Low quality antibody responses in critically ill patients hospitalized with pandemic influenza A(H1N1)pdm09 virus infection

Although some adults infected with influenza 2009 A(H1N1)pdm09 viruses mounted high hemagglutination inhibition (HAI) antibody response, they still suffered from severe disease, or even death. Here, we analyzed antibody profiles in patients (n = 31, 17–65 years) admitted to intensive care units (ICUs) with lung failure and invasive mechanical ventilation use due to infection with A(H1N1)pdm09 viruses during 2009–2011. We performed a comprehensive analysis of the quality and quantity of antibody responses using HAI, virus neutralization, biolayer interferometry, enzyme-linked-lectin and enzyme-linked immunosorbent assays. At time of the ICU admission, 45% (14/31) of the patients had HAI antibody titers ≥ 80 in the first serum (S1), most (13/14) exhibited narrowly-focused HAI and/or anti-HA-head binding antibodies targeting single epitopes in or around the receptor binding site. In contrast, 42% (13/31) of the patients with HAI titers ≤ 10 in S1 had non-neutralizing anti-HA-stem antibodies against A(H1N1)pdm09 viruses. Only 19% (6/31) of the patients showed HA-specific IgG1-dominant antibody responses. Three of 5 fatal patients possessed highly focused cross-type HAI antibodies targeting the (K130 + Q223)-epitopes with extremely low avidity. Our findings suggest that narrowly-focused low-quality antibody responses targeting specific HA-epitopes may have contributed to severe infection of the lower respiratory tract.

These data suggested that infections with CA/09-like viruses induced low quality anti-HA antibody responses during the early clinical course in most (61%) of the ICU patients.
Taken together, we found that most patients who had HAI antibody titers of ≥ 80 at various time points post ICU admission presented or developed focused HAI and/or anti-HA-head binding antibodies targeting different epitopes including the 120-loop, 130-loop 140-loop, 150-loop, 160-loop, 190-helix, and/or 220-loop. Almost all patients who suffered fatal outcomes (except #19) had extremely focused HAI antibody responses.
Lastly, we tested the sera in IgG-ELISA assays using CA/09 rHA-head, BR/60 rHA-head, and 6 purified viruses (Fig. 4c)   We completed two independent antibody adsorption assays. (c) Paired sera were tested by IgG-ELISA using two rHA-heads from CA/09 or BR/60 and 6 purified A(H1N1) viruses. IgG titers are shown in yellow or light blue bars for S1 and orange or dark blue bars for the second sera. We completed two independent ELISA assays. *Patient number (fatal patients in red) with birth year. † Serum collection days post-symptom onset (dpo).  (Fig. 4c). Taken together, our data indicated that infection with CA/09-like viruses induced cross-subtype, even crosstype HAI antibody responses targeting (K130 + Q223)-epitope(s) in some ICU patients. However, only cross-type antibodies displayed lower IgG antibody titers for wt-CA/09 compared to pre-pandemic A(H1N1) viruses and/or rHA-head of IBV. Such low-avidity cross-type antibodies likely contributed to fatal outcomes in some patients.

