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Early presence of ribonucleoprotein antigen on surface of influenza virus-infected cells
Abstract
HOST defence against influenza A virus in experimental animals seems to be mediated mainly by antibody1,2. The mechanism by which antibody protects the host, however, is unknown. Antibody may either neutralise viral particles or recognise viral antigens at the surface of infected cells and then act at an early stage of infection, before viral replication is completed. The latter mechanism is theoretically more efficient, but might cause immunopathological destruction of the infected cells with harmful consequences. Viral envelope antigens (haemagglutinin and neuraminidase) have been shown to be present at the surface of influenza virus-infected cells, by haemadsorption3, electron microscopy4 and immunofluorescence5. Two main type-specific internal antigens have been described in influenza A virus-the matrix protein (MP) antigen6 and the ribonucleoprotein (NP) antigen7, a non-glycosylated polypeptide with a molecular weight of 58,000 (ref. 8). Immunofluorescence studies on fixed cells have shown that NP antigen is first demonstrable in the cell nucleus and then appears as granular masses within the cytoplasm, which become prominent at the, cell borders later in infection, suggesting a close association with the cell membrane9. We report here that NP antigen can be detected by immunofluorescence as early as 2 h after the infection on the surface of unfixed mouse cells infected with influenza A virus.
... FcR-mediated effector functions are a pivotal mechanism for broadly protective noneAbs (45,48,49,52,79). Although internal proteins, patches of typically intracellular NP antigens are transiently expressed on the surface of influenza virus-infected cells, and the M1 has been reported to become readily accessible in cells that die following influenza virus infection (57,77,80). This makes NP and M1 proteins reachable targets for antibody-mediated Fc-receptor functions. ...
... This discrepancy may be due to differences in the experimental conditions used or the inherent biological effect of the divergent anti-NP mAbs. Alternatively, the results might also be skewed by the M1 epitope accessibility on only dead, and not living, influenza virus-infected cells (2,57,77,80). Accordingly, it might be conceivable that the in vitro ADCC and ADCP bioreporter assays were performed in the absence of accessible M1 proteins. Therefore, future experiments with a longer infection period and/or with M1 transfec ted cells might clarify which Fc-effector function(s) is mediated by mAbs targeting the internal M1 protein. ...
Improved broad-spectrum influenza virus vaccines are desperately needed to provide protection against both drifted seasonal and emerging pandemic influenza A viruses (IAVs). Antibody-based protection from influenza A virus-induced morbidity and mortality is traditionally associated with neutralizing antibodies. As such, vaccine efforts have solely focused on the hemagglutinin (HA) as a vaccine target; however, the HA is mutation prone resulting in the need for annual vaccine reformulation. Broad-spectrum vaccines could be achieved through non-neutralizing antibodies that target conserved influenza virus antigens. Here, we describe six human monoclonal antibodies (mAbs) isolated from two H3N2-infected donors that showed robust binding against the conserved internal nucleoprotein (NP) or matrix protein 1 (M1) of IAV strains. Despite the capacity for potent antigen binding, substantial morbidity was observed in mice prophylactically treated with these mAbs and then challenged with A/Netherlands/602/2009 (H1N1) or A/Switzerland/9715293/2013 (H3N2) viruses. While our findings need to be confirmed with a larger number of mAbs and with polyclonal serum, these findings suggest that human NP and M1 antibodies that are elicited following IAV infection/vaccination do not protect from substantial weight loss in the mouse model and imply that protection afforded targeting these antigens following vaccination/infection is most likely the result of cellular-based immunity.
IMPORTANCE
Currently, many groups are focusing on isolating both neutralizing and non-neutralizing antibodies to the mutation-prone hemagglutinin as a tool to treat or prevent influenza virus infection. Less is known about the level of protection induced by non-neutralizing antibodies that target conserved internal influenza virus proteins. Such non-neutralizing antibodies could provide an alternative pathway to induce broad cross-reactive protection against multiple influenza virus serotypes and subtypes by partially overcoming influenza virus escape mediated by antigenic drift and shift. Accordingly, more information about the level of protection and potential mechanism(s) of action of non-neutralizing antibodies targeting internal influenza virus proteins could be useful for the design of broadly protective and universal influenza virus vaccines.
... Several years later Zeller et al. demonstrated that NP of LCMV can be detected on the cell surface of the infected chick embryo cells in vitro [215]. It has been shown that influenza virus NP is expressed on the surface of infected cells for some time and, therefore, can serve as a target for antibody-dependent immune mechanisms [216][217][218][219][220][221]. Influenza virus NP was detected in the airways of infected mice as early as 2-3 d post-infection, it was still present at day 7, and had declined to undetectable levels by day 9, corresponding with the typical time of virus clearance [200]. ...
... Influenza virus NP was detected in the airways of infected mice as early as 2-3 d post-infection, it was still present at day 7, and had declined to undetectable levels by day 9, corresponding with the typical time of virus clearance [200]. Soluble NP was detected in nasal washes [200], supernatants of infected MDCK cells in culture and also on the surface of influenza-infected cells in vitro, along with barely detectable levels of M1 [216,217,220]. Mumps virus NP can be detected at the surface of Vero and A549 cells infected in vitro (Šantak, unpublished results). ...
Negative-stranded RNA viruses (NSVs) are important human pathogens, including emerging and reemerging viruses that cause respiratory, hemorrhagic and other severe illnesses. Vaccine design traditionally relies on the viral surface glycoproteins. However, surface glycoproteins rarely elicit effective long-term immunity due to high variability. Therefore, an alternative approach is to include conserved structural proteins such as nucleoprotein (NP). NP is engaged in myriad processes in the viral life cycle: coating and protection of viral RNA, regulation of transcription/replication processes and induction of immunosuppression of the host. A broad heterosubtypic T-cellular protection was ascribed very early to this protein. In contrast, the understanding of the humoral immunity to NP is very limited in spite of the high titer of non-neutralizing NP-specific antibodies raised upon natural infection or immunization. In this review, the data with important implications for the understanding of the role of NP in the immune response to human NSVs are revisited. Major implications of the elicited T-cell immune responses to NP are evaluated, and the possible multiple mechanisms of the neglected humoral response to NP are discussed. The intention of this review is to remind that NP is a very promising target for the development of future vaccines.
... It is known that almost all synthesized NPs are localized in the nucleus and cytoplasm in infected cells (Bullido et al., 2000). In contrast, only a few NPs are transiently located on the cell surface during the early stage of infection (Patterson et al., 1988;Prokudina & Semenova, 1991;Stitz et al., 1990;Virelizier et al., 1977;Yewdell et al., 1981); therefore, we investigated whether the anti-NP 5C and 6C mAbs bind to NP expressed on the surface of infected cells. MDCK cells were infected with A/whistling swan/Shimane/499/1983 (WS/499, H5N3 subtype) at an m.o.i. of 10, and at 5 h postinfection (p.i.) the cells were subjected to immunofluorescence staining using mouse serum from line 6C (2660). ...
... ADCC and CDC are triggered through interaction of target-bound antibodies on the surface of infected cells. Cell-surface expression of NP has been reported by several studies using different experimental techniques, including immunofluorescence assays (Virelizier et al., 1977;Yewdell et al., 1981), electron microscopy (Patterson et al., 1988), cell-based ELISA (Stitz et al., 1990), and radioimmunoassays (Prokudina & Semenova, 1991;Yewdell et al., 1981), although the mechanism responsible for the penetration of synthesized NP into the lipid bilayer membrane remains unknown. Several anti-NP mAbs that could bind NP expressed on the infected-cell surface were used in the previous studies (Patterson et al., 1988;Prokudina & Semenova, 1991;Stitz et al., 1990;Yewdell et al., 1981); however, there have been no reports on antiviral effects of those antibodies in vivo. ...
The nucleoprotein (NP) possesses the regions that are highly conserved among influenza A viruses, and has therefore been one of the target viral proteins for development of a universal influenza vaccine. It has been expected that human or humanized antibodies are made available for the prophylaxis, preemptive and acute treatment of viral infection. However, it is still unclear whether anti-NP human antibody can confer protection against influenza infections. In this study, we generated transgenic mice expressing anti-NP human monoclonal antibodies (mAbs) derived from lymphocytes of a patient infected with H5N1 highly pathogenic avian influenza (HPAI) virus, and experimental infections were conducted to examine antiviral effects of the anti-NP antibodies against H5N1 HPAI viral infections with a high fatality rate in mammals. Transgenic mouse lines expressing the anti-NP human mAbs of more than 1 mg/mL showed marked resistance to H5N1 virus infections. In addition, resistance to infection with an H1N1 subtype that shows strong pathogenicity to mice was also confirmed. Although the anti-NP mAbs expressed in the transgenic mice did not neutralize the virus, the mAbs could bind to NP located on the cell surface of infected cells. These results suggested a possibility that the non-neutralizing anti-NP human mAbs could induce indirect antiviral effects, such as antibody-dependent cellular cytotoxicity or complement-dependent cytotoxicity. Taken together, these results demonstrated that the anti-NP human mAbs play an important role in heterosubtypic protection against lethal influenza virus infections in vivo.
... Passive transfer A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT 6 of non-neutralising NP Abs can also fully protect against influenza challenge in mice, but the mechanism of protection has yet to be elucidated [41,42]. NP can also be found on the surface of influenza-infected cells in vitro, along with barely detectable levels of M1 [43][44][45]. It is conceivable that non-neutralising Abs to M1 and NP may be involved in clearance of infected cells through Fc mediated effector functions. ...
... Indeed, this study found NK cell activating Abs to highly conserved influenza Ags, but we were not able to detect Ab-mediated killing of target cells infected with rVVs expressing either NP or M1. Internal influenza proteins are present in the extracellular environment following their release from dead or infected cells [42] and NP is present on the surface of influenza-infected cells in vitro as early as one hour post-infection [43][44][45]. In spite of a robust rVV infection system as evidenced by consistently high levels of ADCC against rVV-HA infected cells, we found that only a small percentage (up to ~25%) of target CEM cells infected with rVV-NP had NP on the cell surface, which may be insufficient for detection of Ab-mediated killing in the assays employed. ...
