Pre-existing hemagglutinin stalk antibodies correlate with protection of lower respiratory symptoms in flu-infected transplant patients

Report Pre-existing Hemagglutinin Stalk Antibodies Correlate with Protection of Lower Respiratory Symptoms in Flu-Infected Transplant Patients Graphical Abstract Highlights dSolid organ transplant recipients (SOTRs) had low levels of HAI antibodies at baseline dSOTRs have high levels of pre-existing, broadly crossreactive anti-HA stalk antibodies dAnti-HA stalk antibodies correlate with lack of lower respiratory symptoms in SOTRs dPresence of lower respiratory symptoms is associated with influenza pneumonia Authors Teresa Aydillo, Alba Escalera, Shirin Strohmeier, ..., Florian Krammer, Adolfo Garcı ´a-Sastre, Elisa Cordero Correspondence [email protected] (A.G.-S.), mariae.cordero.sspa@juntadeandalucia. es (E.C.) In Brief Aydillo et al. identify lower respiratory symptoms (LRSs) as a predictor of influenza pneumonia in a cohort of transplant recipients. When pre-existing immunity was characterized, the levels of anti-HA stalk antibodies correlated independently with protection from lower respiratory infection. Aydillo et al., 2020, Cell Reports Medicine 1, 100130 November 17, 2020 ª2020 The Authors. https://doi.org/10.1016/j.xcrm.2020.100130 ll Report Pre-existing Hemagglutinin Stalk Antibodies Correlate with Protection of Lower Respiratory Symptoms in Flu-Infected Transplant Patients Teresa Aydillo, 1,2 Alba Escalera, 1,2 Shirin Strohmeier, 1 Sadaf Aslam, 1,2 Javier Sanchez-Cespedes, 3,13 Juan Ayllon, 1,2,15 Cristina Roca-Oporto, 3,13 Pilar Perez-Romero, 4,13 Miguel Montejo, 5,13 Joan Gavalda, 6,13 Patricia Munoz, 7,13 Francisco Lopez-Medrano, 8,13 Jordi Carratala, 9,13 Florian Krammer, 1 Adolfo Garcı ´a-Sastre, 1,2,10,11,14, * and Elisa Cordero 3,12,13, * 1 Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, NY, USA 2 Global Health and Emerging Pathogens Institute, Icahn School of Medicine at Mount Sinai, New York, NY, USA 3 Clinical Unit of Infectious Diseases, Microbiology and Preventive Medicine, Institute of Biomedicine of Seville, Virgen del Rocı ´o University Hospital/CSIC/University of Seville, Seville, Spain 4 National Center for Microbiology, Instituto de Salud Carlos III, Majadahonda, Madrid, Spain 5 Unit of Infectious Diseases, University Hospital Cruces, Bilbao, Spain 6 Vall d’Hebron University Hospital, Barcelona, Spain 7 Department of Clinical Microbiology and Infectious Diseases, University General Hospital Gregorio Maran ˜o ´n, Instituto de Investigacio ´n Sanitaria Gregorio Maran ˜o ´n, CIBER Respiratory Diseases, Department of Medicine, Complutense University, Madrid, Spain 8 Unit of Infectious Diseases, Research Institute Imas12/University Hospital 12 de Octubre, Complutense University, Madrid, Spain 9 Department of Infectious Diseases, Bellvitge University Hospital, Bellvitge Biomedical Research Institute (IDIBELL), University of Barcelona, L’Hospitalet de Llobregat, Barcelona, Spain 10 Department of Medicine, Division of Infectious Disease, Icahn School of Medicine at Mount Sinai, New York, NY, USA 11 The Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, New York, NY, USA 12 Department of Medicine, University of Seville, Seville, Spain 13 Spanish Network for Research on Infectious Diseases (REIPI), RD16/0016, Carlos III Health Institute, Madrid, Spain 14 Lead Contact 15 Present address: Department of Health Sciences, University of Burgos, Burgos, Spain *Correspondence: [email protected] (A.G.-S.), [email protected] (E.C.) https://doi.org/10.1016/j.xcrm.2020.100130 SUMMARY Hemagglutination-inhibitory antibodies are usually highly strain specific with little effect on infection with drifted or shifted strains. The significance of broadly cross-reactive non-HAI anti-influenza antibodies against conserved domains of virus glycoproteins, such as the hemagglutinin (HA) stalk, is of great interest. We characterize a cohort of 40 H1N1pmd09 influenza-infected patients and identify lower respiratory symptoms (LRSs) as a predictor for development of pneumonia. A binomial logistic regression of log10 pre-existing antibody values shows that the probability of LRS occurrence decreased with increased anti-HA full-length and stalk antibody ELISA titers. However, a multilevel logistic regression model adjusted by other potential serocorrelates demonstrates that only antibodies directed against the stalk of HA correlate with protection from lower respiratory infection, limiting disease progression. Our predictive model indicates that a threshold of protective immunity based on broadly cross-reactive HA stalk antibodies could be feasible. INTRODUCTION The effect of influenza epidemics on society, including morbidity, mortality, and economical consequences, is still a major challenge and a public health concern. 