New research by scientists at the University of Chicago suggests a person’s antibody response to influenza viruses is dramatically shaped by their pre-existing immunity, and that the quality of this response differs in individuals who are vaccinated or naturally infected.
Their results highlight the importance of receiving the annual flu vaccine to induce the most protective immune response.
The researchers found that most of the initial antibodies stimulated after both influenza infections and influenza vaccinations came from old B cells – a type of white blood cell that secretes antibodies – indicating the immune system’s memory plays a major role in how the body responds early on to a viral infection.
These antibodies displayed higher reactivity toward strains of influenza that circulated during an individual’s childhood compared to more recent strains.
The study, published December 10, 2020 in the journal Science Translational Medicine, provides those working on a universal influenza vaccine further understanding of how pre-existing immunity affects the development and performance of neutralizing and non-neutralizing antibodies following infection and vaccination.
Any effective universal influenza vaccine will depend on scientists identifying ‘conserved’ parts of the influenza virus that do not mutate over time and that antibodies can target to prevent infection.
“Most interestingly, we found that people who were actively sick with influenza had old antibodies that predominantly targeted parts of the virus that don’t change—but those antibodies specifically targeted non-neutralizing sites,” said Haley Dugan, co-first author of the study and a Ph.D. candidate in immunology. “When we tested these same antibodies in mice, they weren’t able to protect them from being infected with influenza.”
In contrast, the researchers found that influenza vaccinations boost antibodies that tended to target conserved yet neutralizing regions of the virus, which suggests vaccinations can draw upon pre-existing immunity to prompt more protective responses. Vaccinated individuals also generated many antibodies that targeted new and mutated regions on the virus, suggesting these vaccine-induced antibodies are more adaptable.
Immune system memory ensures a rapid and specific response to previously encountered pathogens. Vaccinations work by exposing the immune system to a small amount of virus, which causes B cells to develop a biological memory to the virus. If the body encounters the same virus later, the immune system is alerted to attack and eliminate the virus.
But in order to be protected, the viral proteins of the infecting strain must typically match those of the strain used in the vaccine. The memory B cells are like keys that fit and bind to the locks—the viral proteins. These memory B cells can survive for decades, providing long-lasting protection from future infections. But if the virus mutates and is significantly different, the memory B cells can no longer recognize the viral proteins, potentially leading to infection.
For this reason, the human body is pitted in an evolutionary arms race with the flu. Because influenza viruses rapidly evolve and mutate each season, our immune system has trouble recognizing the viral surface proteins on new influenza strains. As a result, our bodies often rely on old antibodies to fight new influenza strains; this is possible because some parts of the influenza virus that are critical to its structure or function do not change, remaining familiar to our immune system.
Researchers now understand that specific structural and functional parts of the influenza virus that do not change are better for antibodies to target than others. Antibodies that bind to one of these neutralizing sites are able to prevent infection, while antibodies that target non-neutralizing sites often cannot. Scientists believe a person’s age, history of exposure to the influenza virus and type of exposure – either through infection or vaccination – all shape whether their immune system antibodies target neutralizing or non-neutralizing sites on a virus.
In the UChicago study, scientists sought to address a major knowledge gap: Which conserved viral sites are preferentially targeted following natural infection versus vaccination in people, and how does pre-existing immunity play a role in shaping the landscape of neutralizing and non-neutralizing antibodies?
“For people who have caught the flu, their pre-existing immunity may make them susceptible to infection or increase the severity of their influenza symptoms if their antibodies are targeting ‘bad’ or non-neutralizing viral sites,” said co-first author and Immunology postdoctoral fellow Jenna Guthmiller, Ph.D.
By contrast, vaccination largely induces neutralizing and protective antibodies, old and new, highlighting the importance of receiving the seasonal influenza vaccine.
