Abstract
Influenza causes more than 250,000 deaths annually in the industrialized world and bacterial infections frequently cause secondary illnesses during influenza outbreaks, including pneumonia, bronchitis, sinusitis, and otitis media. Here we demonstrate that cross-reactive immunity to mismatched influenza strains can reduce susceptibility to secondary bacterial infections, even though this fails to prevent influenza infection. Specifically, infecting mice with H3N2 influenza before challenging with mismatched H1N1 influenza reduces susceptibility to either gram-positive Streptococcus pneumoniae or gram-negative Klebsiella pneumoniae. Vaccinating mice with the highly conserved nucleoprotein of influenza also reduces H1N1-induced susceptibility to lethal bacterial infections. Both T cells and antibodies contribute to defense against influenza-induced bacterial diseases; influenza cross-reactive T cells reduce viral titers, whereas antibodies to nucleoprotein suppress induction of inflammation in the lung. These findings suggest that non-neutralizing influenza vaccines that fail to prevent influenza infection may nevertheless protect the public from secondary bacterial diseases when neutralizing vaccines are not available.
INTRODUCTION
Secondary bacterial infections often follow influenza infection and can lead to a variety of illnesses including pneumonia, bronchitis, sinusitis and otitis media (1, 2). Secondary bacterial pneumonia is a particularly serious consequence of influenza infection. It was the primary cause of death during the 1918 influenza pandemic (3) and was associated with significantly higher morbidity and mortality during the 2009 pandemic (4).
Vaccines are the mainstay of public health efforts to prevent influenza epidemics. These vaccines aim to prevent infection by eliciting neutralizing antibodies that bind the hemagglutinin and neuraminidase proteins on the surface of influenza virions. Unfortunately, mutations and reassortments in the surface proteins of influenza viruses allow new strains to emerge and evade neutralizing antibodies (5, 6). Consequently, each year, new vaccines are produced to “match” the most dangerous contemporary strains.
In animal models, mismatched influenza vaccines can prime non-neutralizing immunity that speeds viral clearance and reduces mortality, despite failing to prevent infection (7, 8). Mismatched vaccines may not prevent human pandemics, but they might lessen their severity when matched vaccines are not available. Indeed, several studies suggest humans may benefit from non-neutralizing immunity to influenza (9–12). A recent study demonstrated that the presence of influenza-specific memory in humans correlates with non-neutralizing immunity that significantly reduces the severity of illness (13). These researchers postulated that CD4 T cells confer protection by improving antibody responses to conserved internal viral proteins (13). Unfortunately, many factors confound the interpretation of human studies of influenza and public health campaigns to date have largely neglected the potential for non-neutralizing immunity to combat influenza outbreaks or the associated increase in secondary bacterial infections.
Data from mouse models suggest influenza infection increases susceptibility to secondary bacterial infections by suppressing neutrophil function, decreasing mucociliary flow, desensitizing innate immunity, and creating favorable environments for bacterial adherence and colonization (1). Cytokines, including interleukins and interferons, also affect susceptibility (14–16), suggesting that ongoing immune responses to influenza may facilitate bacterial colonization of the lung. Here, we investigate whether non-neutralizing, mismatched immunity to influenza impacts susceptibility to secondary bacterial infections.
MATERIALS AND METHODS
Mice
Wild type and B cell-deficient μMT C57BL/6 mice were purchased from The Jackson Laboratory (Bar Harbor, ME). Antibody-deficient AID.uS C57BL/6 mice were described previously (17, 18). All mice were bred in the specific pathogen free Trudeau Institute Animal Breeding Facility after embryo rederivation. Experimental mice were matched for age and sex, and cared for according to Trudeau Institute guidelines. Recumbent mice, and mice that lost more than 30% weight, were considered moribund and euthanized.