Discussion
Our findings provide insight into serum antibody profiles of patients with severe influenza A(H1N1)pdm09 virus infection during 2009-2011. At ICU admission, low-quality antibody responses, including extremely focused HAI antibody responses targeting specific epitopes on HA-head, non-neutralizing antibody responses targeting HA-stem, and/or low titers of HA-specific IgG1, were detected in these patients (Figs. 1, 2, 3, 4, 5 and Tables 2,  3). In addition, high titers but very low-avidity RBS-targeted antibodies that cross-reacted with influenza B viruses were detected in most patients with fatal outcome (Tables 1, 2, 3 and Fig. 4). Consistent with previous studies 18,39 , bacterial pneumonia was identified in only 9 patients, including 4 patients at 1-2 dpicu and 5 patients at 8-45 dpicu (Table 1). Therefore, low quality antibody responses particularly at early stage of illness may have contributed to the severe lung infections and fatal outcomes in most of these patients, although secondary bacterial infections also increased morbidity and mortality in some patients. CA/09-like virus infection induced focused HAI and anti-HA-head binding antibody responses targeting in or around the RBS in 65% of patients (Table 3, Fig. 3, and Supplementary Fig. 2). Notably, 4 patients (#2, #7, #12, and #14) exhibited nearly all focused HAI antibodies or completely focused anti-rHA-head binding antibodies as early as 1-3 dpicu (2-5 dpo). Although A(H1N1)pdm09 virus shedding was detected in some patients with high HAI antibody titers (data not shown), these viruses were unfortunately not sequenced. Thus, whether such focused neutralizing antibodies could select escape mutants to evade host immunity is unknown. However, selection of escape mutants by human monoclonal antibodies (hmAbs) targeting the epitopes involving D127, K130, G155, K163, and by human anti-sera possessing focused K163-antibodies has been reported 9,41-44 . Highly focused antibody responses targeting epitopes that are absent on infecting viruses or newly formed escape mutants may not be able to aid in the protection against further infection, virus clearance from infected cells, and attenuation of disease severity 31,32 .
Fulminant influenza with acute respiratory failure as early as 0 dpo were observed in deceased patient #2 with mild obesity and patient #14 without any comorbidity (Table 1). Two patients did not display concurrent or secondary bacterial pneumonia (Table 1). Focused low-avidity HAI antibodies targeting the epitopes possessing HA-(K130 + Q223) were detected in 2 patients as early as 2-3 dpo (Fig. 1, Tables 2, 3). Surprisingly, the (K130 + Q223)-antibodies cross-reacted with both B-Yam and B-Vic IBVs (Fig. 4). The low-avidity (K130 + Q223)-antibodies were also detected in other 2 deceased patients (#1 and #25) ( Tables 1, 2, 3, and Supplementary Table 3). To our knowledge, such RBS-targeting cross-type cross-reactive antibodies that could contribute to detrimental clinical outcomes have not been previously reported 45 , although a few rare human memory B cell clones which produced cross-type IgG have been isolated 27 . Nonetheless, some cross-subtype RBS-targeted hmAbs have been characterized; these hmAbs penetrated the RBS pocket using their unusually long HCDR3 loop to block virus attachment by direct competition with sialic acid host receptors [25][26][27] . If the binding affinity between the antibody and the viral RBS was lower than the binding affinity between the viral RBS and its natural sialic acid receptors on host cells, failure of antibody-mediated protection may occur. Notably, patients #2 and #14 had HAI antibody titers of ≥ 1280 as early as 3 dpo ( Table 2), suggesting that these (K130 + Q223)-antibodies did not prevent infection from CA/09-like viruses. Our data also indicated that HAI and VN assays can effectively detect very low-avidity antibodies likely with no protective function in humans (Table 1 and Fig. 1). Thus, other immunological measures should also be considered to assess the antibody quality and correlates of protection.
Seven critically ill patients displayed focused HAI antibodies targeting the epitopes shielded by glycosylation at HA-127 (Table 3 and Supplementary Fig. 2). The hmAb EM4C04, which was isolated from a previously healthy adult with severe A(H1N1)pdm09 virus infection, can select escape mutants possessing HA-D127E change; such mutant viruses displayed altered receptor specificity and enhanced virulence in mice 24,42 . Any emergence of HA-127 mutation in A(H1N1)pdm09 viruses and their effect on viral pathogenicity should be closely monitored in humans.
Some patients displayed various levels of non-HAI and non-neutralizing antibodies only targeting HA-stem in S1 samples (Fig. 1). One deceased patient #19 exhibited high levels of non-neutralizing antibodies in S1 (7 dpo) targeting both HA-head and HA-stem domains (Fig. 1). Although neutralizing anti-HA-head antibodies can provide protection against influenza in humans, and neutralizing anti-HA-stem antibodies can provide protection against influenza in mice and ferrets in an Fc-receptor mediated manner 6,7,16,22,23 , it has also been reported that some non-neutralizing anti-HA-head and anti-HA-stem antibodies can enhance viral fusion activity and lead to antibody-dependent enhancement (ADE) of pneumonia disease in mice and pigs 11,13 . Therefore, high-affinity neutralizing antibodies often are beneficial, but some non-neutralizing antibodies may exacerbate the disease.
Influenza virus infection and vaccination usually induce dominant-IgG1 responses, which is important for preventing influenza pneumonia 4,[46][47][48] . Surprisingly, only 19% of the patients exhibited dominant-IgG1 responses (Fig. 5). Most patients showed dominant-IgA or IgA co-dominant with IgG1, IgG3 and/or IgM (Fig. 5). Serum IgA cannot be transported into the respiratory secretions 14,49,50 . The role of serum IgA in protection and pathogenesis of lung disease is still poorly understood. Additionally, some patients displayed high levels of IgG3 ( Fig. 5 and Supplementary Table 4), that have been associated with ADE disease in other viral infections 51 .
Anti-NA antibodies can reduce disease severity 2,36 . Unexpectedly, most the patients had high levels of NAI antibodies, even in those with fatal outcomes (Fig. 1 and Table 2). Our previous study indicated that some crossreactive anti-NA antibodies induced by historical A(H1N1) viruses failed to reduce disease severity against novel IAV in mice 52 . More studies are needed to understand why high NAI antibodies failed to prevent severe disease in these patients. www.nature.com/scientificreports/ At ICU admission, comorbidities were present in 81% of the patients, including obese, chronic lung disease, immunosuppression, and/or pregnancy (Table 1). Comorbidities such as obesity can have negative impact on virus-induced innate and adaptive immunity [53][54][55] . However, the 20 obese patients showed similar levels of anti-HA antibody responses as compared to the 11 non-obese patients (Table 1 and Supplementary Fig. 1). Overall, the levels of HAI and VN antibody responses in the most ICU patients with comorbidities were higher than those in the non-ICU patients infected with 2009 A(H1N1)pdm09 virus 40 , and most non-ICU patients did not have comorbidities. These data suggested that antibody quality but not quantity play an important role for disease severity.
There are several limitations in this study. First, it is challenging to collect multiple time-points of sera from ICU patients, therefore we were only able to include a small number of ICU patients in our current study, which did not allow further statistical analysis. Second, serum samples from age-matched mildly ill patients collected during 2009 A(H1N1)pdm09 pandemic were not available for comparison. Third, immune cells and other clinical samples were not collected in 2009. Thus, whether the lack of activation of other antiviral responses, such as reduced levels of IFNs, aberrant cell-mediated immunity, or exaggerated expression of proinflammatory cytokines and chemokines which can play critical roles in disease severity 18,20 , were not investigated here. Finally, the role of ADE should be considered in future antibody-quality study.
In summary, we observed multiple forms of low quality anti-HA antibody responses in severely ill patients infected with A(H1N1)pdm09 virus, especially during early stage of illness onset. Some patients showed extremely immunodominant HAI antibodies with very low-avidity or targeting the specific epitopes that are likely associated with selecting escape mutants. Others displayed dominant non-neutralizing antibodies with possibility of ADE of lung disease. Some patients also displayed IgA-dominant, but not IgG1-dominant antibody responses. Therefore, we conclude that low quality and/or narrowly focused antibody responses to CA/09 HA, especially during the early stage of the clinical course, along with comorbidities have contributed to severe infection of lung and progression to severe influenza. More studies are needed to advance our understanding of overall antibody quality in patients with different severity to inform the development of improved antibodybased immunotherapies and universal vaccines against influenza.

