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Heterosubtypic Immunity to Influenza A Virus Infection Requires B Cells but Not CD8 + Cytotoxic T Lymphocytes
Abstract and Figures
Heterosubtypic immunity (HSI), defined as protective cross-reactivity to lethal infection with influenza A virus of a serotype
different from the virus initially encountered, is thought to be mediated by cross-reactive cytotoxic T lymphocytes (CTL).
This study provides direct evidence for the role of effector CTL versus B cells in HSI in mice with a targeted disruption
in the α chain of CD8 molecule (CD8+ T cell deficient) or the immunoglobulin µ heavy chain (B cell deficient), respectively. CD8+ T cell-deficient mice developed complete HSI. These mice displayed normal humoral immune responses, as determined by titers
of subtype cross-reactive antibodies and virus-neutralizing antibodies specific for the immunizing influenza strain. In contrast,
HSI was not observed in B cell—deficient mice, although these mice could mount cross-reactive CTL responses. These results
show that B cells are required for HSI and provide new insight into the mechanisms of HSI, with significant implications in
vaccine development.
... EF responses. Ag-activated B cells can form EF foci independent of CD4 T cell "help," and such T cell-independent B cell responses can confer some protection against influenza virus infection (33,34). However, most protective B cell responses to influenza, including those generated in EF responses, are T cell-dependent. ...
... The contribution of each adaptive immune compartment to heterosubtypic immunity was evaluated in mice, demonstrating a most potent role for T cell-dependent Ab responses. Genetic ablation of CD4 T cells in mice following a sublethal dose of a H3N2 virus infection resulted in significantly enhanced mortality to subsequent challenge with a lethal dose of H1N1 (33). The same studies with B cell-deficient mice resulted in even greater mortality, although depletion of CD8 T cells had no effect (33). ...
... Genetic ablation of CD4 T cells in mice following a sublethal dose of a H3N2 virus infection resulted in significantly enhanced mortality to subsequent challenge with a lethal dose of H1N1 (33). The same studies with B cell-deficient mice resulted in even greater mortality, although depletion of CD8 T cells had no effect (33). This was despite existing crossreactivity of H3N2-primed CD8 T cells. ...
Protection from yearly recurring, highly acute infections with a pathogen that rapidly and continuously evades previously induced protective neutralizing Abs, as seen during seasonal influenza virus infections, can be expected to require a B cell response that is too highly variable, able to adapt rapidly, and able to reduce morbidity and death when sterile immunity cannot be garnered quickly enough. As we outline in this Brief Review, the influenza-specific B cell response is exactly that: it is multifaceted, involves both innate-like and conventional B cells, provides early and later immune protection, employs B cells with distinct BCR repertoires and distinct modes of activation, and continuously adapts to the ever-changing virus while enhancing overall protection. A formidable response to a formidable pathogen.
... Current flu vaccination strategies elicit protection primarily through the generation of long lasting, type-specific, neutralizing anti-HA antibodies. Influenza virus infection and vaccination both induce antibodies that bind to molecularly similar influenza subtypes, a phenomenon termed heterosubtypic immunity (HSI), and a major reason for the success of seasonal influenza vaccination [4, 5]. Vaccines containing HAs with small antigenic changes from the prior years' influenza strains have a high probability of inducing memory B cells to classswitch and secrete IgG anti-HA antibodies. ...
... One benefit of the large range of detection is that the mPlex-Flu assay only requires 2 dilutions for sample measurements to fall within the linear range of the assay, as opposed to the ~12 dilutions required for the HAI assay. The mPlex-Flu assay is thus able to rapidly measure HSI in a large population in a timely and accurate manner, and allows study of the effect of pre-vaccine anti-influenza antibody levels on human influenza vaccine efficacy [5,383940. This is a significant improvement in both quantitation of anti-influenza Ig activity, as well as high-throughput, multi-dimensional immune responses. ...
... The mPlex-Flu assay would enhance the development and application of mathematical models to predict population immunity and the population antibody repertoire. The mPlex-flu assay will also improve our ability to quantify the influence of pre-vaccine HSI in the evaluation of human influenza vaccine efficacy [5,383940 . Most adults have a spectrum of prior influenza infection and vaccination, and thus have pre-vaccine HSI to strains with antigenic similarity the vaccine strains. ...