Viruses
In uenza virus A/HKx31 (H3N2), in uenza virus A/PR/8/34 (H1N1), cold-adapted influenza virus c.a.A/Alaska/72/CR9 (caH3N2) and the Enders strain of Sendai virus were grown, stored, and titered as previously described (19–21). Influenza infections and vaccinations were administered intranasally to anesthetized mice using 3000 EID-50 for H3N2, 400 EID-50 for H1N1, 350 TCID-50 for caH3N2, and 250 EID-50 for Sendai virus. The viral burden and level of inflammatory cytokines and chemokines in whole lung tissue was determined by real-time PCR measuring acid polymerase copy number (22).
Bacteria
Serotype 4 Streptococcus pneumoniae (ATCC strain 6304) grown on blood agar plates was used to inoculate Tryptic soy broth cultures, which were grown at 37°C without shaking in sealed tubes. After dilution to an OD600nm of 0.15, they were re-grown to an OD600nm of 0.45, washed with saline, and approximately 250 CFU were applied in a volume of 50 μl saline to the nares of lightly anesthetized mice. The number of bacteria in the inoculating dose was confirmed by plating. The intranasal median lethal dose of strain 6304 is approximately 1.5×104 CFU when grown as described above and administered to naïve mice. Additional studies also employed serotype 3 S. pneumoniae strain URF918 (23) and Klebsiella pneumoniae strain IA565 (24). Innocula of strain URF918 were prepared as described for ATCC strain 6304. Working stocks of strain IA565 were prepared by growing in Tryptic/Soy Broth to log phase (OD600=0.900), adding 20% glycerol and freezing aliquots at −70°C. To prepare inocula, 100 μl of working stock was streaked on Tryptic/Soy agar plates, grown overnight at 37°C, and scraped to seed Tryptic/Soy Broth cultures at OD600=0.050. Cultures were grown in 37°C incubator/shaker at 180rpm for approximately 2hr to log phase (OD600=0.900), spun down, and resuspended in PBS to a concentration of 108 CFU per 50 μl infection dose.
Treatments
T cell depletions were performed as described previously (19). The depletion protocols removed more than 90% of the targeted cells from spleen and bronchoalveolar lavage fluid, as determined by flow cytometric analyses of antibody-treated animals that were euthanized at day 5 after H1N1 infection (not shown). Recombinant A/PR/8/34 influenza NP was generated as a C terminal histidine-tagged protein in E. coli and isolated using the ProBond system (Invitrogen), as described (17). Immunizations contained 30 μg NP and employed 20 μg E. coli serotype 0111:B4 lipopolysaccharide (LPS; Enzo Life Sciences) plus alum as adjuvant (17). H3N2 immune serum was collected 21 days after infection with H3N2 and 350 ul was transferred to naïve mice by intraperitoneal injection on the day prior to H1N1 challenge. Passive immunization with mouse IgG2a NP-specific mAb H16-L10-4R5/HB-65 (25) was achieved by administering 350 ug intraperitoneal injections on the day of and the day prior to H1N1 challenge. Control mice received serum from naïve mice or isotype matched mAb C1.18.4. All mAb were Protein G purified and supplied by BioXcell, who reported <2 endotoxin units per mg.
Statistics
Survival curves were analyzed by Log rank tests. CFU and viral titer data that fell below the limit of detection were assigned a value below that limit and, thus, were analyzed by non-parametric Mann Whitney or Kruskal Wallis tests. Bacteremia was scored positive or negative and analyzed by Chi-square tests.