The conserved internal influenza proteins nucleoprotein (NP) and matrix 1 (M1) are well characterised for T cell immunity, but whether they also elicit functional antibodies capable of activating natural killer (NK) cells has not been explored. We studied NP and M1-specific ADCC activity using biochemical, NK cell activation and killing assays with plasma from healthy and influenza-infected subjects. Healthy adults had antibodies to M1 and NP capable of binding dimeric FcγRIIIa and activating NK cells. Natural symptomatic and experimental influenza infections resulted in a rise in antibody dependent NK cell activation post-infection to the hemagglutinin of the infecting strain, but changes in NK cell activation to M1 and NP were variable. Although antibody dependent killing of target cells infected with vaccinia viruses expressing internal influenza proteins was not detected, opsonising antibodies to NP and M1 likely contribute to an antiviral microenvironment by stimulating innate immune cells to secrete cytokines early in infection. We conclude that effector cell activating antibodies to conserved internal influenza proteins are common in healthy and influenza-infected adults. Given the significance of such antibodies in animal models of heterologous influenza infection, the definition of their importance and mechanism of action in human immunity to influenza is essential.
... defined the specificities of clones 11 and F5 (5,10). In view of the fact that NP has been detected at the surface of influenza virus-infected cells (29,30), but not on cells transfected with the NP gene (10), it was of interest to compare purified whole NP with appropriate peptides for its ability to sensitize target cells for lysis in the 5'Cr-release assay. Purified NP was titrated in the assay as described in Materials and Methods. ...
... Influenza NP can be detected with antibody at the surface of influenza virusinfected cells (10,29,30). This is surprising because its sequence contains neither a hydrophobic signal peptide, nor a characteristic membrane anchor sequence . ...
The conserved epitopes of influenza nucleoprotein (NP) recognized by class I MHC-restricted CTL from CBA (H-2k) and C57BL/10 (H-2b) mice have been defined in vitro with synthetic peptides 50-63 and 365-379, respectively. Two Db-restricted clones were described that recognize different epitopes on peptide 365-379. Finally, the recognition of complete NP was shown to be approximately 200-fold less efficient than peptide in the cytotoxicity assay. These phenomena are closely related to results with class II-restricted T cells and they strengthen the hypothesis that influenza proteins are degraded in the infected cell before recognition by class I-restricted CTL.
... Whether such release of this protein is associated with death of infected cells or some other mechanism linked to the virus replication cycle is unknown. Similarly, the role, if any, of plasma membrane-associated NP detected on cultured cells (Virelizier et al., 1977;Yewdell et al., 1981) in virus replication is also unknown. ...
... Whether the less common induction of anti-M1 results from less or differential exposure of the antigen to B cells during infection is unknown. Unlike NP (Virelizier et al., 1977;Yewdell et al., 1981), M1 has not been detected on the surface of infected cells in culture (Mozdzanowska et al., 1999). It is unknown whether anti-M1 antibody is capable of antiviral activity. ...
High-performance neutralizing antibody against influenza virus typically recognizes the globular head region of its hemagglutinin (HA) envelope glycoprotein. To-date, approved human vaccination strategies have been designed to induce such antibodies as a sole means of preventing the consequences of this infection. However, frequent amino-acid changes in the HA globular head allow for efficient immune evasion. Consequently, vaccines inducing such neutralizing antibodies need to be annually re-designed and re-administered at a great expense. These vaccines furthermore provide little-to-no immunity against antigenic-shift strains, which arise from complete replacement of HA or of neuraminidase genes, and pose pandemic risks. To address these issues, laboratory research has focused on inducing immunity effective against all strains, regardless of changes in the HA globular head. Despite prior dogma that such cross-protection needs to be induced by cellular immunity alone, several advances in recent years demonstrate that antibodies of other specificities are capable of cross-strain protection in mice. This review discusses the reactivity, induction, efficacy, and mechanisms of antibodies that react with poorly accessible epitopes in the HA stalk, with the matrix 2 membrane ion channel, and even with the internal nucleoprotein. These advances warrant further investigation of the inducibility and efficacy of such revolutionary antibody strategies in humans.
... In addition, an infected organism typically produces a significant amount of anti-NP antibodies with high affinity that do not exhibit neutralizing activity but are associated with a reduction in disease severity [18]. Although this antigen is not exposed on the surface of virions, it has been detected on the surface of virus-infected cells [19,20], making it a potential target for antibody-mediated immune responses, such as antibody-dependent cellular cytotoxicity (ADCC) or complement activation [21,22]. ...
Annual vaccination is considered as the main preventive strategy against seasonal influenza. Due to the highly variable nature of major viral antigens, such as hemagglutinin (HA) and neuraminidase (NA), influenza vaccine strains should be regularly updated to antigenically match the circulating viruses. The influenza virus nucleoprotein (NP) is much more conserved than HA and NA, and thus seems to be a promising target for the design of improved influenza vaccines with broad cross-reactivity against antigenically diverse influenza viruses. Traditional subunit or recombinant protein influenza vaccines do not contain the NP antigen, whereas live-attenuated influenza vaccines (LAIVs) express the viral NP within infected cells, thus inducing strong NP-specific antibodies and T-cell responses. Many strategies have been explored to design broadly protective NP-based vaccines, mostly targeted at the T-cell mode of immunity. Although the NP is highly conserved, it still undergoes slow evolutionary changes due to selective immune pressure, meaning that the particular NP antigen selected for vaccine design may have a significant impact on the overall immunogenicity and efficacy of the vaccine candidate. In this review, we summarize existing data on the conservation of the influenza A viral nucleoprotein and review the results of preclinical and clinical trials of NP-targeting influenza vaccine prototypes, focusing on the ability of NP-specific immune responses to protect against diverse influenza viruses.
... The cell surface expression of viral RNA-and DNA-binding proteins dates to initial polyclonal Ab detection of retrovirus gag (11) and polyoma virus T antigen in the 1970s (12). These findings were extended to influenza virus N using polyclonal Abs (13), and definitively established with monoclonal Abs (mAbs) (14). Similar findings were made using mAbs specific for surface N expressed by vesicular stomatitis (15), lymphocytic choriomeningitis (16), human immunodeficiency (17), respiratory syncytial (18), and measles viruses (19). ...
We recently reported that SARS-CoV-2 nucleocapsid (N) protein is abundantly expressed on the surface of both infected and neighboring uninfected cells, where it enables activation of Fc receptor-bearing immune cells with anti-N antibodies (Abs) and inhibits leukocyte chemotaxis by binding chemokines (CHKs). Here, we extend these findings to N from the common cold human coronavirus (HCoV)-OC43, which is also robustly expressed on the surface of infected and noninfected cells by binding heparan sulfate/heparin (HS/H). HCoV-OC43 N binds with high affinity to the same set of 11 human CHKs as SARS-CoV-2 N, but also to a nonoverlapping set of six cytokines. As with SARS-CoV-2 N, HCoV-OC43 N inhibits CXCL12β-mediated leukocyte migration in chemotaxis assays, as do all highly pathogenic and common cold HCoV N proteins. Together, our findings indicate that cell surface HCoV N plays important evolutionarily conserved roles in manipulating host innate immunity and as a target for adaptive immunity.
... Therefore, DM-C may provide an additional layer of protection by ADCC function targeting both M2 and NA. It was shown that NP, previously recognized as a strictly internal protein, could be displayed on the surface of infected cells (69). However, whether NP is an additional target for ADCC remains to be investigated (39). ...
Influenza virus infections can cause a broad range of symptoms, form mild respiratory problems to severe and fatal complications. While influenza virus poses a global health threat, the frequent antigenic change often significantly compromises the protective efficacy of seasonal vaccines, further increasing the vulnerability to viral infection. Therefore, it is in great need to employ strategies for the development of universal influenza vaccines (UIVs) which can elicit broad protection against diverse influenza viruses. Using a mouse infection model, we examined the breadth of protection of the caspase-triggered live attenuated influenza vaccine (ctLAIV), which was self-attenuated by the host caspase-dependent cleavage of internal viral proteins. A single vaccination in mice induced a broad reactive antibody response against four different influenza viruses, H1 and rH5 (HA group 1) and H3 and rH7 subtypes (HA group 2). Notably, despite the lack of detectable neutralizing antibodies, the vaccination provided heterosubtypic protection against the lethal challenge with the viruses. Sterile protection was confirmed by the complete absence of viral titers in the lungs and nasal turbinates after the challenge. Antibody-dependent cellular cytotoxicity (ADCC) activities of non-neutralizing antibodies contributed to cross-protection. The cross-protection remained robust even after in vivo depletion of T cells or NK cells, reflecting the strength and breadth of the antibody-dependent effector function. The robust mucosal secretion of sIgA reflects an additional level of cross-protection. Our data show that the host-restricted designer vaccine serves an option for developing a UIV, providing pan-influenza A protection against both group 1 and 2 influenza viruses. The present results of potency and breadth of protection from wild type and reassortant viruses addressed in the mouse model by single immunization merits further confirmation and validation, preferably in clinically relevant ferret models with wild type challenges.
... Mice immunized with recombinant NP proteins demonstrated significantly reduced morbidity upon heterologous influenza infections in an antibody Fc-dependent manner [63,64]. Although it has been shown that NP can be expressed on cell membrane of virus-infected cells [65], whether NP antibodies exert antibody effector functions remains controversial. A study showed that human NP and M1 antibodies were capable of activating NK cells but did not kill target cells in vitro [66]. ...
Influenza virus infection remains a major public health challenge, causing significant morbidity and mortality by annual epidemics and intermittent pandemics. Although current seasonal influenza vaccines provide efficient protection, antigenic changes of the viruses often significantly compromise the protection efficacy of vaccines, rendering most populations vulnerable to the viral infection. Considerable efforts have been made to develop a universal influenza vaccine (UIV) able to confer long-lasting and broad protection. Recent studies have characterized multiple immune correlates required for providing broad protection against influenza viruses, including neutralizing antibodies, non-neutralizing antibodies, antibody effector functions, T cell responses, and mucosal immunity. To induce broadly protective immune responses by vaccination, various strategies using live attenuated influenza vaccines (LAIVs) and novel vaccine platforms are under investigation. Despite superior cross-protection ability, very little attention has been paid to LAIVs for the development of UIV. This review focuses on immune responses induced by LAIVs, with special emphasis placed on the breadth and the potency of individual immune correlates. The promising prospect of LAIVs to serve as an attractive and reliable vaccine platforms for a UIV is also discussed. Several important issues that should be addressed with respect to the use of LAIVs as UIV are also reviewed.