We describe the use of classical and metric multidimensional scaling methods for graphical representation of the proximity between collections of data consisting of cases characterized by multidimensional attributes. These methods can preserve metric differences between cases, while allowing for dimensional reduction and projection to two or three dimensions ideal for data exploration. We demonstrate these methods with three datasets for: (i) the immunological similarity of influenza proteins measured by a multidimensional assay; (ii) influenza protein sequence similarity; and (iii) reconstruction of airport-relative locations from paired proximity measurements. These examples highlight the use of proximity matrices, eigenvalues, eigenvectors, and linear and nonlinear mappings using numerical minimization methods. Some considerations and caveats for each method are also discussed, and compact Mathematica programs are provided.
... Current flu vaccination strategies elicit protection primarily through the generation of long lasting, type-specific, neutralizing anti-HA antibodies. Influenza virus infection and vaccination both induce antibodies that bind to molecularly similar influenza subtypes, a phenomenon termed heterosubtypic immunity (HSI), and a major reason for the success of seasonal influenza vaccination [4,5]. Vaccines containing HAs with small antigenic changes from the prior years' influenza strains have a high probability of inducing memory B cells to classswitch and secrete IgG anti-HA antibodies. ...
... One benefit of the large range of detection is that the mPlex-Flu assay only requires 2 dilutions for sample measurements to fall within the linear range of the assay, as opposed to the~12 dilutions required for the HAI assay. The mPlex-Flu assay is thus able to rapidly measure HSI in a large population in a timely and accurate manner, and allows study of the effect of pre-vaccine anti-influenza antibody levels on human influenza vaccine efficacy [5,[38][39][40]. This is a significant improvement in both quantitation of anti-influenza Ig activity, as well as high-throughput, multi-dimensional immune responses. ...
... The mPlex-flu assay will also improve our ability to quantify the influence of pre-vaccine HSI in the evaluation of human influenza vaccine efficacy [5,[38][39][40]. Most adults have a spectrum of prior influenza infection and vaccination, and thus have pre-vaccine HSI to strains with antigenic similarity the vaccine strains. ...
The human immune response to influenza vaccination depends in part on preexisting cross-reactive (heterosubtypic) immunity from previous infection by, and/or vaccination with, influenza strains that share antigenic determinants with the vaccine strains. However, current methods for assessing heterosubtypic antibody responses against influenza, including the hemagglutination-inhibition (HAI) assay and ELISA, are time and labor intensive, and require moderate amounts of serum and reagents. To address these issues we have developed a fluorescent multiplex assay, mPlex-Flu, that rapidly and simultaneously measures strain specific IgG, IgA, and IgM antibodies against influenza hemagglutinin (HA) from multiple viral strains. We cloned, expressed and purified HA proteins from 12 influenza strains, and coupled them to multiplex beads. Assay validation showed that minimal sample volumes (
... Similarly, data from the Cleveland Family Study show that adults recently recovered from H1N1 infection were much less susceptible to infection with H2N2 virus than children in the same households who were not previously exposed to H1N1 (13). Although the interpretation of these data is still being debated, multiple lines of evidence demonstrate that cross-reactive T cells and even antibodies are important for resistance to heterosubtypic strains of influenza (14)(15)(16)(17). In part, the difference between humans and laboratory animals in the effectiveness of heterosubtypic immunity may be due to the short-lived nature of heterosubtypic effector mechanisms (15) and to the fact that experiments in animals are performed on timescales of months, whereas studies in humans test resistance over several seasons. ...
... Moreover, immunization with conserved proteins, like nucleoprotein (NP) or matrix-2 (M2) leads to demonstrable heterosubtypic immunity (17,21,23,24), as does immunization with peptide antigens that elicit influenzaspecific memory CD8 T cells (22,(25)(26)(27). However, CD8 T cells appear to be dispensable for heterosubtypic immunity in some studies (16,28), possibly because CD4, CD8, NKT and γδT cells play partially redundant roles in heterosubtypic immunity and elimination of any one of these populations does not substantially impair the response as a whole (4). ...
... Despite the well-established idea that heterosubtypic immunity to influenza is mediated by cross-reactive T cells, isolated reports suggest that B cells are also important for resistance to heterosubtypic strains of influenza and may be more important than CD8 T cells (16,28). However, it is not clear how B cells might mediate their protective effects. ...