RESULTS
Non-neutralizing immunity to influenza protects from secondary bacterial infection
Fig 1A depicts our general experimental approach to assessing the impact of prior immunity to influenza on susceptibility to secondary bacterial infection. Naive mice readily survived low dose intranasal challenge with 250 CFU of S. pneumoniae (Fig. 1B). Naive mice also survived low dose intranasal challenge with H1N1 influenza (Fig. 1B). However, consistent with prior reports (26), we observed that mice succumbed to bacterial infection when challenged with low dose S. pneumoniae following a sublethal influenza challenge (Fig. 1B). To investigate the impact of non-neutralizing, mismatched immunity to influenza, we infected mice with low dose H3N2 influenza, challenged 5–6 months later with low dose H1N1 influenza, and then measured susceptibility to S. pneumoniae. We observed that prior exposure to H3N2 influenza improved survival (Fig. 1B), reduced pneumococcal colonization of lung tissue (Fig. 1C), and largely prevented bacteremia (Fig. 1D). Notably, susceptibility to bacterial infection did not simply correlate with viral titers at the time of challenge. For example, mice challenged with S. pneumoniae on days 5 and 14 after H1N1 infection exhibited similar bacterial burden (Fig. 1C), despite more than a 10,000-fold difference in viral titers at those time points (Fig. 1E).
To investigate the specificity of the H3N2-induced protection from secondary bacterial infection, we evaluated protection conferred by Sendai virus, a parainfluenza virus that causes an acute pulmonary infection similar to influenza, but does not prime cross-reactive immunity to influenza (20). In parallel, we examined protection conferred by a cold-adapted H3N2 (caH3N2) vaccine strain (19, 21, 27, 28). We found that exposure to either the H3N2 influenza virus or the live attenuated caH3N2 vaccine protected against H1N1-induced susceptibility to pneumococcal infection as early as 3 weeks after exposure (Fig. 2A). The protection was associated with reduced bacterial burden in the lungs (Fig. 2B) and reduced H1N1 titers (Fig. 2C). In contrast, prior exposure to Sendai virus had no significant impact on H1N1-induced susceptibility to pneumococcal infection (Fig. 2A). Infection with Sendai virus, like H1N1 infection, induced susceptibility to S. pneumoniae when challenged on day 7 (29), but this susceptibility waned by 26 days after infection (Fig. 3A and B), indicating that residual impacts of primary Sendai infection did not account for the pneumococcal susceptibility observed when Sendai-exposed mice were infected with H1N1 influenza. Thus, specific cross-reactive immunity to influenza, not just conditioning of the lung by any viral infection, reduces susceptibility to secondary bacterial infection. Importantly, the cross-reactive immunity to influenza reduced susceptibility to diverse types of bacterial infections: the bacterial challenge studies described above used a serotype 4 strain of gram-positive S. pneumoniae but similar results were observed when mice were challenged with serotype 3 S. pneumoniae (Fig. 4A, B, C) or Klebsiella pneumoniae (Fig. 4D, E, F), a gram-negative bacterium.
It has been shown that non-neutralizing immunity to influenza can accelerate viral clearance (7, 8, 30). Thus, preexisting immunity to influenza may have shifted the period of H1N1-induced susceptibility to pneumococcal infection, such that mice became susceptible prior to day 5 after H1N1 challenge. To investigate this possibility, we examined the kinetics of susceptibility in greater detail. We found that mice were susceptible to H1N1-induced pneumococcal infection when bacteria were administered on days 3, 5 and 7, but not day 1, after H1N1 infection, and that prior infection or vaccination with H3N2 suppressed pneumococcal susceptibility at these same times (Fig. 2D). Again, susceptibility correlated with increased bacterial burden (Fig. 2E) and higher viral titers (Fig. 2F). Thus, prior exposure to H3N2 influenza did not accelerate the time of susceptibility. Rather, preexisting mismatched immunity to influenza reduced overall susceptibility to pneumococcal infection.