... Although antibodies targeting these antigens can clearly bind infected cells, using similar approaches to the present study we recently showed that non-structural accessory proteins were more potent ADCC targets during HCMV infection 26 Nucleocapsid is therefore likely to be the major target for ADCC during natural SARS-CoV2 infection in most people. Influenza nucleoprotein is also detected at the plasma membrane [58][59][60] , and antibodies targeting nucleoprotein can mediate ADCC 61,62 . Structural proteins involved in genome packaging may therefore represent a common target for ADCC across multiple virus families. ...
SARS-CoV-2 antagonises the cellular interferon response, but whether the virus manipulates cellular immunity is unclear. An unbiased proteomic approach to determine how cell surface protein expression is altered on SARS-CoV-2-infected lung epithelial cells showed downregulation of activating NK cell ligands: B7-H6, MICA, ULBP2, and Nectin1, but no effect on surface MHC-I expression. NK ligand downregulation correlated with a reduction in NK cell activation by infected cells, and was overcome by antibody-dependent NK cell activation (ADNKA). Depletion of spike-specific antibodies confirmed their dominant role in virus neutralisation, but these antibodies played only a minor role in ADNKA compared to antibodies to other viral proteins, including ORF3a, Membrane, and Nucleocapsid. In contrast, ADNKA induced following vaccination was focussed solely on spike, was weaker than ADNKA following natural infection, and was not boosted by the second dose. These insights have important implications for understanding disease progression, vaccine efficacy, and vaccine design.
... Unlike neutralizing antibodies, NP-specific antibodies cannot neutralize the virus and prevent the virus entry into host cells [32,33]. NP in various forms can be detected in the supernatant of MDCK cells infected with influenza virus and in the nasal wash of mice infected with influenza virus, including monomer, polymer and complex formed with RNA or polymerase, so that NP-specific antibodies have sufficient opportunity to interact with NP to exert antiviral immune responses [34]. In addition, investigators also found NP expression on the surface of influenza virus-infected cells [35]. ...
The nucleoprotein (NP) is a highly conserved internal protein of the influenza virus, a major target for universal influenza vaccine. Our previous studies have proven NP-based subunit vaccine can provide partial protection in mice. It is reported that the protein transduction domain (PTD) TAT protein from human immunodeficiency virus-1 (HIV-1) is able to penetrate cells when added exogenous protein and could effectively enhance the immune response induced by the exogenous protein. In present study, the recombinant protein TAT-NP, a fusion of TAT and NP was effectively expressed in Escherichia coli and purified as a candidate component for an influenza vaccine. We evaluated the immunogenicity and protective efficacy of recombinant influenza TAT-NP vaccine by intranasal immunization. In vitro experiments showed that TAT-NP could efficiently penetrate into cells. Animal results showed that mice vaccinated with TAT-NP could not only induce higher levels of IgG and mucosal IgA, but also elicit a robust cellular immune response. Moreover, the TAT-NP fusion protein could significantly increase the protection of mice against lethal doses of homologous influenza virus PR8 and could also provide mice protection against a lethal dose challenge against heterosubtypic H9N2 and H3N2 influenza virus. In conclusion, the recombinant TAT-NP might be a universal vaccine candidate against influenza virus.
... In addition to those proteins expressed on the viral surface, influenza nucleoprotein (NP) possesses highly conserved regions and is also detected on influenza virus-infected host cells surface [123,124]. This makes NP reachable by antibody and substantial efforts have been focused on the antiviral effects of anti-NP mAbs. ...
Influenza causes millions of cases of hospitalizations annually and remains a public health concern on a global scale. Vaccines are developed and have proven to be the most effective countermeasures against influenza infection. Their efficacy has been largely evaluated by hemagglutinin inhibition (HI) titers exhibited by vaccine-induced neutralizing antibodies, which correlate fairly well with vaccine-conferred protection. Contrarily, non-neutralizing antibodies and their therapeutic potential are less well defined, yet, recent advances in anti-influenza antibody research indicate that non-neutralizing Fc-effector activities, especially antibody-dependent cellular cytotoxicity (ADCC), also serve as a critical mechanism in antibody-mediated anti-influenza host response. Monoclonal antibodies (mAbs) with Fc-effector activities have the potential for prophylactic and therapeutic treatment of influenza infection. Inducing mAbs mediated Fc-effector functions could be a complementary or alternative approach to the existing neutralizing antibody-based prevention and therapy. This review mainly discusses recent advances in Fc-effector functions, especially ADCC and their potential role in influenza countermeasures. Considering the complexity of anti-influenza approaches, future vaccines may need a cocktail of immunogens in order to elicit antibodies with broad-spectrum protection via multiple protective mechanisms.
... Although this type of immunity cannot prevent infection entirely, it can considerably impede its development. For example, influenza virus nucleoprotein (NP) could be detected on the surface of infected cells at the early stages of infection and neutral ized by antibodies against this protein [3]. ...
Recently we obtained complexes between genetically modified Tobacco Mosaic Virus (TMV) particles and proteins carrying conserved influenza antigen such as M2e epitope. Viral vector TMV-N-lys based on TMV-U1 genome was constructed by insertion of chemically active lysine into the exposed N-terminal part of the coat protein. Nicotiana benthamiana plants were agroinjected and TMV-N-lys virions were purified from non-inoculated leaves. Preparation was analyzed by SDS-PAGE/Coomassie staining; main protein with electrophoretic mobility of 21 kDa was detected. Electron microscopy confirmed the stability of modified particles. Chemical conjugation of TMV-N-lys virions and target influenza antigen M2e expressed in E. coli was performed using 5 mM 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide and 1 mM N-hydroxysuccinimide. The efficiency of chemical conjugation was confirmed by Western blotting. For additional characterization we used conventional electron microscopy. The diameter of the complexes did not differ significantly from the initial TMV-N-lys virions, but complexes formed highly organized and extensive network with dense “grains” on the surface. Dynamic light scattering demonstrated that the single peaks, reflecting the complexes TMV-N-lys/DHFR-M2e were significantly shifted relative to the control TMV-N-lys virions. The indirect enzymelinked immunosorbent assay with TMV- and DHFR-M2e-specific antibodies showed that the complexes retain stability during overnight adsorption. Thus, the results allow using these complexes for immunization of animals with the subsequent preparation of a candidate universal vaccine against the influenza virus.
... Previous studies have shown that NP can be expressed on the surface of influenza virus-infected cells (123)(124)(125), however evidence for Fc-mediated effector functions of anti-NP Abs is controversial. Regarding ADCC, despite Varderven et al reported that healthy individuals had anti-NP and anti-M1 Abs capable of activating NK cells through FCγRIII, these Abs had no killing activity on target cells in vitro (116). ...
The influenza A virus infection continues to be a threat to the human population. The seasonal variation of the virus and the likelihood of periodical pandemics caused by completely new virus strains make it difficult to produce vaccines that efficiently protect against this infection. Antibodies (Abs) are very important in preventing the infection and in blocking virus propagation once the infection has taken place. However, the precise protection mechanism provided by these Abs still needs to be established. Furthermore, most research has focused on Abs directed to the globular head domain of hemagglutinin (HA). However, other domains of HA (like the stem) and other proteins are also able to elicit protective Ab responses. In this article, we review the current knowledge about the role of both neutralizing and non-neutralizing anti-influenza proteins Abs that play a protective role during infection or vaccination.
... Query filters: positive assays only, organism: influenza A virus (ID:11320, influenza A), MHC restriction type: class I or class II, host: Homo sapiens (human) [18]. d There is evidence of transitory expression of NP on the cell surface, confirmed by NP-specific antibody staining [19,20]. A possible role of anti-NP antibodies in influenza protection has been demonstrated recently [21]. ...
Introduction:
One of the essential mechanisms of virus infection control is cell-mediated cytotoxicity, which can act in an antibody-dependent or -independent fashion and is provided by different effector cells. The role of CD8 T-cells in infection control and in affecting the pathological outcome of different types of infection has been demonstrated in numerous animal studies. Despite this, their role in controlling human influenza infection is not fully understood. Especially, knowledge about their induction and turnover in human influenza infection is limited. Differences in the development of CD8 T-cells after influenza infection or immunizations should be explored in detail, in relation to the bioaccessibility of influenza antigens, site of application and distribution routes. Areas covered: This review focuses on the basics of CD8 T-cell immune response both in human influenza infection and after administration of inactivated or live attenuated influenza vaccine. Some aspects of the accessibility, distribution and presentation of influenza antigens to CD8 T-cells are described. Expert commentary: The CD8 T-cell response is an essential connection between innate and antibody-mediated responses, which are all-important for influenza control. We hypothesize that immunization with live influenza vaccine is the most straightforward artificial way to induce an efficient influenza-specific CD8 T-cell response.
... Furthermore, passive transfer of anti-NP antibodies and vaccine-elicited antibodies against the extracellular domain of M2 (M2e) protects mice from lethal influenza challenge [41][42][43]. How anti-NP antibody mediates protection remains unclear, but NP is found on the surface of influenza-infected cells in vitro [38,44]. IAV infection induces robust ADCC antibodies in humans and nonhuman primates (reviewed in [45]). ...
Background:
Influenza A virus (IAV) vaccines offer little protection from mismatched viruses with antigenically distant hemagglutinin (HA) glycoproteins. We sought to determine if a cationic lipid/DNA complex (CLDC) adjuvant could induce heterosubtypic protection if added to a whole-inactivated influenza A virus vaccine (WIV).
Methods:
Adult rhesus macaques (RM) were vaccinated and at two weeks boosted with either a H1N1-WIV or a H3N2-WIV, with and without CLDC adjuvant. Four weeks post-boost, animals were challenged with a H1N1 influenza A virus matched to the H1N1-WIV vaccine.