1,2 The World Health Organization (WHO) estimates that about 3–5 million cases of severe disease and 290,000–650,000 deaths annually are caused by influenza disease. Vulnerable populations, such as solid organ transplant recipients (SOTRs), are at high risk of severe outcomes with mortality rates from 5%–8% and complications in 5%–20% of cases. 3,4 Although annual influenza vaccination can help reduce the burden of influenza disease, 4,5 its efficacy varies widely. 6–8 Currently licensed influenza vaccines are formulated to mainly induce strain-specific antibody responses against the major surface glycoprotein of influenza virus, hemagglutinin (HA). However, influenza viruses have the ability to accumulate yearly mutations in HA, favoring emergence of antigenic variants in a phenomenon known as antigenic drift. This leads to the regular need to update influenza vaccines according to the circulating viruses in humans. 9 Although cross-reactivity between antigenically related influenza viruses can happen, no protection would be provided by seasonal influenza vaccines in the case of emergence of a shifted pandemic strain, like what happened in 2009. 10,11 Although the globular HA head domain Cell Reports Medicine 1, 100130, November 17, 2020 ª2020 The Authors. 1 This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). ll OPEN ACCESS is immunodominant, eliciting potent neutralizing antibodies that can be measured by a hemagglutination inhibition (HAI) assay, 12 it is rather tolerant and permissive to mutations, 13 enabling seasonal influenza viruses to escape pre-existing immunity in humans. Paradoxically, the HA head is the main target of influenza vaccines, and HAI titers in serum are traditionally used as a correlate of influenza vaccine-induced protection; a HAI titer of more than 40 is considered to provide protection in humans. 14 In contrast, antibodies targeting other conserved regions of the HA surface protein, such as the stalk domain, can bind to a variety of influenza subtypes. 12,15 In addition to being cross-reac- tive, HA stalk antibodies can also mediate Fc-Fc receptor (FcR) effector functions, such as antibody-dependent cell cytotoxicity (ADCC), potentially contributing to protection from disease through Fc-FcR interactions and engagement that results in apoptosis of infected cells and secretion of antiviral cytokines and chemokines. 16,17 Antibodies targeting neuraminidase (NA), the second surface glycoprotein of influenza viruses, might also be more cross-protective than traditional HAI antibodies. 18,19 A growing interest regarding development of a universal influenza vaccine capable to provide long-term protection in humans against multiple strains and subtypes of the virus, including potential emerging pandemic strains, is guiding intense research efforts to identify new vaccine candidates and, therefore, new correlates of protection for influenza infection and disease. 9,20–22 Several approaches have been proposed, including targeting conserved regions of the HA surface protein, such as the stalk domain. 15,21,23 Although a few human studies have suggested that HA stalk and NA antibodies are additional independent correlates of protection from influenza infection, different from HAI antibodies, 18,22,24 more clinical and epidemiological data are needed to demonstrate antibody association with severe/mild disease versus infection and transmission. Studies in SOTRs might provide valid information regarding correlates of immune protection against influenza disease. First, severe disease outcomes are more frequent in such patients, 3,4 which reduces the number of individuals in the cohort required to identify a significant number of cases with different disease outcomes. Second, the need to constantly monitor the health status of SOTRs allows frequent clinical assessment of infectious diseases, including influenza virus infection, and monitoring of immune responses to influenza vaccine and/or infection at baseline and post-vaccination/infection. Baseline antibody responses in humans depend on the first encounter with influenza virus antigens, usually at an early age, and on repeated exposure during the lifetime through repeated infection and vaccination. 