“This study provides a major framework for understanding how pre-existing immunity shapes protective antibody responses to influenza in humans,” said Patrick Wilson, Ph.D., a professor of immunology and lead author of the study. “We need more studies to determine whether the targeting of specific neutralizing and non-neutralizing viral sites directly impacts a person’s likelihood of becoming ill.”
The researchers are now examining how early exposure to the influenza virus in children shapes their immune response later in life as a follow-up to this work.
Innate Effector Cell Types in the Response to Influenza Infection
During influenza virus infections, innate signaling and adaptive immune responses both play important roles in protecting against the virus and achieving viral clearance. At the site of infection, infected cells produce chemokines and cytokines such as type I IFNs, IL-6, IL-8, TNF-α, CCL2 (MCP-1), RANTES, and MIP-1α that recruit immune cells including NK cells, neutrophils, macrophages and DCs to the site of infection where they initiate the innate immune response [94,95,96]. These immune cells are responsible for both protection and immunopathology following influenza virus infection.
4.1. Natural Killer Cells, Neutrophils, and Macrophages
Innate cells are initially recruited to the site of infection from the systemic circulation by the proinflammatory cytokines that are released by infected lung epithelial cells. NK cells recognize HA on influenza-infected cells via NKp44 and NKp46 receptors [97,98,99] or detect antibody-coated infected cells via CD16 . Then, activated NK cells lyse the infected cells by secreting perforin and granzyme [101,102]. Defects in NK cells lead to increased susceptibility to influenza infection and uncontrolled viral growth in the lungs [103,104]. However, excessive NK cell-induced inflammation can also contribute to pathology following high dose influenza virus infection [105,106]. Neutrophils, monocytes, and macrophages inhibit the spread of infection by phagocytosing infected apoptotic cells . Neutrophils rapidly migrate to the site of infection and initiate phagocytosis, degranulation, and the formation of neutrophil extracellular traps . The importance of neutrophils has been demonstrated in animal studies showing that neutrophil depletion leads to increased influenza mortality due to impaired viral clearance [109,110]. Neutropenic mice display reduced flu-specific CD8+ T cell responses in the respiratory tract . Further to this, neutrophils guide CD8+ T cells to the infection site by leaving a trail of CXCL12 . These studies indicate that neutrophils play a protective role in influenza infection, but that excessive recruitment of neutrophils can also contribute to immunopathology .
Alveolar macrophages (AMs) are lung-resident macrophages that control lung homeostasis at steady state and phagocytose opsonized pathogens and infected cells . AMs are also the main producer of type I IFN in the lung during infection with RNA viruses . There are several lines of evidence supporting the protective role of AMs during influenza infection. Clodronate-induced depletion of AMs increases the mortality of mice and enhances pulmonary inflammation during influenza infection . Csf2−/− mice, which are deficient in AM development, experience respiratory failure and reduced resistance to influenza infection despite having normal adaptive immune responses against influenza virus . Consistently, an AM depletion model in CD169-DTR mice led to aggressive inflammatory responses and severe lung pathology after influenza infection . AMs control lung dysfunction by increasing the antiviral resistance of type-1 alveolar epithelial cells, which are responsible for gas exchange . These results indicate that AMs play a crucial role in maintaining pulmonary homeostasis during influenza infection.
DCs, which belong to the innate immune response system, are known as professional antigen-presenting cells. Because T cells only recognize foreign antigens that are associated with MHC molecules, DCs play a critical role in the priming and activation of naïve T cells. The specialized antigen-presenting function of DCs allows them to serve as the link between innate and adaptive immunity (Figure 3).
Many studies have been conducted to clarify the role of DCs during influenza virus infection. CD11c-DTR transgenic mice express a high-affinity diphtheria toxin receptor (DTR) in CD11c+ cells , allowing targeted depletion of DCs by the administration of diphtheria toxin (DT). CD11c-DTR mice have been used to study the function of DCs in vivo. Following influenza virus infection, DT-treated mice experience significantly more severe weight loss than controls with intact DCs .