Results:
After challenge, vRNA levels in the trachea of control RM and RM vaccinated with the unadjuvanted H1 or H3 WIV vaccines were similar. However, vRNA levels in the trachea of both the H1-WIV/CLDC (p<0.01) and the H3-WIV/CLDC (p<0.05) vaccinated RM were significantly lower than in unvaccinated control RM. Heterosubtypic protection in H3-WIV/CLDC RM was associated with significantly higher levels of nucleoprotein (NP) and matrix-1 (M1)-specific IgG antibodies (p<0.05 or greater) and NP-specific non-neutralizing antibody dependent natural killer (NK) cell activation (p<0.01) compared to unprotected H3-WIV RM.
Conclusions:
Addition of the CLDC adjuvant to a simple WIV elicited immunity to conserved virus structural proteins in RM that correlate with protection from uncontrolled virus replication after heterosubtypic influenza virus challenge.
... The antibodies directed to the conservative proteins PB2, PB1, PA, NP, and M1 do not have neutralizing activity but could play an important role in virus elimination by means of ADCC. It was shown that the NP protein could be temporary expressed on the surface of the cell and the antibodies induced by this protein could also possess the neutralizing activity [66]. Moreover, the NP protein peptides, presented on MHC class I molecules, represent the most important targets for the cytotoxic CD8 + cells [28]. ...
The lack of population immunity to the periodically emerging pandemic influenza strains makes influenza infection especially dangerous. The fragmented nature of the influenza virus genome contributes to the formation of influenza virus reassortants containing genomic fragments from different strains. This mechanism is the main reason for the natural influenza virus antigenic diversity as well as for the occurrence of influenza pandemics. Vaccination is the best measure to prevent the spread of influenza infection, but the efficacy of existing vaccines is not sufficient, especially for the elderly and small children. Specific immunity, developed after disease or immunization, poorly protects against infection by influenza viruses of another subtype. In this regard, there is an urgent need for a more effective universal influenza vaccine that provides a long-lasting broad cross-protective immunity, and is able to protect against influenza A and B viruses of all known subtypes. The basic approaches to as well as challenges of creating such a vaccine are discussed in this review.
... Антитела к консервативным внутренним белкам PB2, PB1, PA, NP и M1 не относятся к нейтрализующим, но могут играть важную роль в обеспечении элиминации вируса за счет ADCC. Для белка NP показано, что он может быть временно экспрессирован на клеточной поверхности и поэтому антитела, индуцируемые этим белком, могут также обладать нейтрализующей активностью [66]. Кроме того, пептиды белка NP, презентированные на молекулах МНС I класса, представляют собой важнейшие мишени для цитотоксических клеток CD8 + [28]. ...
Периодически появляющиеся новые пандемические штаммы вируса гриппа А, к которым отсутствует популяционный иммунитет, превращают грипп в особо опасную инфекцию. Сегментированная природа генома вируса гриппа способствует образованию реассортантов – вирусов, в состав которых входят геномные сегменты разных штаммов, принадлежащих одному роду. Именно механизм реассортации является основной причиной антигенного разнообразия вирусов гриппа в природе и появления штаммов, вызывающих пандемии в человеческой популяции. Лучшим средством предотвращения распространения гриппозной инфекции считается вакцинация. Однако эффективность известных на сегодняшний день вакцин недостаточна, особенно при иммунизации пожилых людей и маленьких детей. Специфический иммунитет, вырабатываемый после перенесенного заболевания или вакцинации одним подтипом вируса гриппа, слабо защищает от инфекции вирусом другого подтипа. В связи с этим не потерял актуальности вопрос разработки эффективной универсальной гриппозной вакцины, которая будет индуцировать широкий кросс-протективный длительный иммунитет как к вирусам гриппа А различных подтипов, так и к вирусам гриппа В. Основные подходы к созданию такой вакцины и проблемы их реализации рассматриваются в данном обзоре.
... Non-neutralizing antibodies mounted against highly conserved internal proteins, such as NP [80][81][82], M1, PA, PB1 or PB2, may contribute to clearing influenza virus-infected cells, although the exact mechanism through which this clearance is mediated remains largely unclear. It was shown that NP may be transiently expressed on the cellular surface [83], thus offering a target for antibody binding and possible subsequent neutralization or ADCC. Further studies are required to elucidate the exact mechanisms of these interactions in the context of influenza virus infections or protection from infection. ...
Influenza viruses have a huge impact on public health. Current influenza vaccines need to be updated annually and protect poorly against antigenic drift variants or novel emerging subtypes. Vaccination against influenza can be improved in two important ways, either by inducing more broadly protective immune responses or by decreasing the time of vaccine production, which is relevant especially during a pandemic outbreak. In this review, we outline the current efforts to develop so-called "universal influenza vaccines", describing antigens that may induce broadly protective immunity and novel vaccine production platforms that facilitate timely availability of vaccines.
... Passive transfer of nonneutralizing polyclonal Abs or mAbs toward NP was associated with protection from lethal influenza challenge in mice (64,68), although the mechanism of Ab-mediated protection remains unclear. Of interest, studies showed that intracellular NP Ag is transiently expressed on the surface of influenza virus-infected cells (69,70) and, therefore, could represent a target for ADCC. Bodewes et al. (71) investigated the in vitro Fc-mediated effector functions of anti-NP Abs, demonstrating that a human anti-NP Ab did not mediate neutralization or complement-dependent cytotoxicity or improve presentation by opsonization. ...
There is an urgent need for universal influenza vaccines that can control emerging pandemic influenza virus threats without the need to generate new vaccines for each strain. Neutralizing Abs to the influenza virus hemagglutinin glycoprotein are effective at controlling influenza infection but generally target highly variable regions. Abs that can mediate other functions, such as killing influenza-infected cells and activating innate immune responses (termed "Ab-dependent cellular cytotoxicity [ADCC]-mediating Abs"), may assist in protective immunity to influenza. ADCC-mediating Abs can target more conserved regions of influenza virus proteins and recognize a broader array of influenza strains. We review recent research on influenza-specific ADCC Abs and their potential role in improved influenza-vaccination strategies.
... Unexpectedly, Abs specific for the viral nucleoprotein (NP), which surrounds the genome, have also shown passive protection in mouse models at very high doses (Carragher et al., 2008;Lamere et al., 2011) and can be found in human serum (Sukeno et al., 1979). In vitro studies demonstrated that influenza-infected cells express low levels of NP on their surface (Virelizier et al., 1977;Yewdell et al., 1981), which may enable NP recognition by immune effectors, or alternatively, it is possible that NP-specific Abs are internalized and interrupt virus replication. Utilization of non-neutralizing NP and M2e Abs might be beneficial when combined with additional protective immune mechanisms. ...
Although an influenza vaccine has been available for 70 years, influenza virus still causes seasonal epidemics and worldwide pandemics. Currently available vaccines elicit strain-specific antibody responses to the surface haemagglutinin (HA) and neuraminidase (NA) proteins, but these can be ineffective against serologically-distinct viral variants and novel subtypes. Thus, there is a need for cross-protective or “universal” influenza vaccines to overcome the necessity for annual immunisation against seasonal influenza and to provide immunity to reduce the severity of infection with pandemic or outbreak viruses. It is well established that natural influenza infection can provide cross-reactive immunity that can reduce the impact of infection with distinct influenza type A strains and subtypes, including H1N1, H3N2, H2N2, H5N1 and H7N9. The key to generating universal influenza immunity via vaccination is to target functionally-conserved regions of the virus, which include epitopes on the internal proteins for cross-reactive T cell immunity or on the HA stem for broadly reactive antibody responses. In the wake of the 2009 H1N1 pandemic, broadly neutralizing antibodies have been characterized and isolated from convalescent and vaccinated individuals, inspiring development of new vaccination techniques to elicit such responses. Induction of influenza-specific T cell responses through vaccination has also been examined in clinical trials. Strong evidence is available from human and animal models of influenza to show that established influenza-specific T cell memory can reduce viral shedding and symptom severity. However, the published evidence also shows that CD8+ T cells can efficiently select immune escape mutants early after influenza virus infection. Here, we discuss universal immunity to influenza viruses mediated by both cross-reactive T cells and antibodies, the mechanisms of immune evasion in influenza, and how to counteract commonly occurring-escape variants.
... Previous studies have shown that passive transfer of NP-specific antibodies into mice provided protection from heterologous challenge (45,50,51). Unlike, other internal proteins, influenza virus NP is at least transiently expressed on the surfaces of virus-infected cells (52,53). The mechanism by which these antibodies mediate their activity is unclear, with recent studies suggesting that antibody-mediated phagocytosis or complementmediated lysis is unlikely to be the mechanism (54). ...
Yearly vaccination with the trivalent inactivated influenza vaccine (TIV) is recommended since current vaccines induce little cross neutralization to divergent influenza strains. Whether the TIV can induce ADCC responses that can cross-recognize divergent influenza strains is unknown. We immunized 6 influenza-naïve pigtail macaques twice with the 2011-2012 season TIV and then challenged macaques, along with 12 control macaques, serially with H1N1 and H3N2 viruses. We measured ADCC responses in plasma to a panel of H1 and H3 hemagglutinin (HA) proteins and influenza-specific CD8 T cell (CTL) responses using a sensitive MHC-tetramer reagent. The TIV was weakly immunogenic and, although binding antibodies were detected by ELISA, did not induce detectable influenza-specific ADCC or CTLs responses. The H1N1 challenge elicited robust ADCC to both homologous and heterologous H1 HA proteins but not influenza HA proteins from different subtypes (H2-H7). There was no anamnestic influenza-specific ADCC or CTL response in vaccinated animals. The subsequent H3N2 challenge did not induce or boost ADCC either to H1 HA proteins or divergent H3 proteins but did boost CTL responses. ADCC or CTLs responses are not induced by TIV vaccination in influenza-naïve macaques. There was a marked difference in the ability of infection compared to vaccination in inducing cross-reactive ADCC and CTL responses. Improved vaccination strategies are needed to induce broad-based ADCC immunity to influenza.
... Adsorption of NP released by necrotic or killed infected cells onto the cell surface of intact cells or virions may represent one possible explanation for these findings. Interestingly, presence of influenza virus NP epitopes on the surface of infected cells has also been described long time ago but the underlying mechanism is nonetheless still obscure [26,27]. Hence, in both viral systems, epitopes of internal proteins usually associated with the viral RNA can be found on the surface of infected cells and corresponding Abs facilitate viral elimination in vivo although they are unable to directly prevent virus entry into host cells. ...