25,26 Because of the age of SOTRs, one expects diverse antibody responses in these individuals against different viral strains because of their differences regarding virus and vaccine exposure. Antibody responses induced by natural infection compared with vaccination can differ, with induction of broader and longer-lived antibody responses in the first. Here we aimed to identify whether antibodies against conserved or non-conserved antigens of influenza virus glycoproteins (HA head, HA stalk, and NA) correlate with the burden of influenza H1N1pdm09 disease in a clinical cohort of naturally influenza virus-infected SOTRs. We sought to identify anti-influenza antibody levels of protection by characterizing risk factors of severe outcomes, such as development of lower respiratory infection in SOTRs and determining how broadly and non-broadly pre-existing specific influenza antibodies correlate with clinical outcome. Last, we investigated which antibody levels can predict protection from severe clinical outcomes after adjustment for confounders, including other potential serocorrelates. RESULTS SOTRs Show High Levels of Pre-existing Anti-HA Antibodies A total of 127 SOTRs were included in the naturally influenza infected cohort between 2010 and 2013 (Figure S1A). Of the 127 SOTRs, 118 had a positive RT-PCR for influenza virus, and a total of 74 (62.7%) had paired serum samples at enrollment (baseline) and convalescent time points. Of those, 40 (54%) were H1N1pdm09, 23 (30%) were H3N2, and 11 (16%) were influenza B virus infections. The levels of pre-existing HAI active antibodies at onset of the infection in the influenza A cases were 14 (35%) and 12 (53%) for H1N1pdm09 and H3N2, respectively. For this report, only H1N1pdm09-infected SOTRs with a full set of serum samples were considered. High levels of HAI antibodies in SOTRs with H3N2 infection limited assessment of the potential role of non-HAI and NA antibodies; low levels of HAI in H1 influenza cases allowed us to investigate the effect of other potential serocorrelates. No influenza B cases were included because of the limited sample size and change of predominantly circulating influenza B virus strains between seasons. Serum samples from 9 SOTRs with suspected influenza virus infection (but who tested negative by RT-PCR) were included for comparison. No follow-up samples were collected from these patients. Most of the H1N1pdm09- infected cohort represented individuals enrolled in the 2010– 2011 post-pandemic season (34, 85%); four (10%) and two (5%) patients were enrolled in the 2011–2012 and 2012–2013 influenza seasons, respectively. More SOTRs were men (57.7%), and the median age was 57 years (interquartile range, 27–77 years). The type of transplant was kidney in 22 cases (55%), liver in 10 (25%), and heart in 6 (15%). Only 2 (5%) had a lung transplantation. Comorbidities such as diabetes or other chronic diseases were present in 72.5% of cases (Figure S1B). Baseline antibody titers (Figure 1A) demonstrated high levels of pre-existing anti-influenza stalk and full-length HA; only a few SOTRs (9, 22.5%) had HAI titers of 40 or higher. Interestingly, pre-existing antibody titers against the HA and NA protein showed high associated ADCC activity (Figure 1B). HA Stalk Antibodies Correlate with Protection against LRSs in Influenza Virus-Infected SOTRs Twenty-five (62.5%) SOTRs required hospitalization. All naturally influenza-infected SOTRs received antiviral therapy with oseltamivir. Most of them (33, 82.5%) started treatment within 48 h of onset of symptoms (mean; 95% confidence interval [CI], 1.7; 0.2–3.1 days). A high proportion of patients reported upper respiratory and systemic symptoms, such as headache, fever, or 2Cell Reports Medicine 1, 100130, November 17, 2020 Report ll OPEN ACCESS myalgia (26, 65% and 25, 62.5%, respectively) at enrollment. Acute lower respiratory symptoms, such as dyspnea or tachypnea, were noted in 18 (45%) SOTRs. Twelve (30%) patients developed pneumonia. Of those, four (33.3%) required admission to an intensive care unit (ICU), and two (16.6%) died. Coinfection was detected in one patient with pneumonia and one without: Streptococcus pneumoniae and Pseudomonas aeruginosa, respectively. Clinical characteristics, treatment, and outcomes of the naturally influenza virus-infected SOTRs are shown in Figure S2A. The presence of upper respiratory and systemic symptoms was less common in pneumonia influenza virus-in- fected SOTRs. Conversely, SOTRs with acute LRSs had a higher risk of worsening influenza virus infection and pneumonia development; 50% (9 of 18) SOTRs with LRSs had pneumonia, and only 13.6% (3 of 22) of SOTRs without LRSs developed viral pneumonia (odds ratio [OR] 95% CI, 6.3; 1.3–29.2; p = 0.01). No other risk factors for development of pneumonia were found (Figure