Further to this, in DC-depleted mice, influenza-specific CD8+ T cell populations are significantly decreased in the lung, and viral clearance is impaired, suggesting that CD11c+ DCs are responsible for the activation of CD8+ T cells and T cell-mediated viral clearance during influenza virus infection.
In the lungs, DCs are classified into migratory conventional DCs (cDCs), and pDCs at a steady state, and monocyte-derived DCs (MoDCs) are produced after exposure to inflammatory stimuli [122,123,124]. CD11chi-MHC class II+ cDCs are distinguished by the surface expression of markers CD103+ migratory cDC1, and CD11b+ migratory cDC2 in mice, and CD141+ (BDCA-3), cDC1, and CD1c+ (BDCA-1), cDC2 in humans .
Several studies have suggested that the various DC subsets have specialized functions in the response to influenza virus infection. Remarkably, respiratory DC subsets display different susceptibility to influenza virus infection. cDC1 is more susceptible to influenza virus infection than cDC2 and pDCs , but influenza infection of cDC1 does not lead to productive viral replication .
Following influenza infection, lung-migratory cDCs take up viral antigen and migrate to the draining mediastinal lymph nodes (mLNs) where they present antigens to naïve T cells and induce adaptive immune responses. In the mLNs, migratory and resident cDCs can both prime T cells, and are subdivided into CD8+ cDC1 and CD11b+ cDC2. While resident CD8+ cDC1s in the mLNs have the potential to activate CD8+ T cells , mLN-resident cDCs seem to be less effective at inducing anti-influenza CD8+ T cell responses [127,129]. More research is needed to clarify the specific role of mLN-resident cDCs in the anti-influenza response.
Most studies determining the role of DCs in influenza virus infection have focused on the cDC1 subset. CD103+ cDC1s are well known as specialized cells that initiate CD8+ T cell activation via MHC class I-mediated cross-presentation of antigens. Lung-migratory cDC1s migrate into the mLNs to prime CD8+ T cells, and this process reaches its peak within 48–72 h after influenza virus infection .
There have been several reports that have suggested that CD103+ cDC1 is the dominant population for activation of effective CD8+ T cell responses against the influenza virus [121,127,131,132]. For example, Batf3−/− mice, which lack cDC1s , cannot mount efficient anti-influenza virus CD8+ T cell responses [127,128].
Further to this, DT-treated Clec9A-DTR mice, which have been modified to express DTR in C-type lectin receptor-expressing cells such as cDC1, are highly susceptible to influenza virus infection after treatment with DT . These cDC1-depleted mice were unable to recover from weight loss and had severe pulmonary inflammation and lung damage.
In addition to reductions in effector CD8+ T cell function, the development of effective memory CD8+ T cell responses was severely impaired after cDC1 depletion. Indeed, cDC1s are involved in CD8+ T cell exit from the lymph node and CD8+ T cell survival.
Taken together, cDC1s play a crucial role in enabling protective CD8+ T cell responses. Surprisingly, there is evidence supporting the capability of cDC1s to induce CD4+ T cell responses to IAV. In an in vitro T cell proliferation assay, cDC1s had superior CD4 and CD8 T cell expansion efficacy relative to pDCs and double-negative (CD8- CD205-) DCs .
Furthermore, both human CD141+ cDC1 and CD1c+ cDC2 elicited CD4+ T cell proliferation and Th1 responses after stimulation with live-attenuated influenza virus , indicating that cDC1 can be responsible for both MHC class I- and class II-mediated antigen presentation.
cDC2 subsets are considered to be potent presenters of MHC-II-loaded antigens to CD4+ T cells . While cDC1 subsets are the dominant DC population that transports antigens to lymph nodes that drain from the lung, cDC2s remain the dominant source of proinflammatory cytokines produced in the lung .