CD8(+) T cells have an essential role in controlling LCMV infection in mice. Here, we examined the contribution of humoral immunity, including non-neutralizing antibodies (Abs), in this infection induced by low virus inoculation doses. Mice with impaired humoral immunity readily terminated infection with the slowly replicating LCMV strain Armstrong but showed delayed virus elimination after inoculation with the faster replicating LCMV strain WE and failed to clear the rapidly replicating LCMV strain Docile, which is in contrast to the results obtained with wild-type mice. Thus, the requirement for adaptive humoral immunity to control the infection was dependent on the replication speed of the LCMV strains used. Ab transfers further showed that LCMV-specific IgG Abs isolated from LCMV immune serum accelerated virus elimination. These Abs were mainly directed against the viral nucleoprotein and completely lacked virus neutralizing activity. Moreover, mAbs specific for the LCMV nucleoprotein were also able to decrease viral titers after transfer into infected hosts. Intriguingly, neither C3 nor FcγR were required for the antiviral activity of the transferred Abs. In conclusion, our study suggests that rapidly generated non-neutralizing Ab specific for the viral nucleoprotein speed up virus elimination and thereby may counteract T-cell exhaustion. This article is protected by copyright. All rights reserved.
... Many moons ago (371, to be precise), while investigating the basis for cross-subtype recognition by CD8+ T cells, I helped to show that nucleoprotein is expressed on the surface of infected cells [4,5] (sadly, a red herring for T cell recognition), and can serve as a target of antibody-targeted cellular cytotoxicity (ADCC) [6]. Though the pathway that traffics NP to the surface remains an intriguing mystery (that likely functions for many viral and host " internal " proteins), the potential importance of surface of NP as a target for protective Abs was resurrected by Randall and colleagues789, who demonstrated a clear protective effect in mice following immunization with NP. ...
Rapid antigenic evolution of the influenza A virus hemagglutinin has precluded developing vaccines that provide durable protection. The yearly costs of influenza (circa $10(11) in the USA alone) easily justify investments in better understanding the interaction of influenza with antibodies and other inducible elements of the immune system that potentially limit or circumvent antigenic variation. Here, I summarize exciting new findings that offer the possibility of a quantum improvement in vaccine efficacy, focusing on studies clearly documenting robust neutralizing antibody responses to the conserved stem region of the hemagglutinin.
... Alternatively, antiviral immune responses might be induced by forming a complex between anti-N antibody and N proteins released from dead cells or infected cells in vivo [146]. If N proteins are present in cell surface as reported in influenza virus-infected cells [147,148], complement-mediated cell lysis could be another mechanism to support the elimination of infected cells [146]. ...
Rift Valley fever (RVF) is an emerging zoonotic disease distributed in sub-Saharan African countries and the Arabian Peninsula. The disease is caused by the Rift Valley fever virus (RVFV) of the family Bunyaviridae and the genus Phlebovirus. The virus is transmitted by mosquitoes, and virus replication in domestic ruminant results in high rates of mortality and abortion. RVFV infection in humans usually causes a self-limiting, acute and febrile illness; however, a small number of cases progress to neurological disorders, partial or complete blindness, hemorrhagic fever, or thrombosis. This review describes the pathology of RVF in human patients and several animal models, and summarizes the role of viral virulence factors and host factors that affect RVFV pathogenesis.
... [101][102][103] However, for sustained and complete protection, vaccines would also need to comprehensively and combinatorially include conserved signatures of the highly conserved intra-virion proteins PB2, NP, M1, and so on (Figure 1, Supplementary Information S1). [104][105][106][107][108][109][110][111][112] Therefore, universal heterosubtypic antibody-based vaccine strategies would be complemented by alternate less-universal heterosubtypic, perhaps other non-coincident, antibody-based approaches, using panels of less-widely conserved more subtype-selective libraries for determinants of neutralization and pathogenicity that would include constructs with conserved discontinuous epitopes (for example, globular-head HA and NA domains) displayed within conformation-maintained low to neutral Ag scaffolds, 113 with the latter directed at subtype-specific conserved elements, in HA and NA, to promote neutralizing subtype-specific antibody responses. DNA and recombinant approaches may be best suited to deliver the complex array of conserved linear and discontinuous and conformational Ag signatures required for universal seasoned (for example, Figure 2 and Supplementary Information S2) or subtype-selective (for example, Figure 3 and Supplementary Information S2) antibody-based vaccine protection. ...
Fundamentally new approaches are required for the development of vaccines to pre-empt and protect against emerging and pandemic influenzas. Current strategies involve post-emergent homotypic vaccines that are modelled upon select circulating 'seasonal' influenzas, but cannot induce cross-strain protection against newly evolved or zoonotically introduced highly pathogenic influenza (HPI). Avian H5N1 and the less-lethal 2009 H1N1 and their reassortants loom as candidates to seed a future HPI pandemic. Therefore, more universal 'seasoned' vaccine approaches are urgently needed for heterotypic protection ahead of time. Pivotal to this is the need to understand mechanisms that can deliver broad strain protection. Heterotypic and heterosubtypic humoral immunities have largely been overlooked for influenza cross-protection, with most 'seasoned' vaccine efforts for humans focussed on heterotypic cellular immunity. However, 5 years ago we began to identify direct and indirect indicators of humoral-herd immunity to protein sites preserved among H1N1, H3N2 and H5N1 influenzas. Since then the evidence for cross-protective antibodies in humans has been accumulating. Now proposed is a rationale to stimulate and enhance pre-existing heterotypic humoral responses that, together with cell-mediated initiatives, will deliver pre-emptive and universal human protection against emerging epidemic and pandemic influenzas.
... Although NP antibody may not protect cells against influenza infection (39), the substantial antibody response generated against NP protein by natural infection or vaccination indicates a potential role for anti-NP antibody in disease control and virus clearance (14). Considering that NP is also expressed on the surface of influenza-infected cells, and is a major target for CTL (35,39,48), it is reasonable to propose a significant role for the NP antibody in a crosssubtype immune response, perhaps even in humans. For the in vivo CTL assay, conserved HA and NP epitope-pulsed splenocytes were used as target cells to represent a general CTL response against influenza A virus. ...
Influenza A virus is highly variable and a major viral respiratory pathogen that can cause severe illness in humans. Therefore it is important to induce a sufficient immune response specific to current strains and to heterosubtypic viruses with vaccines. In this study, we developed a dual-promoter-based bivalent DNA vaccine that encodes both hemagglutinin (HA) and nucleoprotein (NP) proteins from a highly pathogenic A/Chicken/Henan/12/2004 (H5N1) virus. Our results show that the expression levels of HA and NP genes from the dual-promoter plasmid are similar to those seen when they are expressed individually in independent plasmids. When the bivalent DNA vaccine was inoculated via intramuscular injection and in vivo electroporation, high levels of both humoral and cellular immune responses were elicited against homologous H5N1 virus and heterosubtypic H9N2 virus. Furthermore, no obvious antigenic competition was observed between HA and NP proteins in the dual-promoter-based bivalent vaccine compared to monovalent vaccines. Our data suggest that a combination of influenza surface and internal viral genes in a dual-promoter-expressing plasmid may provide a new approach for developing a DNA vaccine that may protect not only specifically against a currently circulating strain, but also may cross-protect broadly against new heterosubtypic viruses.
... Virus could not be detected in animals vaccinated with AAV-H1 alone or with a combination of AAV-H1, AAV-NP and AAV-M1 (Fig. 4). Since antibodies against the internal proteins NP and M1 do not neutralize influenza virus particles [27][28][29] this lack of detectable virus replication can probably be attributed to the induction of neutralizing antibodies specific for the surface glycoprotein hemagglutinin. In the group vaccinated with AAV-NP alone, the viral loads in the lungs were only slightly lower than in the control group (statistically not significant, p = 0.48), indicating that a pre-existing NP-specific T-cell response does little to limit the initial spread of the virus. ...
The recent H1N1 influenza pandemic and the inevitable delay between identification of the virus and production of the specific vaccine have highlighted the urgent need for new generation influenza vaccines that can preemptively induce broad immunity to different strains of the virus. In this study we have produced AAV-based vectors expressing the A/Mexico/4603/2009 (H1N1) hemagglutinin (HA), nucleocapsid (NP) and the matrix protein M1 and have evaluated their ability to induce specific immune response and protect mice against homologous and heterologous challenge. Each of the vaccine vectors elicited potent cellular and humoral immune responses in mice. Although immunization with AAV-M1 did not improve survival after challenge with the homologous strain, immunization with the AAV-H1 and AAV-NP vectors resulted in survival of all mice, as did inoculation with a combination of all three vectors. Furthermore, trivalent vaccination also conferred partial protection against challenge with the highly heterologous and virulent A/PR/8/34 strain of H1N1 influenza.
... More contentious is what role if any do antibodies directed at internal influenza antigens, such as in the highly-conserved NP protein, play in hetero(sub)typic protection. NP is expressed on the surface of influenza infected cells (Virelizier et al, 1977; Stitz et al, 1990; Prokudina and Semenova, 1991) and is a major target for cytotoxic T-lymphocytes (Stitz et al, 1990; Prokudina and Semenova, 1991). However, early studies indicated that NP did not protect against influenza infection (Stitz et al, 1990). ...
Well understood are the adaptive and dramatic neutralizing homosubtypic antibody responses to hypervariable, immunodominant sites of the hemagglutinin (HA) and neuraminidase (NA) of individual influenza strains. These define influenza subtypes and vaccines modelled upon their HA and NA antigens provide seasonal neutralizing antibody protection against subsequent exposure to the strain and its close relatives, but give little if any protection against antigenically drifted or shifted strains. Contrasting to this is a different form of acquired antibody response, called heterosubtypic immunity. This provides a more seasoned adaptive antibody response to immune-recessive epitopes that are highly-conserved amongst strains. Although, such responses are of lower individual amplitudes than seasonal mechanisms they are active across influenza subtypes, and may give pre-emptive protection against new strains yet to emerge. Heterosubtypic immunities have been well studied in animals, but surprisingly there is minimal evidence for this type of antibody immunity in humans. Thus championed is the notion that seasoned humoral responses can through repeated exposure to sites widely conserved across different strains, cumulatively provide humans with a level of broad protection against emergent novel strains, such as H5N1, that is not afforded by seasonal humoral responses.