S2B). Bivariate analysis of the multiple serological surrogates demonstrated no statistical differences in levels of circulating pre-existing antibodies at baseline in SOTRs with upper respiratory or systemic symptoms, but an inverse correlation was found when we compared geometric mean titers (GMTs) in SOTRs with LRSs. As shown in Figures 1C–1F and Table 1, patients with LRSs had lower GMTs against the full-length (6,170 versus 15,746; p = 0.006) and stalk (12,834 versus 21,558; p = 0.03) HA protein, as measured by ELISA. More importantly, the currently accepted correlate of protection, HAI titer, was similar in patients regardless of symptomatology. Although 25 patients (62.5%) received the corresponding season-matched influenza vaccine at least 6 weeks before the episode of influenza virus infection, only 14 (56%) of the influenza vaccinated SOTRs had pre-existing detectable levels of HAI antibodies, and only 9 (36%) of them were scored as seroprotected (HAI titers R40) (Figure 1A). AB CDE F Figure 1. Immunological Features in the H1N1 Influenza-Infected SOTR Cohort (A and B) Pre-existing antibody titers in SOTRs with H1N1pdm09 influenza virus infection (n = 40) compared with non-infected matched SOTRs controls (n = 9). The antibody level for each patient, GMT, and 95% CI are shown (A) for the indicated ELISA assay on the left y axis (full-length HA, stalk HA, and NA recombinant proteins as antigen) and HA-inhibiting (HAI) antibody titers on the right y axis. The HAI assay was performed using the whole H1N1pdm09 reference strain virus. (B) shows antibody-dependent cellular cytotoxicity (ADCC), measured by a reporter assay for full-length HA, stalk HA, and NA. (C–F) Pre-existing antibody responses in SOTRs with LRSs at baseline. The antibody level for each patient and time point, GMT, and 95% CI are shown for (C) fulllength HA ELISA, (D) stalk HA ELISA, (E) HAI, and (F) NA ELISA (ns, not significant; ***p = 0.01; **p = 0.006; *p = 0.03). Experiments were performed in triplicates. Cell Reports Medicine 1, 100130, November 17, 2020 3 Report ll OPEN ACCESS We then selected presence of LRSs at baseline as a predictor of disease outcome in SOTRs to discriminate HA full length and stalk cutoff points correlating with protection from lower respiratory tract influenza virus infection. The plotted predicted probability curve for HA full length and stalk (OR, 0.13; 95% CI, 0.02– 0.74; p = 0.02 and OR, 0.06; 95% CI, 0.004–0.85; p = 0.03, respectively) demonstrated that the probability of developing LRSs decreased with increasing log10 values for titers, and 10 4 full-length and stalk antibodies showed 50% predicted protection in SOTRs (Figures 2A and 2B). The fitted curves of the relationship between log10 assay values and the probability of protection were next used to estimate protective thresholds and intervals of antibody titers for HA full length and stalk as 0– 10,000, 10,000–25,000, and more than 25,000. We then defined intervals of protection according to the proportion of patients with LRSs in those selected cutoffs. As shown in Figures 2C and 2D, only 25% of SOTRs (3 of 12) with HA stalk antibodies higher than 25,000 had LRSs; most SOTRs (80%, 8 of 10) with antibodies against the HA stalk lower than 10,000 presented LRSs at onset of the influenza episode. This was similar for antibodies against HA full length higher than 25,000. Receiver operating characteristic (ROC) curve analysis was also performed to estimate the sensitivity and specificity of HA full length and stalk ELISA titers for predicting LRSs. The results showed an area under the curve (AUC) of 77% and 76% for HA full length and stalk, with a sensitivity and specificity of predicting LRS of 60% and 70%, respectively (p < 0.005). We next performed a multivariate logistic regression analysis including our potential serological predictors (HA full length and stalk antibody titers) and adjusted by HAI and NA ELISA titers to assess the balance of the antibody responses that correlated independently with the protection afforded. Age, type of transplant, and influenza vaccination status were also included in the model to control variability between SOTRs (Figure S3). Immunosuppressive drugs were included in an alternative model, but no predictive value was found (data not shown). In this multilevel analysis, we categorized antibodies for HA full length and stalk antibody titers in the levels of protection observed (0–10,000; 10,000–25,000, and >25,000). Because mean NA antibody levels were 10 times lower than those for the HA protein at baseline (Figure 1A), we defined the intervals as 0–1,000, 1,000–2,500, and