOT, or incubated with an H3N2 mono-bulk vaccine subunit and stained with rH1, and anti-CD20 plus anti-human IgG antibodies. The scatter plot depicts paired values of H1⁺ IgG⁺ B-cell frequencies measured by flow-cytometry (y-axis) and by ELISPOT (x-axis) across three different experimental sessions. Shown are: the regression line with the related 95% confidence interval (gray areas), slope, intercepts, R² and p-value. <b>E.</b> Variability plot showing mean standard deviations of the measurements done by ELISPOT and flow-cytometry. The three dotted lines mark the grand mean and the upper and lower control limits.</p

Simultaneous identification of B lymphocytes specific for HA from different influenza strains in <i>ex vivo</i> PBMCs samples.

<p><b>A.</b> PBMCs from an anonymous blood donor were pre-incubated with subunits from either B/Brisbane/60/2008 or A/Panama/2007/1999 (H3N2), and then stained with anti-CD20 mAb, HSA conjugated with A488 and A647, with A647-rH3 (from A/Brisbane/10/2007) and A488-rH1 (from A/California/07/09), or with A647-rB/HA (from B/Brisbane/60/2008) and A488-rH1 (from A/California/07/09). The staining patterns observed in the CD20⁺ B-cell gate are shown. <b>B.</b> PBMCs from 16 anonymous blood donors were pre-saturated with B/Brisbane/60/2008 and then stained with anti-CD20 mAb, A647-rH3 (from A/Brisbane/10/2007) and A488-rH1 (from A/California/07/09). The scatter plot depicts paired values of H1⁺ (y-axis) and H3⁺ (x-axis) B-cells. The insert box plot depicts the distribution of H1⁺, H3⁺ and H1⁺H3⁺ B-cells in the same 16 donors. Mean values are indicated by dotted lines.</p

Vaccination induced changes in the pool of H1+ B-cells.

<p>PBMCs samples collected before (day 0) and at 3 and 6 weeks after vaccination from four seasonal influenza vaccinees, were pre-incubated with H3N2 subunit (from A/Panama/2007/1999) and then stained with rH1 A/Solomon Island/3/06) and mAbs anti-CD20, anti-CD27 and anti-human IgG. <b>A.</b> Dot plots gated on CD20⁺ B-cells showing the distribution of H1⁺ (middle panels) and H1^neg (bottom panels) B-cells from donor #a across: the mature memory (CD27⁺) and putatively naive (CD27^neg) CD20+ B-cell subsets (upper panels); un-switched mature memory (CD27⁺IgG^neg), IgG-switched mature (CD27⁺IgG⁺) and immature (CD27^negIgG⁺) memory B-cells. <b>B.</b> Numbers of circulating H1⁺ CD20⁺ B-cells in 4 vaccinees before and at 3 and 6 weeks after seasonal vaccination are overlaid with paired titers of antibodies inhibiting virus-induced hemmaglutination measured in their blood. The frequencies of H1⁺ B-cells are normalized according to the frequencies of CD20⁺ B-cells in 10⁶ PBMCs. <b>C.</b> Distribution of circulating H1⁺ and H1^neg B-cells across same subsets identified in <b>B</b> in all vaccinees.</p

Identification of B lymphocytes specific for HA from A and B influenza strains in <i>ex vivo</i> PBMCs samples.

<p>PBMCs from different anonymous blood donors were pre-incubated with vaccine mono-bulk subunits from the B/Brisbane/60/2008 (B/HA pretreatment), or the H3N2 A/Panama/2007/1999 strain (A/HA pretreatment) and then stained HSA, rH3 (from A/Brisbane/10/2007), rH1 (from A/California/07/2009), or B/HA (from B/Brisbane/60/2008), as indicated. <b>A.</b> Staining pattern observed on CD20⁺ cells in PBMCs stained with the different rHA bait. The rectangular gates identify brilliant HA+ B-cells; the dotted vertical lines mark the gates used to sort HA⁺ B-cells for the ELISPOT assays. <b>B.</b> Expression of the CD27 memory marker on HA⁺ and HA^neg B cells identified based on the sorting gates. <b>C.</b> H3⁺ (n = 15,234), H1⁺ (n = 6482) and B/HA⁺ (n = 26,803) B-cells identified in A were sorted, mixed with autologous CD20^neg cells (in the ratio of 1∶20, 1∶100 and 1∶33) and activated with CpG and IL-2 for 5 days <i>in vitro</i>. Unsorted PBMCs and CD20^neg cells mixed with HA^neg B cells were also cultured in the same manner, as controls. After 5 days cultured cells were harvested and assayed by ELISPOT for the number of cells secreting IgG and IgG specific for mono-bulk subunits from the vaccine strain homologous to the sorting bait. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070620#s2" target="_blank">Results</a> are expressed as numbers of antibody secreting cells (ASC) normalized to 10⁶ cultured cells assayed by ELISPOT. Nd indicates undetectable ASC.</p

Molecular cloning of HA⁺ B lymphocytes.

<p>PBMCs from 4 anonymous blood bank donors were stained as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070620#pone-0070620-g004" target="_blank">Figure 4B</a>. Single H1⁺, H3⁺, or H1^negH3^neg CD20⁺ B-cells were sorted to perform molecular cloning and analysis of their paired V_HV_L Ig regions as described in Material and Methods section. <b>A–E.</b> Distribution of V_H (A), D_H (B), J_H (C), V_k (D) and J_k (E) gene use across arrays of B-cells sorted from each donor (16 and 18 HA⁺ clones from donors #1 and #2; 35 HA⁺ and 20 HA^neg clones from donor 3; 16 HA⁺ and 16 HA^neg clones from donor #4. <b>F–I.</b> Number of mutations in H1⁺ and H1^neg CD20⁺ B-cells from donors #3 and #4, which cause dissimilar (F, H) or similar (G, I) amino acid substitutions in V_H (G,I) and V_L (F,H). NS and ** indicate not significant, or significant (p<0.036) difference between mean numbers of mutations by one-way Wilcoxon non-parametric test.</p