... Nonetheless, anti-NP Abs are eventually generated during natural influenza virus infection (18,19), indicating that this Ag is somehow exposed to the humoral immune system. This exposure may be via NP released from dying infected cells (52) or by its expression on the plasma membrane (53,54). Therefore, interaction of vaccine-induced anti-NP Abs with this NP early in the infection likely triggers downstream effector mechanisms that blunt virus replication, slow progression of the infection, and reduce morbidity. ...
Current influenza vaccines elicit Abs to the hemagglutinin and neuraminidase envelope proteins. Due to antigenic drift, these vaccines must be reformulated annually to include the envelope proteins predicted to dominate in the following season. By contrast, vaccination with the conserved nucleoprotein (NP) elicits immunity against multiple serotypes (heterosubtypic immunity). NP vaccination is generally thought to convey protection primarily via CD8 effector mechanisms. However, significant titers of anti-NP Abs are also induced, yet the involvement of Abs in protection has largely been disregarded. To investigate how Ab responses might contribute to heterosubtypic immunity, we vaccinated C57BL/6 mice with soluble rNP. This approach induced high titers of NP-specific serum Ab, but only poorly detectable NP-specific T cell responses. Nevertheless, rNP immunization significantly reduced morbidity and viral titers after influenza challenge. Importantly, Ab-deficient mice were not protected by this vaccination strategy. Furthermore, rNP-immune serum could transfer protection to naive hosts in an Ab-dependent manner. Therefore, Ab to conserved, internal viral proteins, such as NP, provides an unexpected, yet important mechanism of protection against influenza. These results suggest that vaccines designed to elicit optimal heterosubtypic immunity to influenza should promote both Ab and T cell responses to conserved internal proteins.
We recently reported that SARS-CoV-2 Nucleocapsid (N) protein is abundantly expressed on the surface of both infected and neighboring uninfected cells, where it enables activation of Fc receptor-bearing immune cells with anti-N antibodies (Abs) and inhibits leukocyte chemotaxis by binding chemokines (CHKs). Here, we extend these findings to N from the seasonal human coronavirus (HCoV)-OC43, which is also robustly expressed on the surface of infected and non-infected cells by binding heparan-sulfate/heparin (HS/H). HCoV-OC43 N binds with high affinity to the same set of 11 human CHKs as SARS-CoV-2 N, but also to a non-overlapping set of 6 cytokines (CKs). As with SARS-CoV-2 N, HCoV-OC43 N inhibits CXCL12β-mediated leukocyte migration in chemotaxis assays, as do all highly pathogenic and endemic HCoV N proteins. Together, our findings indicate that cell surface HCoV N plays important evolutionary conserved roles in manipulating host innate immunity and as a target for adaptive immunity.
The outcome of infection is dependent on the ability of viruses to manipulate the infected cell to evade immunity, and the ability of the immune response to overcome this evasion. Understanding this process is key to understanding pathogenesis, genetic risk factors, and both natural and vaccine-induced immunity. SARS-CoV-2 antagonises the innate interferon response, but whether it manipulates innate cellular immunity is unclear. An unbiased proteomic analysis determined how cell surface protein expression is altered on SARS-CoV-2-infected lung epithelial cells, showing downregulation of activating NK ligands B7-H6, MICA, ULBP2, and Nectin1, with minimal effects on MHC-I. This occurred at the level of protein synthesis, could be mediated by Nsp1 and Nsp14, and correlated with a reduction in NK cell activation. This identifies a novel mechanism by which SARS-CoV-2 host-shutoff antagonises innate immunity. Later in the disease process, strong antibody-dependent NK cell activation (ADNKA) developed. These responses were sustained for at least 6 months in most patients, and led to high levels of pro-inflammatory cytokine production. Depletion of spike-specific antibodies confirmed their dominant role in neutralisation, but these antibodies played only a minor role in ADNKA compared to antibodies to other proteins, including ORF3a, Membrane, and Nucleocapsid. In contrast, ADNKA induced following vaccination was focussed solely on spike, was weaker than ADNKA following natural infection, and was not boosted by the second dose. These insights have important implications for understanding disease progression, vaccine efficacy, and vaccine design.
Humans are exposed to influenza virus through periodic infections. Due to these repeated exposures, human populations commonly have elevated antibody titres targeting the conserved internal influenza virus nucleoprotein (NP). Despite the presence of anti‐NP antibodies, humans are acutely susceptible to drifted influenza viruses with antigenically different surface proteins and the protective potential of human NP antibodies is unclear. In this study, high levels of anti‐NP antibody and NP‐specific B cells were detected in both adult humans and influenza‐infected mice, confirming that NP is a major target of humoral immunity. Through sorting single B cells from influenza‐exposed human adults, we generated a panel of 11 anti‐NP monoclonal antibodies (mAbs). The majority of anti‐NP human mAbs generated were capable of engaging cellular Fc receptors and bound NP on the surface of influenza‐infected cell lines in vitro, suggesting that anti‐NP mAbs have the potential to mediate downstream Fc effector functions such as antibody‐dependent cellular cytotoxicity and antibody‐dependent phagocytosis. However, human anti‐NP mAbs were not protective in vivo when passively transferred into a murine influenza challenge model. Future in vivo studies examining the synergistic effect of anti‐NP mAbs infused with other influenza‐specific mAbs are warranted.
Significance
CD8 ⁺ T cells eliminate infections and cancers through recognition of antigen-derived peptides displayed at the cell surface in combination with MHC class I molecules. We show that a single glycoprotein-derived epitope is generated from two sources: 1) the conventional cohort that is delivered to the endoplasmic reticulum, a fraction failing quality control and undergoing ERAD, and 2) an exceedingly minor fraction that is mislocalized to the cytosol during translation and immediately degraded. Notably, peptide derived from mislocalized antigen is delivered to the cell surface with faster kinetics and drives greater CD8 ⁺ T cell expansion and functionality. These findings provide key insights for development of vaccines intended to elicit CD8 ⁺ T cell-mediated protection.
At the moment, developing new broad-spectrum influenza vaccines which would help avoid annual changes in a vaccine's strain set is urgency. In addition, developing new vaccines based on highly conserved influenza virus proteins could allow us to better prepare for potential pandemics and significantly reduce the damage they cause. Evaluation of the humoral response to vaccine administration is a key aspect of the characterization of the effectiveness of influenza vaccines. In the development of new broad-spectrum influenza vaccines, it is important to study the mechanisms of action of various antibodies, including non-neutralizing ones, as well as to be in the possession of methods for quantifying these antibodies after immunization with new vaccines against influenza. In this review, we focused on the mechanisms of anti-influenza action of non-neutralizing antibodies, such as antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and antibody-mediated complement-dependent cytotoxicity (CDC). The influenza virus antigens that trigger these reactions are hemagglutinin (HA) and neuraminidase (NA), as well as highly conserved antigens, such as M2 (ion channel), M1 (matrix protein), and NP (nucleoprotein). In addition, the mechanisms of action and methods for detecting antibodies to neuraminidase (NA) and to the stem domain of hemagglutinin (HA) of the influenza virus are considered.
Transmission of influenza virus between susceptible hosts mediates spread of infection in the population and can occur via direct-contact or airborne routes. Mathematical models suggest that vaccines that reduce viral transmission from infected individuals could substantially reduce viral spread in an epidemic or pandemic, even if they do not completely protect against infection. Vaccines targeting conserved nucleoprotein (A/NP) and matrix 2 (M2) antigens of influenza virus do not completely prevent infection upon influenza virus challenge, but reduce viral replication, morbidity, and mortality. Using a mouse model of influenza virus transmission, we have previously shown that immunization with recombinant adenovirus vectors expressing the combination of A/NP and M2 can reduce viral transmission to unimmunized contacts. Here we demonstrate that transmission reduction is more effective when mice are immunized against A/NP and M2 intranasally than via the intramuscular route. We show that immunization against the combination of A/NP and M2 is more effective at reducing transmission than either antigen alone, with a clear hierarchy of effectiveness (A/NP + M2 > A/NP > M2). Transmission reduction is seen to a similar degree under both direct-contact and airborne transmission conditions. Finally, using seroconversion as an indicator of infection, we show that immunizing contact mice against A/NP and M2 prevents a significant fraction (∼50%) from becoming infected under direct-contact conditions. These findings suggest that when strain-matched vaccines are unavailable, conserved antigen vaccines could not only reduce severity of disease in vaccinated individuals but also limit the spread of virus during influenza epidemics or pandemics.
This chapter discusses some major emerging bunyaviruses in the genus of phlebovirus, hantavirus, nairovirus, and orthobunyavirus. The phlebotomus fever viruses are transmitted by phlebotomine sand flies except for the Rift Valley fever virus (RVFV) which is primarily associated with mosquitoes, whereas uukuviruses are transmitted by ticks. Humans or host animals develop protective immune responses against highly virulent wt RVFV. As an example of novel emerging pathogens, severe fever with thrombocytopenia syndrome virus (SFTSV), which is responsible for severe thrombocytopenia and multiorgan dysfunction with high morbidity and mortality, is the newly identified phlebovirus isolated in China. In infected humans, hantaviruses can cause acute infections that result in the often fatal hemorrhagic fever with renal syndrome (HFRS) or hantavirus pulmonary syndrome (HPS) diseases which are not completely understood with respect to pathogenesis. An important and emerging orthobunyavirus is La Crosse virus (LACV), a California serogroup bunyavirus.
To survive as a species, each virus family has had to use a particiliar strategy of replication and dissemination adapted to both the intrinsic properties of the viral genome and the abilities of the host’s immune system to mount efficient immune responses aimed at eradicating the virus from the body. It is likely that during evolution the virus genomes which have survived have done so because of either versatility permitting antigenic variation, or an ability to integrate into the host genome without transcribing viral genes or else a capacity to take advantage for their replication of transcriptional mechanisms which immunocompetent cells use for their own activation. The present communication is not intended to exhaustively review the varied situations where viruses survive immune attack in single hosts or in whole populations, but rather to discuss a few examples of host-virus relationships suggestive of general strategies of viruses to cope with their host’s environment, to remain infective and to ensure their transmission from one individual to another. Instead of the usual description of the arsenal of host defense mechanisms, we propose a reflection about failures of the immune system to eradicate virus diseases, with the hope that such a salubrious exercise will help in designing better, future vaccines.