Material and methods
Patient enrollment and serum collection. During 2009 and 2011, Canadian ICU physicians established a multicenter cohort of critically-ill adolescents and adults hospitalized with laboratory-confirmed influenza A(H1N1)pdm09 virus infection 39 . Blood samples were collected when patients stayed at the ICUs (Table 1). All thirty-one patients (17-65 years old) admitted to ICUs with RT-PCR or serology confirmed influenza A(H1N1)pdm09 virus infection with available stored sera (at − 20 °C) were included in the current study. All patients provided informed consent for specimen collection and storage of sera for future analysis, informed consent was obtained from legal guardian(s) of the deceased. The study was approved by the National Center for Immunization and Respiratory Diseases, Centers for Disease Control and Prevention human subject research determination ethic committee review. All methods were carried out in accordance with relevant guidelines and regulations 56,57 . Influenza viruses. All viruses were propagated in embryonated eggs. Some viruses were purified on a liner sucrose gradient. Four viruses were generated by reverse genetics (RG), including the virus possessing wt-CA/09 HA, NA and 6 A/Puerto Rico/8/1934 (PR8) internal genes 30 . All viruses were sequenced, details of RG-viruses information are described in Supplementary Table 1.
Hemagglutination inhibition (HAI) assay. Sera were treated with receptor-destroying enzyme (RDE, Denke-Seiken, Japan) to remove non-specific inhibitors, and adsorbed with packed turkey red blood cells (TRBCs) to remove non-specific agglutinins prior to testing with 4 HA units of virus and 0.5% TRBCs (World Health Organization manual).
Traditional virus neutralization (VN) assay. Two-fold dilutions of RDE-treated sera were incubated with 100 TCID 50 of virus at 37° for 1 h. Madin-Darby Canine Kidney (MDCK) cell monolayers in 96-well plates were washed 3 times with phosphate buffered saline (PBS). After 1 h, the virus-serum mixtures were supplemented with 1 µg/ml Tosyl phenylalanyl chloromethyl ketone (TPCK)-treated trypsin and 1% bovine serum albumin (BSA) were added to MDCK cells and incubated at 37 °C for 3 days. The neutralization antibody titer is the highest serum dilution demonstrating complete neutralization in which no HA titer in supernatants was detected.
Antibody adsorption. Serum was mixed with ~ 10 5 HAU of purified virus or PBS as a control. After incubating for about 2 h at 4 °C, the virus-serum mixture was centrifuged for 45 min at 100,000g to remove virusantibody complexes and most of the unbound viruses. Residual viruses were removed by the addition of 100 µl of packed TRBCs 30 .
Enzyme-linked lectin assay (ELLA). NA inhibition (NAI) antibodies were detected using ELLA as described previously 58 . H6N1 reassortant virus with N1 from CA/09 and a mismatched HA from A/turkey/Massachusetts/3740/1975 H6 virus was used. Briefly, sera were first heat inactivated. Serial twofold diluted sera were then incubated with A(H6N1) virus in plates coated with fetuin for 16-18 h. Following incubation, HRP-labeled peanut agglutinin (lectin) was added to the reaction and incubated for 2 h, followed by tetramethylbenzidine (TMB) substrate to reveal enzymatic cleavage of fetuin by viral NA. The percent inhibition of NA enzymatic activity was calculated by comparing with values from virus control wells. Endpoint NAI antibody titers were calculated as the reciprocal of the highest dilution with at least 50% inhibition.

Disclaimer
The findings and conclusions in this report are those of the authors and do not necessarily represent the official position of the Centers for Disease Control and Prevention.