more than 2,500. HAI titer was categorized at two levels: less than 40 (no seroprotection) and 40 or more (seroprotection). The adjusted model shown in Figure 2D demonstrates that anti-HA stalk antibodies were associated independently with protection from development of acute LRSs (10,000–25,000; OR, 0.03; 95% CI, 0.01–0.83; p = 0.03 and >25,000; OR, 0.005; 95% CI, 0.00–0.62); no protection was observed for stalk titers lower than 10,000. Likewise, HA full length pre-existing titers showed no significant protection when adjusted by levels of stalk titers. A previous history of vaccination was also associated with lower risk of LRSs (OR, 0.02; 95% CI, 0.001–0.52; p = 0.01). Remarkably, a linear regression analysis demonstrated good correlation between values of anti-HA stalk antibodies measured by ELISA and stalk-mediated ADCC activity (Figure 2E; p < 0.0001), an important mechanism of antibody-mediated protection in humans. 27 Despite the fact that whole HA- and HA stalk-binding antibodies at baseline correlated inversely with LRSs and that LRS symptoms at onset were the best predictors of pneumonia, when the analysis of serocorrelates was restricted to prevention of development of pneumonia during the course of influenza virus infection, we found no direct association of any of the potential serological surrogates, suggesting that some other events, such as colonization with pathogenic bacteria, might also influence the outcome of pneumonia. Alternatively, our study did not have enough power to find correlations associated with development of pneumonia. Nevertheless, there was a clear correlation between HA stalk antibody titers and lack of LRSs. To determine whether influenza vaccination can also induce HA stalk antibodies in SOTRs, we compared the effect of vaccination versus infection in a second cohort of seasonal influenza-vaccinated Table 1. Pre-existing Antibody Levels in SOTRs According to Symptomatology Yes GMT (95% CI) No GMT (95% CI) p Value Upper Respiratory Symptoms HAI 10 (6–16) 15 (6–40) 0.62 HA hull length ELISA 8,918 (5,598–14,208) 13,568 (6,759–27,238) 0.45 HA stalk ELISA 16,325 (11,729–22,724) 18,545 (13,575–25,334) 0.68 NA ELISA 134 (48–368) 37 (7–196) 0.22 Systemic Symptoms HAI 12 (7–20) 12 (6–25) 0.9 HA full length ELISA 10,136 (5,700–18,023) 10,571 (63,04–17,725) 0.84 HA stalk ELISA 14,713 (10,371–20,873) 20,470 (15,030–27,877) 0.14 NA ELISA 109 (45–263) 63 (12–340) 0.77 Lower Respiratory Symptoms HAI 8 (5–15) 15 (7–29) 0.21 HA full length ELISA 6,170 (3,327–11,440) 15,746 (10,341–23,974) 0.006 HA stalk ELISA 12,834 (8,380–19,655) 21,558 (17,165–27,075) 0.03 NA ELISA 106 (27–408) 70 (21–237) 0.67 4Cell Reports Medicine 1, 100130, November 17, 2020 Report ll OPEN ACCESS SOTRs enrolled during the 2010–2011 and 2011–2012 influenza seasons and randomly selected 20 seasonal influenza-vacci- nated SOTRs for both influenza seasons (10 each). Both cohorts matched in age, type of transplant, comorbidities, immunosuppressive drug regimen, and time from transplantation. Overall, the humoral immune response was greater after natural influenza virus infection compared with vaccination (Figure S4). Although infection induced HAI activity, full-length HA and stalk binding, NA binding, and ADCC responses against influenza virus antigens, vaccination failed to significantly induce stalk- and NA- specific antibodies. SOTRs Show a Significant Increase of Anti-Influenza Antibodies after Infection To characterize the immune response after influenza disease, we also measured HAI and non-HAI antibody levels and associated ADCC activity in convalescent sera of SOTRs. As expected, a significant increase compared with baseline levels was shown after influenza infection for all potential serocorrelates (Figure 3A). To quantify induction of de novo antibody response according to disease outcome, we calculated fold induction log10 values in SOTRs. Overall, patients with LRSs, and likely lower respiratory infection, at hospital admission had higher induction of anti-influenza antibodies (Figures 3B–3E), although this was only statistically significant for de novo NA antibodies produced after influenza virus infection (Figure 3E). These antibodies showed a good correlation with NA-mediated ADCC activity (Figure 3F). In contrast, SOTRs that had HAI titers of 40 or higher A C EF D BFigure 2. Anti-Stalk HA Antibodies as an Independent Correlate of Protection against Development of LRSs (A and B) Binomial logistic regression of log10- transformed values of full-length and stalk HA: fitted curves of the relationship between log10 assay values and the probability of