A current obsession of cellular immunology is the definition of the T-cell receptor repertoire. Many of the questions that have been raised derive from experiments with virus systems (Doherty et al., 1976a; Zinkernagel and Doherty, 1979). How do we explain the apparent dual specificity for major histocompatibility (MHC) components and for virus?
My research in the area of virus and tumor immunology began in the 1960s, when I was working in the Division of Virology of the National Institute for Medical Research, Mill Hill. The Institute was at that time a major world centre of immunology research, and since most other virologists were becoming molecular biologists I chose (as usual) to do something different: using contemporary immunological methods to define major mechanisms of resistance to infectious agents, especially viruses and virus-induced tumors. At the same time we began research on adjuvants, which had long been used empirically to increase humoral and cell-mediated immune responses to a variety of antigens. The whole program was motivated partly by scientific curiosity about mechanisms of immunity and immunore-gulation, and partly by the practical objective of using viral subunits and defined bacterial antigens in vaccines. In this way I hoped to contribute to the development of a new generation of vaccines which would avoid the problems associated with live virus vaccines (severe or persistent infections in some persons) and with crude bacterial vaccines such as that used for pertussis. Now, twenty years later, that goal is at last being realized and I am becoming involved in molecular biology research after all. It is the only way to produce many antigens in quantity and to dissect mechanisms of immunoregulation. One cannot escape destiny.
Over the past 20 years, it has become increasingly clear that thymus-derived lymphocytes (T lymphocytes) play a pivotal role in immune responsiveness. Perhaps nowhere is this more apparent than in antiviral immunity, in which T lymphocytes provide a critical helper function in antibody responses and also function directly to reduce viral replication. Of the large number of viruses known to elicit T-lymphocyte responses, influenza virus has been the most extensively studied.
Influenza virus (Kilbourne, 1975) is an enveloped, segmented single-stranded RNA virus. The virus derives its lipid envelope during the process of maturation (budding) from the plasma membrane of the host cell. Two types of viral glycoproteins, the hemagglutinin (HA) and the neuraminidase (NA), form a dense array of surface projections (spikes) on the outside of the lipid envelope. Inside the envelope are five virus-coded proteins: the matrix (M) protein, which is thought to form an internal protein sheet beneath the lipid envelope; the nu-cleoprotein (NP), which is associated with the viral RNA; and three proteins of large molecular weight with proven or suspected polymerase activity (P1, P2, P3). The viral proteins constitute 70–75% of the dry weight of the virion and are present in the following approximate proportions: M (40%), HA (32%), NP (21%), NA (5%), P1, -2, -3 (2%). In addition to these seven structural proteins, two nonstructural viral proteins are produced in the infected cell. Furthermore, the initial virus inoculum contains components (glycolipids, carbohydrates) that are not coded for by the viral genome but are intimately associated with the virion. Although present in small quantity, the immunogenicity of these components is often potentiated as a result of their association with the virus (Lindenmann, 1977). Consequently, infection or immunization with influenza virus elicits highly heterogeneous and polyspecific immune sera that are not suitable, in general, for studies concerned with the delineation of the antigenic structure of individual viral components.
We have used the technique of DNA-mediated gene transfer to examine cytotoxic T lymphocyte (CTL) recognition of the product of the cloned A/JAPAN/305/57 hemagglutinin (HA) gene in murine (L929) cells. Using both heterogeneous and homogeneous (clonal) populations of type A influenza-specific CTL, we have demonstrated that the HA molecule can serve as a target antigen for both the subtype-specific and the cross-reactive subpopulations of influenza-specific CTL. Our results also raise the possibility that other virus-specified polypeptides may serve as target molecules for cross-reactive CTL.
We purified the major influenza virus nonstructural protein, designated NS1, from cytoplasmic inclusions that were solubilized and used to raise antisera in rabbits. One of the antisera was found to be specific for NS1 by complement fixation tests and analyses of immune precipitates. Antiserum to NS1 isolated from cells infected with A/WSN/33 virus specifically precipitated NS1 from extracts of cells infected with seven distinct isolates of influenza A virus representing five different antigenic subtypes. These included A/WSN/33, A/PR/8/34, A/FW/5/50, A/USSR/90/77, A/RI/5+/57, A/Victoria/3/75, and A/Swine /1977/31; however, NS1 from cells infected with B/Lee/40 virus was not precipitated. Radioimmunoassays using radioiodinated NS1 protein from A/WSN virus-infected cells and unlabeled cytoplasmic extracts of cells infected with various strains of influenza virus as competitors indicated significant antigenic cross-reactivities for the NS1 proteins of all influenza A viruses tested. The results suggest a gradual antigenic drift over the 45 yr separating the earliest and most recent virus isolates examined. Thus, compared with the virion neuraminidase and hemagglutinin antigens, NS1 appears to be highly conserved in different influenza A virus isolates.
L929 or P815 cells were infected with WSN (H0/N1), JAP (H2/N2) and Port Chalmers (H3/N2) A strains or the B/LEE strain of influenza virus and pulsed for and at different times after infection with either 35S-methionine or 3H-amino acids. Viral antigens expressed at the cell surface were identified by reacting the infected labeled cells at 0°C with hyperimmune rabbit or convalescent mouse serum, lysing the cells with detergent and recovering the antigen-antibody complexes by immuno-precipitation. The washed immuno-precipitates were analysed by polyacrylamide gel electrophoresis (PAGE). The antigen profiles seen were more complex than anticipated due to recovery of non-viral coded components in the immuno-precipitates. These contaminating proteins were mainly disulphide bonded, so examination of the immuno-precipitates under non-reducing conditions on PAGE allowed non-disulphide bonded viral antigens to be identified. A peak of mol. wt 25,000 was identified as matrix protein A (A strain infected cells) because of co-migration with purified matrix protein and recovery from the cell surface using specific anti-matrix protein A whole serum or isolated immuno-globulin. A component of similar mol. wt occuring at the cell surface after injection with B/LEE virus is also most likely to be matrix protein B although the identification is not so complete. The expression of matrix protein at the surface of influenza virus-infected cells, which previously was thought not to occur, has several implications. One of them is that the recognition of matrix protein A (an antigen which is present in all A strain viruses) by cytotoxic T cells would provide a simple explanation for the observed killing of target cells infected with a wide variety of A strain viruses by cytotoxic T cells isolated from mice inoculated with any single A strain virus.
This paper deals with the generation and specificity of cytotoxic T cells directed to cells infected with type A influenza viruses. Mice were primed in vivo and their spleen cells restimulated in vitro. Four type A viruses with serologically distinct surface proteins are equally effective in secondary stimulation of cytotoxicity regardless of the viral strain used for priming. The resulting cytotoxic cells will lyse cells infected with any of the 4 type A viruses. Cross-reactivity between type A and B influenza viruses does not occur.
Competition experiments show that the cross-reactive cytotoxic T cells contain a minor population of cells with increased affinity for the type A virus strain used for priming, but the majority of the cells do not distinguish between the type A virus strains.
Virus replication is not required for the secondary generation of cytotoxic T cells; inactivated virus and viral hemagglutinin can serve as stimulators. Hemagglutinin derived from type A/Jap/Bel selects cytotoxic cells with specificity restricted to A/Jap/Bel originally used for priming and A/Jap which shares the hemagglutinin. This secondary stimulation can be achieved only when either of those two type A viruses are used for priming.
The results indicate that hemagglutinin is one of the viral proteins recognized by cytotoxic T cells; however, it is not clear which viral protein(s) are responsible for cross-reactivity.
Cytotoxic T-cell clones were raised in CBA mice that recognised both A/X31 and A/JAP/305/1957 influenza virus. Here, we describe one CTL clone that recognises target cells infected with a recombinant vaccinia virus expressing influenza PB1.
An attempt has been made to generate monoclonal antibodies which recognize the same target structures on influenza-infected cells as those seen by cytotoxic T lymphocyte (CTL) receptors. Such antibodies, if they mimicked the T cell receptor specificity, would be expected to be both virus specific and restricted in their binding by the major histocompatibility complex (MHC) antigens. Approximately 200 hybridomas from C57BL/6 (H-2b) mice primed and boosted with influenza virus (X-31)-infected EL4 (a C57BL/6 T cell lymphoma) were screened for reactivity on infected and uninfected cells of different MHC haplotypes. Of the 10 hybridoma antibodies which were identified as being reactive with X-31-infected EL4, but not uninfected EL4, all reacted equally well with X-31-infected cells of H-2b, H-2d and H-2k haplotypes, indicating a lack of MHC restriction in their recognition of the infected cells. Unexpectedly, 7 of the 10 monoclonal antibodies were found to react specifically with the purified influenza virus nucleoprotein (NP), a predominant viral antigen in CTL recognition of infected cells. Fluorescence-activated flow cytometry confirmed that these antibodies were able to recognize NP serological determinants on the surface of viable, infected cells, but the anti-NP antibodies were unable to block the lytic activity of an NP-specific CTL clone.
Influenza A virus causes recurring seasonal epidemics and occasional influenza pandemics. Because of changes in envelope glycoprotein Ags, neutralizing Abs induced by inactivated vaccines provide limited cross-protection against new viral serotypes. However, prior influenza infection induces heterosubtypic immunity that accelerates viral clearance of a second strain, even if the external proteins are distinct. In mice, cross-protection can also be elicited by systemic immunization with the highly conserved internal nucleoprotein (NP). Both T lymphocytes and Ab contribute to such cross-protection. In this paper, we demonstrate that anti-NP IgG specifically promoted influenza virus clearance in mice by using a mechanism involving both FcRs and CD8(+) cells. Furthermore, anti-NP IgG rescued poor heterosubtypic immunity in B cell-deficient mice, correlating with enhanced NP-specific CD8 T cell responses. Thus, Ab against this conserved Ag has potent antiviral activity both in naive and in influenza-immune subjects. Such antiviral activity was not seen when mice were vaccinated with another internal influenza protein, nonstructural 1. The high conservation of NP Ag and the known longevity of Ab responses suggest that anti-NP IgG may provide a critically needed component of a universal influenza vaccine.