Cross-reactive T cells and antibody contribute to protection
Cross-reactive CD8 T cells can facilitate non-neutralizing protection against mismatched influenza strains (7, 8), and influenza cross-reactive memory T cells produce interferon-gamma, one of the cytokines that contributes to H1N1-induced susceptibility to pneumococcal infection (15). Thus, cellular immunity to influenza might be predicted to exacerbate susceptibility to pneumococcal disease. However, depletion of all T cells (anti-Thy1 treatment) or depletion of only CD8 T cells from H3N2-immune mice immediately prior to H1N1 challenge modestly diminished the protection conferred by prior exposure to H3N2 (Fig. 5A) and slightly elevated both the bacterial burden (Fig. 5B) and viral titer (Fig. 5C). Thus, the presence of cross-reactive memory T cells did not exacerbate pneumococcal infection and, rather, contributed to cross-reactive defense against bacterial disease, at least in part, by reducing H1N1 titers.
Antibodies to conserved viral proteins also contribute to cross-reactive immunity to influenza (7, 17, 18, 31, 32). To test the role of antibody in defense against H1N1-induced pneumococcal susceptibility, we administered H3N2-immune serum or control serum to naïve mice prior to infection with H1N1 influenza. The mismatched H3N2-immune serum significantly decreased susceptibility to secondary pneumococcal infection (Fig. 5D). Despite conferring significant protection from lethality, passive immunization with mismatched serum did not significantly reduce bacterial burden (Fig. 5E) or viral titers (Fig. 5F) in the lung. However, H3N2-immune serum did significantly reduce levels of bacteremia and the number of mice with detectable bacteria in blood cultures (Fig. 5E).
Influenza cross-reactive non-neutralizing immunity typically recognizes conserved, internal proteins of influenza, and immunity to nucleoprotein (NP), a highly conserved internal protein, confers significant protection from lethal influenza challenge (7, 8, 17, 18, 32–38). We observed that vaccinating mice with purified recombinant NP conferred robust protection from H1N1-induced susceptibility to secondary pneumococcal infection (Fig. 5G). NP vaccination markedly reduced pneumococcal burden in the lung (Fig. 5H), despite only modestly reducing viral titers (Fig. 5I). NP vaccination also significantly reduced H1N1-induced susceptibility to Klebsiella pneumoniae and serotype 3 S. pneumoniae (Fig. 4).
Depletion of CD8 T cells alone, or both CD4 and CD8 T cells, at the time of H1N1 challenge did not significantly impair the protection conferred by NP vaccination (Fig. 6A), suggesting the involvement of alternative effector mechanisms. Other studies have demonstrated that antibodies to NP can confer significant protection from lethal influenza challenge (17, 18, 32). Consistent with a critical role for antibodies, NP vaccination poorly protected B cell-deficient μMT mice and antibody-deficient AID/μS mice from death following influenza and S. pneumoniae infections (Fig. 6B and C). To definitively assess the protective capacity of NP-specific antibodies, we passively transferred NP-specific mAb into naïve mice and then challenged with influenza followed by S. pneumoniae. Remarkably, administration of NP-specific mAb conferred robust protection from H1N1-induced secondary pneumococcal disease (Fig. 5J). As with passive immunization with H3N2-immune serum, NP-specific mAb did not significantly reduce viral titers in the lung (Fig. 5L), but did reduce the number of bacteremic mice and levels of bacteremia (Fig. 5K). Notably, passive immunization with NP-specific mAb did not did not suppress bacterial infection directly since it did not affect the median lethal dose of S. pneumoniae in naïve mice (data not shown) and only reduced pneumococcal lethality in mice infected previously with influenza.