protection. A red dot indicates predicted protection of ~50%. (C and D) Bars shows the proportion of SOTRs with LRSs according to defined antibody cutoffs. Each bar shows the number and percentage of SOTRs with LRSs among the total number of SOTRs with each antibody interval. **p = 0.02. (E) Multivariate logistic regression model of factors correlated with LRSs at baseline on SOTRs with influenza infection. The model is adjusted by potential confounding factors to estimate the independent effect of antibody titers on disease progression and the serological predictors (Figure S3). The adjusted odds ratio (OR; 95% CI) for each assay and each level are shown. (F) Linear regression of log10-values of antistalk antibodies measured by ELISA and antistalk antibodies with mediated ADCC activity measured in a bioreporter assay. ADCC experiments were performed in triplicate. at baseline had lower levels of de novo antibodies against the HA and NA protein of influenza virus. Unexpectedly, a negative correlation between pre-exist- ing antibodies against the HA head with HAI activity and induction of full-length and stalk HA and NA titers after influenza virus infection was found (Figures 4A and 4B). Although HAI titers higher than 40 at hospital admission did not prevent severe disease in SOTRs, we found that SOTRs scored as seroprotected (HAI R40) at baseline had lower induction of HA (full length and stalk) and NA antibodies, measured by ELISA (Figures 4C and 4D) after the influenza episode. DISCUSSION Influenza virus HA-inhibitory activity has been traditionally acknowledged as a correlate of protection for influenza vaccine efficacy. However, head-specific HA-neutralizing antibodies have been only modestly associated with protection against infection with drifted and shifted influenza viruses in humans, and the role of heterosubtypic antibodies capable of inducing additional immune functions is not clear. Although the influenza virus continues to evolve every year because of antigenic drift, humans face new exposure to the influenza virus through infection and repeated vaccination, making immune responses complex and multifaceted. Here we present a detailed analysis of humoral immunological correlates associated with protection from disease progression in a clinically characterized cohort of 40 SOTRs naturally infected with the H1N1 influenza virus. We analyzed the levels of anti-influenza virus antibodies targeting the two surface glycoproteins HA and NA and the HA stalk domain in patient sera and their associated ADCC activity. Our Cell Reports Medicine 1, 100130, November 17, 2020 5 Report ll OPEN ACCESS results showed that pre-existing HA-binding antibodies (ELISA) were associated with a lower risk of development of LRSs in the setting of influenza virus infection; as the antibody titer increased, the frequency of symptoms decreased (Figures 2B and 2C). Indeed, our predictive model suggests that, among non-HAI antibodies, a stalk antibody titer higher than 10 4 ,as measured by ELISA, would suffice to protect at least 50% of patients (as shown in the predicted probability curve in Figures 2A and 2B). Even though the presence of LRSs at hospital admission was a potent predictor of development of pneumonia during the influenza episode, no differences in antibody titers were found when comparing pneumonia and SOTRs without pneumonia. It is likely that progression to pneumonia, especially in SOTRs with substantial morbidity and chronic immunosuppression, involves other immunological or predisposing factors that are not accounted for in our study, such as T cell immunity or bacterial coinfection. In fact, 3 patients (13.6%) developed pneumonia regardless of the absence of LRSs at the enrollment. Likewise, the rate of previous influenza vaccination was more than 60% among SOTRs, but we found no evidence that HAI antibodies protected from disease burden, perhaps in part because of the titers being in the low range, suggesting that, when HAI antibodies fail to confer protection from symptomatic influenza infection, pre-existing HA stalk antibodies protect against severe disease in SOTRs. Our results agree with recent data where HA stem antibodies correlated with protection from infection in naturally exposed individuals and experimental human challenges independent of HAI antibodies. 