The respective roles of cell-mediated immunity and humoral immunity in host defence were investigated in mice infected with influenza A/PR8 virus. Transferred immune spleen cells were shown to provide full protection only when they were actually secreting antibody. Serum antibody transferred in 'physiological' amounts was found to be protective in immunologically intact or in immunosuppressed animals. The specificity of the transferred antibody was shown to be critical, since antibody to internal components of the virus was inefficient while antibody to haemagglutimin, especially that to the strain-specific determinants of the haemagglutinin molecule, was highly efficient.
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Electron microscopic examination of thin sections through infected cells has resulted in the accumulation of considerable information not only about viral structure and development but also regarding associated alterations in host cell architecture (Rose and Morgan, 1960). Unfortunately, however, most of these studies apply of necessity to terminal stages in the formation of the virus, since it has rarely been possible to distinguish viral components before their assembly into recognizable particles. In order to identify viral constituents which are either in process of synthesis or have accumulated during the course of infection it is necessary to label them in some manner. One method of accomplishing this is to utilize specific antibody conjugated with ferritin (Singer, 1959). Ferritin consists of protein (mol. wt. c. 750,000) enclosing an iron core which is about 55 Å in diameter and readily visible in the electron microscope. The procedure of conjugation employing m-xylelene diisocyanate has...
There is much evidence to indicate that viruses, in certain cases at least, have an antigenic structure of comparable complexity to that of the bacteria. Hughes (1933) found that the serum of animals immunized with the yellow-fever virus contained two independent antibodies—precipitins and protective antibodies; the precipitinogen was distinct from the virus. Craigie & Wishart (1936) in investigations of the vaccinia virus have shown that, in addition to the elementary bodies, virus suspensions contain two soluble precipitable substances, the “L” antigen which is labile at 56° C. and the “S” antigen which is stable at 95° C. These antigens were readily demonstrated by precipitation, agglutination and complement fixation. Their nature and origin have not, however, been precisely determined. Bedson (1936), working with the psittacosis virus, prepared a soluble antigen, which was independent of the elementary bodies. It was most satisfactorily demonstrated by complement fixation.
In CBA mice the protection provided by transferred immune spleen cells or by antibody has been investigated in immunologically intact, cyclophosphamide-treated and thymus-deprived animals infected with A/PR8 virus. The degree of protection was more closely related to serum antibody levels than to the presence of immune lymphocytes in recipients. Comparison of the protective efficiency of various anti-influenza antisera with different specificities within an influenza A subtype indicated that antibodies recognizing the strain-specific determinants of the influenza hemagglutinin have an important role in protection. Physiologic amounts of transferred antibodies were shown to protect immunodepressed mice, suggesting that, provided a sufficient amount of specific antibodies is secreted, the participation of effectors of cell-mediated immunity is not essential. However, our results suggest that thymus-derived lymphocytes have an indirect role in protection by enhancing, through their helper effect, the secretion of anti-influenza antibodies.
Immunization of mice with influenza virus A/Japan/305/57 (H2N2) gives protection against infection with the homologous strain,
but not against infection with A/Hong Kong/1/68 (H3N2) and A/Equi/Miami/1/63 (Heq2Neq2). Immunizations with these two strains
result in a protection against homologous and cross infections with A/68 or A/Equi-2/63, although there is hardly a cross-hemagglutination
or neuraminidase-inhibiting antibody level. These immunizations do not protect against infection with A/57.
The presence of an unknown antigenic component (Mice Protecting Antigen: MPA) is suggested and some of its characteristics
are discussed.
In the process of cell infection by myxo- or paramyxoviruses, new virus specific antigens are demonstrable at the cell surface. It is assumed that at these antigenically modified sites new virus particles arise by budding processes and that the modified cell membrane becomes the envelope of new virions. Recently, it has been postulated and experimentally shown in lymphocytes that different structural elements of the plasma membrane are mobile in the plane of the cell surface. The present work investigates whether the newly occurring influenza virus induced antigens are mobile or firmly attached to the surface membrane of Hela cells.
Three virus-coded structural antigens of the influenza virus have been previously described. The ribonucleoprotein (RNP) antigen, identified as the ‘soluble’ antigen of influenza by Hoyle & Fairbrother (1937), is located internally in the virus particle and is antigenically invariant and type-specific, providing the basis for the division of influenza viruses into types A, B and C. The haemagglutinin and neuraminidase antigens occur at the virus surface and are immunologically and morphologically (Laver & Valentine, 1969) distinct proteins which show a considerable degree of antigenic variation. In addition, Dimmock & Watson (1969) have presented evidence for a non-structural antigen induced by the influenza virus which appeared to be present in the nucleoli of cells early after infection with influenza. This report describes studies on an additional antigen of the virus which appears to be located internally in the particle.
The effect of hormonal bursectomy and neonatal surgical thymectomy on the course of an avian influenza virus infection in chickens was studied. Analysis of the immunologic status of the chickens prior to infection included assay of natural agglutinins to rabbit red blood cells and induced agglutinins to sheep red blood cells, serum immunoelectrophoresis patterns, and in vitro effects of phytohemagglutinin on lymphocyte transformation. At 6 wk of age the chickens received the influenza virus intratracheally. Daily temperatures and mortality were recorded and HAI antibody titers were measured 7 and 14 days later. Completely thymectomized chickens were characterized by a failure of lymphocyte transformation to take place in two successive studies and absence of thymic remnants at autopsy. Bursectomy was associated with a significantly higher occurrence of temperature elevation (P < 0.05) and mortality (P < 0.001). Thymectomy had no significant effect on the course of the virus infection. Preliminary findings with passive administration of serum from immune animals also suggested a role for antibody in host resistance. These studies suggest cell-mediated immunity is less important than humoral immunity in recovery from avian influenza virus infection.
Analyses of the polypeptides synthesized in fowl plague virus-infected chick embryo fibroblasts are reported. The results indicate that both the amount of each individual polypeptide synthesized and the time at which each is produced during infection is specifically controlled. Early in infection only three types of infected cell specific polypeptide were detected and it is suggested, from the results of experiments involving cycloheximide and actinomycin D, that this is a reflection of the selective transcription of three of the virus genes by the virion polymerase.
Specific antisera for hemagglutinin (HA) and neuraminidase antigens of influenza A(2) virus (A(2)E) were produced through the segregation of the two proteins in reciprocal viral recombinants of A(2)E and A(0)e viruses. Gamma globulin fractions of these specific antisera and of antiserum specific for the nucleoprotein (NP) antigen of A(0)e virus were conjugated with fluorescein isothiocyanate and employed to follow the synthesis of the three structural proteins in clone 1-5C-4 human aneuploid cells, with parallel measurement of serological and biological activity of the antigens by other techniques. In this system, NP antigen appeared first (at 3 hr) in the cell nucleus, whereas HA and neuraminidase appeared coincidentally, at 4 hr after infection, in the cytoplasm. The initial detectability of biological or complement-fixing activity of the proteins coincided with their demonstrability as stainable antigens. Late in infection, all three antigens were detected at the cell surface. Antibody specific for HA partially blocked the intracellular staining of neuraminidase and inhibited the enzymatic activity of both extracted and intact extracellular virus. These observations suggest the close intracytoplasmic proximity of the two envelope antigens and perhaps their initial association in a larger protein.
Hyperimmune peritoneal fluids were obtained from mice which were infected with the WSN strain of influenza A0 and subsequently inoculated intraperitoneally with infected mouse tissue. Up to 12 precipitin lines were obtained in agar immunodiffusion tests with extracts of chick embryo fibroblast cells infected with WSN. In similar tests using a hyperimmune peritoneal fluid which had been absorbed with purified virus until no antibody against haemagglutinin, neuraminidase, or internal antigen could be detected, up to five precipitin lines were observed. At least three precipitin lines were also detected in tests using this absorbed peritoneal fluid and extracts of cells infected with an influenza A1 or an A2 strain. Extracts from WSN-infected cells which had been pretreated with actinomycin D failed to give precipitin lines with the virus-absorbed peritoneal fluid.Nucleoli of infected cells which were reacted with fluorescent antibody against the internal antigen of the virion were not stained whereas the nucleoli of cells which were reacted with the virus particle-absorbed peritoneal fluid stained brilliantly. Faint staining was also present in the nucleus and the cytoplasm. The same distribution of fluorescence was observed in cells infected with an A1 or an A2 strain. No fluorescence was obtained with the virus-absorbed peritoneal fluid in WSN-infected cells which had been pretreated with actinomycin D.
Analyses of the polypeptide composition of three influenza viruses, X-31, X-7F1, and AO/BEL/42, and of the isolated antigenic components of these viruses are reported. The viruses contain polypeptides of seven or eight species which are separable by polyacrylamide gel electrophoresis and range in molecular weight from 25,000 to 94,000. At least two of these polypeptides are glycopeptides. The ribonucleoprotein antigen of X-31 contain polypeptides of a single species of molecular weight 53,000. The haemagglutinin of AO/BEL/42 virus is made up of polypeptides of two species, one a glycopeptide. These have molecular weights of 58,000 and 28,000. The neuraminidase of X-7F1 virus is also made up of two polypeptides of molecular weights 80,000 and 70,000.
Schulman, Jerome L. (Cornell University Medical College, New York, N.Y.), and Edwin D. Kilbourne. Induction of partial specific heterotypic immunity in mice by a single infection with influenza A virus. J. Bacteriol.
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170–174. 1965.—Mice infected 4 weeks previously with influenza A virus were found to be partially immune when challenged with influenza A2 virus. This partial immunity was demonstrated by reduced titers of pulmonary virus, decreased mortality, and less extensive lung lesions. A specific immunological basis for this protection was suggested by the absence of any protection in animals previously infected with influenza B virus when challenged with A2 virus, or in animals previously infected with influenza A virus when challenged with influenza B virus. Parenteral inoculation with inactivated influenza A virus did not induce partial immunity to A2 virus challenge. An accelerated rise of hemagglutinating-inhibiting antibody after A2 virus challenge was demonstrated in animals previously infected with influenza A virus.
- J J Skehel
- JJ Skehel