Immunity to NP reduces lung inflammation
NP-specific antibodies can help to reduce H1N1 titers in lethal influenza challenge models (17, 18, 31, 32), but did not appear to reduce H1N1 titers in our model of sublethal influenza challenge. Antibodies are known to play diverse roles during host defense, including the suppression of inflammation (39). Indeed, in our studies, passive immunization with NP-specific mAb markedly suppressed levels of inflammatory cytokines and chemokines in lung tissue of H1N1-infected mice, including IL-6, CCL2/MCP-1, and CXCL1/KC (Fig. 7B, C and D). Notably, levels of interferon-gamma were not affected (Fig. 7A). Treatment with H3N2 serum likewise reduced levels of pulmonary inflammation, as did active vaccination with H3N2 virus or NP (Fig. 7B, C and D). In addition, the non-neutralizing immunity induced by infection, immunization or antibody transfer also significantly reduced the expression of platelet activating factor receptor (Fig 7E). Elevated expression of platelet activating factor receptor has been associated with enhanced inflammation and lung pathology during influenza infection (40, 41). These findings suggest non-neutralizing immunity, and cross-reactive NP-specific antibodies in particular, may reduce susceptibility to bacterial infection by reducing inflammation and pathological damage to lung epithelium, events that facilitate colonization of the lung by bacteria (1).
DISCUSSION
Secondary bacterial infections are a common complication of influenza infection and cause significant morbidity and mortality (1, 2). Because of this important clinical problem, we have employed a murine model to examine the influence of influenza infection on susceptibility to secondary bacterial infections and how this can be prevented. Using this model, we have shown that the susceptibility to secondary infection applies to several strains of bacteria and results in significant lung colonization, bacterial dissemination and death. Consistent with prior studies (1, 14–16), we also found that the window of susceptibility to secondary infection begins at day 3 of influenza infection and extends until at least day 14, when the vast majority of virus has been cleared. These results suggest that susceptibility to secondary infection is not just due to influenza infection but may also be associated with the immune response to the virus, including inflammation in the lungs. In fact, it has been postulated that the extensive mortality observed in the 1918 influenza pandemic was the result of extensive immunopathology that increased susceptibility to secondary bacterial pneumonia (42).
This model has also allowed us to explore how this susceptibility to secondary infections can be overcome. The most common way to manipulate immunity to influenza is via vaccination. Neutralizing immunity to influenza, which is the goal of the yearly influenza vaccine, can prevent infection and illness. While this is the most desirable goal of vaccination, it is not always possible due to rapid antigenic changes in the virus or the appearance of new viral strains. Prior studies have demonstrated that non-neutralizing immunity can reduce influenza illness and mortality in mice. Our observations demonstrate that non-neutralizing immunity to influenza also confers remarkable protection from secondary bacterial infections. Non-neutralizing immunity can be conferred by mismatched live attenuated vaccines (such as Flumist) or by prior influenza infection (19, 43). It can also be conferred by vaccination with conserved internal proteins from the influenza virus, such as NP (17, 18, 32). This non-neutralizing immunity protects from secondary bacterial infection by reducing lung colonization, bacterial dissemination and death.
Our results demonstrate that antibody is a main effector mechanism of the non-neutralizing immunity that protects from bacterial infection. Vaccination of mice that lack B cells or secreted antibody could not confer protection from secondary bacterial infection. In contrast, passive transfer of serum collected from mice that had been previously infected with a mismatched, heterosubtypic influenza strain, or a monoclonal antibody to influenza NP, could protect from secondary bacterial infection. The presence of non-neutralizing antibodies was associated with significant reduction in inflammatory molecules in the lungs of influenza-infected mice. As discussed in a recent review (45), the ability of antibodies to modulate inflammation during an immune response has been appreciated for a long while. Under certain conditions, antibodies can dampen the inflammatory response and they have been used clinically as an anti-inflammatory agent. While the mechanism of this action most likely involves Fc receptors, precisely how this activity works has yet to be elucidated. Importantly, the use of prophylactic antibody treatment to reduce an inflammatory response is not unprecedented in the clinic (46, 47).