Despite the increased presence of cDC2s in lymph nodes that drain from the lungs during influenza virus infection, these cells rarely carried intact viral antigens . Consistently, migratory cDC2s are less efficient at priming and expanding antigen-specific CD8+ T cells compared to migratory cDC1s [127,129]. However, cDC2s have the potential to cross-present antigens to CD8+ T cells , and a recent study demonstrated that IRF4-dependent DCs that express CD11b+CD24hi contribute to the development of CD8+ T cell memory responses following IAV infection, suggesting that cDC2s promote the differentiation of memory CD8+ T cells . Further investigation is required to better understand the role of cDC1s and cDC2s in the response to influenza virus infection.
pDCs, which are primarily known as producers of type I IFNs , seem to play a minor role in the defense against influenza virus infection. Despite the accumulation of pDCs in the lung following influenza virus infection, pDC-depleted mice showed identical survival rate and body weight loss compared to healthy controls [121,139].
Further to this, depletion of pDCs using the 120G8 antibody did not affect body weight loss, effector CD8+ T cell responses, or viral clearance after influenza virus infection in mice . Consistently, both wild-type and pDC-deficient IkarosL/L mice showed comparable weight loss, viral burden in lung, influenza virus-specific antibody production, and lung pathology following viral infection . However, there is some evidence that pDCs may cross-present influenza virus antigens to CD8+ T cells [140,141].
Although the role of MoDCs during influenza virus infection has not been well established, it has been reported that the number of CCR2-expressing MoDCs are increased in influenza virus-infected lungs . These cells induce the robust production of nitric oxide synthase 2, and deficiency in CCR2 leads to attenuation in weight loss, pulmonary pathology, and mortality after a lethal dose of the influenza virus.
While the specific mechanisms remain unknown, these results indicate that MoDCs are associated with influenza virus-induced pulmonary pathology. On the other hand, other work has suggested a protective role for MoDCs during secondary influenza virus infection . Researchers found that MoDC-deficient CCR2−/− mice displayed an impairment of antigen-specific CD8+ T cell formation following primary influenza challenge, and lack of MoDCs during primary influenza infection reduced host resistance to secondary virus infection, suggesting that MoDCs could be considered as putative targets of influenza vaccine.
Protective Adaptive Immune Responses against Influenza Infections
Although innate immunity contributes to early viral control, adaptive immune responses are crucial for eventual viral clearance, recovery, and protection from reinfection. As expected, immunodeficient mice that are infected with influenza experience a significantly higher rate of mortality than healthy controls, confirming that adaptive immunity is crucial for the anti-influenza response [143,144]. In particular, the HA and NA proteins are primary targets of protective immunity.
CD4+ Helper T Cell Response
Naïve CD4+ cells recognize the viral antigens presented by the major histocompatibility complex (MHC) class II proteins on antigen-presenting cells, and subsequently differentiate into several types of helper T cells depending on the cytokine milieu. Activated CD4+ T cells support the activation and differentiation of antibody-producing B cells and also promote CD8+ T cells responses. CD4+ T cell responses peak at 10 days after influenza infection in the mouse lung .
Adaptive transfer of effector CD4+ T cells isolated from mice infected with influenza lead to increased survival of recipient mice after influenza challenge . The cytokine milieu generated during influenza virus infection is polarized to support the generation of Th1 cells .
Th1 cells produce IFN-γ, IL-2, and tumor necrosis factor α (TNFα). These cytokines activate macrophages, promote B cells to produce IgG2a and IgG3 isotype antibodies , and mediate cellular immune responses. Th2 responses are also induced following influenza virus infection.
Th2 cells secrete IL-4, IL-5, and IL-13 and promote isotype switching in B cells to produce other isotypes of antibodies such as IgG1 and IgE . However, Th1 cells are more closely associated with survival after influenza virus infection compared with Th2 cells . Some CD4+ T cell populations in influenza-infected mice show evidence of perforin/granzyme-mediated cytolytic activity [146,149,150].