24,28–31 In addition to direct antiviral activity, some broadly influenza cross-reactive antibodies can mediate FcR functions against infected cells. Our data showed a good correlation (Spearman correlation coefficient, r 2 = 0.64; Figure 2F) between levels of anti- HA stalk antibodies and stalk ADCC activity, measured by a bioluminescence reporter assay, 27 suggesting that the ability to recruit cell-mediated functions can also contribute to protection from LRSs and, therefore, disease progression. A higher increase in HA and NA antibodies with ADCC activity was also noted after influenza virus infection compared with after vaccination (Figures 3C–3E), which was expected because natural infection is known to induce anti-stalk antibodies whereas vaccination with currently licensed vaccines does not. 12 Although current influenza vaccinations are potent inducers of anti-HA head antibodies 9 with anti-agglutinating function, our data show limited induction of antibodies with other specificities by the seasonal influenza vaccine (Figure S4). This is not necessarily surprising because influenza vaccines are optimized for HA content, and the HA head is immunodominant. Nonetheless, a previous history of vaccination was associated independently with a lower incidence of LRSs in multivariate logistic regression (Figure S3); however, no differences were found in antibody titers between patients with influenza vaccination compared with unvaccinated ones (Figure S5). Whether this is due to the need A B C DE F Figure 3. Antibody Responses after Influenza Infection in SOTRs (A) Paired antibody levels for each patient, GMT, and 95% CI are shown at baseline and convalescence for the indicated ELISA assay on the left y axis (full-length HA, stalk HA, and NA) and hemagglutination inhibition (HAI) antibody titers on the right y axis. (B–E) Fold induction log values after influenza virus infection according to presence of lower respiratory symptoms (LRSs; yes or no), GMT, and 95% CI are shown for (B) stalk HA, (C) full-length HA, (D) HAI, and (E) NA. (F) Linear regression of log fold induction values of anti-NA antibodies measured by ELISA and anti-NA antibodies with ADCC activity. Experiments were performed in triplicate. 6Cell Reports Medicine 1, 100130, November 17, 2020 Report ll OPEN ACCESS for additional immunological assays to further characterize the correlates of protection associated with the seasonal influenza vaccine or due to the sample size of our study, which might be underpowered to address this question, remains unanswered. In summary, our results have significant implications for prevention and control of influenza virus infection. We provide a more granular characterization of the specificity of antibodies after natural influenza virus infection and vaccination in SOTRs. In addition, we provide evidence that supports the ability of crossreactive antibodies in limiting clinical disease progression in naturally influenza virus-infected individuals. We also confirmed that natural infection is a better inducer of cross-reactive protective antibodies than seasonal vaccination, underscoring the need for improved influenza vaccines. This study contributes to a better understanding of the mechanisms that mediate the pathogenesis of influenza disease and induction of broad humoral immune responses, which will aid the design of better, perhaps even universal influenza vaccines. Our data indicate that a threshold of protective immunity based on broadly cross-reactive anti HA-stalk antibodies could be feasible. Limitations of Study Some limitations of our study need to be acknowledged. First, the number of subjects with available paired serum samples at the end of the enrollment period is relatively small compared with the total SOTR cohort included in the study. Different influenza virus strains circulated during three consecutive seasons: pandemic H1N1pdm09 and seasonal H3N2 and influenza B. As expected, the levels of pre-existing HAI active antibodies against the H1 pandemic strain were low compared with the seasonal H3 strain. Indeed, only 35% of SOTRs with pandemic influenza infection had some detectable levels of HAI active antibodies, which allows assessment of the protective role of other non-HAI cross-reactive antibodies in disease burden. On the other hand, the numbers of H3N2 and influenza B cases were low compared with H1N1pdm09, limiting conclusions regarding the role of influenza A group 2 and influenza B stalk antibodies. Our study shows that the presence of stalk antibodies against H1N1pdm09 group 1 HA correlated with lack of LRSs in SOTRs with influenza infection. Whether group 2 HA or influenza B stalk antibodies have a protective effect on influenza disease outcome is still not clear. Second, we defined stalk antibodies levels of protection in a complex population of SOTRs with different ages, types of transplants, and immunosuppressive drug therapies. Although