The efficient generation of high affinity NP-specific antibodies presumably requires CD4 T helper cells specific for influenza NP (48). A recent study in humans demonstrated an important role for CD4 T cells specific for internal influenza proteins in protection from severe influenza-induced illness (13). In individuals with preexisting CD4 T cells with specificities for NP or matrix protein, there was less illness following influenza challenge. The CD4 T cells identified following viral challenge could respond to a number of different influenza strains, indicating that they would be useful during a mismatched, heterosubtypic infection. While it could not be formally demonstrated in these human studies, the authors speculated that these influenza-specific CD4 T cells were exerting this protective influence by acting as helper cells for a humoral response directed at internal influenza proteins. Moreover, in concordance with our findings, the authors of the human study proposed that the reduction in illness resulted from a reduction in immunopathology, which should result in a reduced susceptibility to secondary bacterial infections (13).
While antibody could suffice to provide significant protection in our mouse model, more robust protection was observed after H3N2 infection (Fig 3A) or rNP vaccination (Fig 3G), suggesting that additional components of the immune system, presumably T cells, also contributed to an optimal protective response. Indeed, we observed that depleting T cells modestly impacted H1N1 titers (Fig 5C), bacterial burden (Fig 5B), and survival (Fig 5A and 6A). Another study using a similar mouse model of secondary bacterial infection recently suggested dominant protective roles for CD4 T cells (44). Specifically, that study demonstrated that seasonal FluMist vaccine could protect against mismatched H1N1 influenza infection and secondary bacterial infection, with CD4 T cells participating in the control of viral titers. Notably, the authors of that study concluded that antibody did not contribute to protection. They came to that conclusion after observing that immune serum from FluMist immunized mice could not neutralize the infectivity of the mismatched H1N1 virus. We obtained analogous results after passive transfer of H3N2 immune serum (Fig 5F) or NP-specific mAb (Fig 5L). Nevertheless, we found that the H3N2 immune serum and NP-specific mAb significantly improved survival and reduced burden after secondary bacterial infection, despite their failure to impact viral titers. Thus, our study reveals a previously unappreciated mechanism of protection in this model, namely that non-neutralizing cross-reactive antibody to influenza can provide significant protection from secondary bacterial infection.
Our decisive findings in a well-controlled animal model substantially strengthen the conclusions of prior studies reporting that mismatched immunity to influenza confers clinical efficacy (11, 12, 49–51). Together, these clinical and animal studies provide compelling evidence that certain mismatched vaccines may benefit public health when matched vaccines are not available. Moreover, our studies suggest that boosting NP immunity may suffice to provide clinical benefit. Boosting NP immunity may require use of live attenuated influenza vaccines or NP-containing subunit vaccines since classical inactivated influenza vaccines only contain low quantities of NP and weakly boost NP antibody responses (18). Passive immunotherapy using NP-specific antibody also may be useful, particularly for those who respond inadequately to active immunization regimen, such as the immunocompromised and elderly. Finally, by demonstrating that preexisting immunity to influenza NP impacts susceptibility to secondary pneumococcal infection, this report opens new lines of investigation for those studying the pathology, epidemiology, treatment and prevention of pneumonia, bronchitis, sinusitis, otitis media, and other bacterial diseases commonly associated with influenza infections (1, 3, 6, 52–54).
Acknowledgments
The authors thank Drs. Markus Mohrs, Marcia Blackman, and Elizabeth Leadbetter for reading our manuscript and the employees of the Trudeau Institute Animal Breeding and Maintenance Facilities for care of the mice used in these studies. We also thank Drs. Richard Dutton and Brian Murphy for providing influenza strain c.a.A/Alaska/72/CR9, and Drs. Kazuyoshi Kawakami and Thomas Moore for S. pneumoniae strain URF918 and Klebsiella pneumoniae strain IA565, respectively.
Abbreviations used
- ca
cold adapted influenza
- NP
influenza nucleoprotein
- Spn
Streptococcus pneumoniae
Footnotes
This work was supported by funds from The Trudeau Institute and National Institutes of Health grants AI071295 (S.T.S.), AG02160 (L.H.), AI072689 (T.D.R), AI83610 (J.E.K), AI67967 (D.L.W), and AI76499 (D.L.W.) and Department of Defense contract W911QY-10-C-0184 (L.H.).
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