In addition, IAV infection leads to robust regulatory T cells (Tregs) that mediate immunosuppression and tissue repair via IL-10 and amphiregulin [151,152], among other effector molecules. Notably, Treg depletion results in a reduction of the influenza-specific follicular helper T cell response . These results indicate that Tregs play an important role in host protection during and after infection with the influenza virus.
Cytotoxic CD8+ T Cell Response
CD8+ T cells are important for viral clearance and host protection during influenza virus infection. CD8+ T cells recognize viral antigens loaded onto MHC class I proteins on the surface of viral antigen-presenting cells. CD8+ responses reach a maximum at 8 days after infection in the draining mLN and at 10 days post-infection in bronchoalveolar lavage fluid (BALF) [121,154]. β2-Microglobulin-deficient mice, which lack CD8+ T cells, display delayed viral clearance and severe mortality following influenza virus infection .
Activated cytotoxic CD8+ T cells (CTLs) eliminate virus-infected cells via cytolysis. They produce perforin to permeabilize the membranes of infected host cells and secrete granzyme into cells to induce apoptosis . Additionally, CTLs can kill infected host cells via TNF receptor family-dependent pathways. CTLs express the Fas ligand (FasL) that binds to Fas on target cells, and the Fas–FasL interaction induces apoptosis via activation of a caspase cascade.
TNF-related apoptosis-inducing ligand, also expressed on CD8+ T cells, is another mechanism for CD8+ T cell-mediated cytotoxicity . Effector CD8+ T cells in the lung produce IFN-γ and TNFα, which contribute to viral defense mechanisms [158,159,160]. Remarkably, IL-10 is also produced by effector CD8+ T cells and is responsible for the regulation of pulmonary inflammation during the response to influenza virus infections. Previous studies have suggested that CD8+ effector cells are the major producers of IL-10 in the lungs of mice infected with the influenza virus . Further, blockade of IL-10 signaling increases pulmonary inflammation and lethal injury following sublethal influenza virus challenge .
The protective role of B cells in the anti-influenza virus immune response has been extensively studied in recent years. During influenza virus infection, naïve B cells in the mLNs encounter the influenza virus antigen and differentiate into antibody-forming cells (AFCs). B lymphocyte-deficient μMT mice are more susceptible to influenza virus infection compared to wild-type mice [162,163]. The B cell response against influenza virus begins approximately 3 days after infection, and B cells begin to secrete anti-influenza virus IgG by day 7 .
The total number of B cells in BALF peaks around 10 days post-infection in mice . In mice, the majority of influenza virus antigen-specific AFCs in the lung produce IgG and IgM, but AFCs in the upper respiratory tract primarily produce IgA . Systemic AFCs are first detected 6–7 days after infection. Antibodies specific for HA and NA are important for protective immunity because these proteins are responsible for viral entry and release.
HA-specific antibodies bind to the HA globular head and inhibit the attachment of the virus to the host cell’s surface [166,167,168]. This mechanism of viral inactivation is called neutralization. NA-specific antibodies do not neutralize the virus, but instead block viral replication by inhibiting the enzymatic activity of NA [156,169]. M2 proteins are also the target of specific antibodies.
Passive transfer of M2-specific antibodies provide protection against viral replication . Surprisingly, NP-specific antibodies are protective in nature despite having an internal viral protein as their target . In addition to the previously described functions, influenza virus-specific antibodies mediate antibody-dependent cell cytotoxicity and Fc receptor-mediated phagocytosis.
Therefore, these antibodies also contribute significantly to the clearance of infected cells . Moreover, B-1 cells that produce natural IgM, independent of antigenic priming, contribute to protection against influenza virus infection. Natural IgM in the airway has neutralizing activity and mediates early immune responses 
reference link: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7291249/
More information: Haley L. Dugan et al. Preexisting immunity shapes distinct antibody landscapes after influenza virus infection and vaccination in humans. Science Translational Medicine. 09 Dec 2020: Vol. 12, Issue 573, eabd3601 DOI: 10.1126/scitranslmed.abd3601