immunosuppression has been associated with a lower immune response after influenza infection and vaccination in SOTRs, 3,7,32 no correlation was found among the different combinations of drug therapies or individual treatments and disease outcome. It is likely that the complexity of treatment, with concomitant drugs according to time from transplantation, variability of combinations, and different drug doses, limits possibilities to assess the influence of specific therapies (e.g., mammalian target of rapamycin [m-TOR] inhibitors). Although our results cannot be fully translated to other populations, such as healthy adults, they provide a better understanding of new serocorrelates that can mediate influenza disease protection in AB CD E Figure 4. Influence of Pre-existing Immunity on Subsequent Immune Response in Influenza-Infected SOTRs (A and B) Correlation matrix showing Spearman’s correlation coefficient between levels of HAI antibodies at onset and fold induction ELISA titers and ADCC activity, respectively, at convalescence after influenza infection. Strength of the association of HAI at the onset with ELISA full-length HA (p = 0.02), ELISA stalk HA (p = 0.01), ELISA NA (p = 0.002), ADCC full-length HA (p = 0.009), ADCC stalk HA (p = 0.02), and ADCC NA (p = 0.002). *p = 0.04. (C) Fold induction log10 values after influenza infection. GMT and 95% CI are shown for patients with a pre-existing HAI titer of 40 or higher (seroprotection) in comparison with those with an HAI titer of less than 40 (no seroprotection). Experiments were performed in triplicate. Cell Reports Medicine 1, 100130, November 17, 2020 7 Report ll OPEN ACCESS immunosuppressed subjects, especially important because of the high frequency of severe outcomes. Last, it is likely that some SOTRs with very mild or asymptomatic influenza infection did not go to the hospital despite having been advised to seek care in the case of influenza-compatible respiratory symptoms. Cases of influenza infection that required hospital admission were included in this study, allowing characterization of the immune response and risk factors of severe outcome in SOTRs with influenza infection. STAR+METHODS Detailed methods are provided in the online version of this paper and include the following: dKEY RESOURCES TABLE dRESOURCE AVAILABILITY BLead Contact BMaterials Availability BData and Code Availability dEXPERIMENTAL MODEL AND SUBJECTS DETAILS dMETHOD DETAILS BHemagglutination Inhibition (HAI) Assay BEnzyme-linked Immunosorbent Assay (ELISA) BAntibody-dependent Cell Cytotoxicity (ADCC) Bioassay 27 . dQUANTIFICATION AND STATISTICAL ANALYSIS SUPPLEMENTAL INFORMATION Supplemental Information can be found online at https://doi.org/10.1016/j. xcrm.2020.100130. ACKNOWLEDGMENTS This work was supported by the Programa de Investigacio ´n sobre gripe A/ H1N1, Instituto de Salud Carlos III, Ministerio. de Ciencia e Innovacio ´n (GR09/0041); the Ministerio de Ciencia e Innovacio ´n, Instituto de Salud Carlos III, co-financed by the European Development Regional Fund ‘‘A way to Achieve Europe’’ ERDF; the Spanish Network for Research in Infectious Diseases (REIPI RD06/0008 to E.C.); NIAID grants P01AI097092 and U19AI135972; CRIP (Center for Research on Influenza Pathogenesis), an NIAID-funded Center of Excellence on Influenza Research and Surveillance (CEIRS; contract HHSN272201400008C); and SEM-CIVIC, an NIAID- funded Collaborative Influenza Vaccine Innovation Center (contract 75N93019C00051 to A.G.-S. and F.K.). AUTHOR CONTRIBUTIONS T.A., E.C., and A.G.-S. conceived and designed the study. T.A. collected samples and data, performed experiments, analyzed data, and wrote the report. A.E. performed experiments. S.S., S.A., J.A., and F.K. provided reagents, methods, and expertise. P.P.-R., C.R.-O., M.M., J.G., P.M., F.L.-M., J.S.-C., and J.C. collected samples and data. T.A., E.C., and A.G.-S. supervised the study. All authors reviewed and edited the paper. DECLARATION OF INTERESTS A.G.-S. is an inventor of patents owned by the Icahn School of Medicine at Mount Sinai in the field of influenza virus vaccines. The A.G.-S. lab has received research funds from Avimex, GSK, and 7Hills to investigate novel influenza virus vaccines. F.K. is a named inventor of technology in the field of human influenza vaccines and a named co-inventor on patents to develop chimeric HA technology to develop a universal influenza vaccine. Received: April 14, 2020 Revised: May 27, 2020 Accepted: October 12, 2020 Published: November 3, 2020 REFERENCES 1. GBD 2017 Influenza Collaborators (2019). 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