Immunity to heterosubtypic strains of influenza is thought to be mediated primarily by memory T cells, which recognize epitopes in conserved proteins. However, the involvement of B cells in this process is controversial. We show here that influenza-specific memory T cells are insufficient to protect mice against a lethal challenge with a virulent strain of influenza in the absence of B cells. B cells contribute to protection in multiple ways. First, although non-neutralizing antibodies by themselves do not provide any protection to challenge infection, they do reduce weight loss, lower viral titers and promote recovery of mice challenged with a virulent heterosubtypic virus in the presence of memory T cells. Non-neutralizing antibodies also facilitate the expansion of responding memory CD8 T cells. Furthermore, in cooperation with memory T cells, naïve B cells also promote recovery from infection with a virulent heterosubtypic virus by generating new neutralizing antibodies. These data demonstrate that B cells use multiple mechanisms to promote resistance to heterosubtypic strains of influenza and suggest that vaccines that elicit both memory T cells and antibodies to conserved epitopes of influenza may be an effective defense against a wide range of influenza serotypes.
... Egg-grown influenza virus strains A/PR/8/34 (H1N1) (A/ PR8), A/Philippines/2/82/X-79 (H3N2) (A/Philippines) were prepared as previously reported [32]. Mouse-adapted viruses A/ PR8 and A/Philippines prepared as lung homogenates of IN infected mice were used for challenge as previously described [32]. ...
... Egg-grown influenza virus strains A/PR/8/34 (H1N1) (A/ PR8), A/Philippines/2/82/X-79 (H3N2) (A/Philippines) were prepared as previously reported [32]. Mouse-adapted viruses A/ PR8 and A/Philippines prepared as lung homogenates of IN infected mice were used for challenge as previously described [32]. Choi, Chungbuk University, Korea, was adapted by multiple passages (15 times) in BALB/c mice. ...
... Protection was associated with antigen-specific IgG Ab levels in blood (Fig. 2). Although the dose used for SL immunization was significantly higher than that used for N or IN immunization, it is important that SL immunization with DelNS1 viruses induced antigen specific IgG at levels comparable to those induced by administration of the vaccine to different sites of the respiratory tract, a finding which is consistent with earlier reports [32,35]. (m H1N1) or wt live virus A/PR8/34 (PR8) H1N1 (WT) via the sublingual (SL) or intranasal (IN) route. ...
The nonstructural protein 1 (NS1) of influenza A virus (IAV) enables the virus to disarm the host cell type 1 IFN defense system. Mutation or deletion of the NS1 gene leads to attenuation of the virus and enhances host antiviral response making such live-attenuated influenza viruses attractive vaccine candidates. Sublingual (SL) immunization with live influenza virus has been found to be safe and effective for inducing protective immune responses in mucosal and systemic compartments. Here we demonstrate that SL immunization with NS1 deleted IAV (DeltaNS1 H1N1 or DeltaNS1 H5N1) induced protection against challenge with homologous as well as heterosubtypic influenza viruses. Protection was comparable with that induced by intranasal (IN) immunization and was associated with high levels of virus-specific antibodies (Abs). SL immunization with DeltaNS1 virus induced broad Ab responses in mucosal and systemic compartments and stimulated immune cells in mucosa-associated and systemic lymphoid organs. Thus, SL immunization with DeltaNS1 offers a novel potential vaccination strategy for the control of influenza outbreaks including pandemics.
... In rodent models, primary infection with a sublethal dose of influenza A virus induces cross-protective immunity against lethal infection with a heterosubtypic virus strain434445464748. To date, a majority of research into the mechanisms of cross protective immunity against lethal influenza A viruses has been conducted using female BALB/c or C57BL/6 mice [32, 33,4344454950515253. There are few studies detailing heterosubtypic immunity in males [54], but many studies that either do not report the sex of the mice or combine the responses of males and females555657585960. ...
... Primary infection with a sublethal dose of influenza A virus provides cross protection against secondary challenge with a heterosubtypic virus primarily through increased activity and numbers of virus-specific CD8 + cytotoxic T lymphocytes (CTLs) that recognize shared epitopes of internal proteins [44, 56, 57]. There are, however, data illustrating that CTLs are not the sole mechanism mediating heterosubtypic immunity as mice depleted of CD8 + and CD4 + T cells, deficient in β2-microglobulin, or deficient in IFN-γ still have cross-protective immunity against heterosubtypic challenge49505152 54]. Further, mice that are depleted of B cells (i.e., µMT mice) are not protected against heterosubtypic challenge with lethal doses of influenza A viruses [47,505152. ...
... There are, however, data illustrating that CTLs are not the sole mechanism mediating heterosubtypic immunity as mice depleted of CD8 + and CD4 + T cells, deficient in β2-microglobulin, or deficient in IFN-γ still have cross-protective immunity against heterosubtypic challenge49505152 54]. Further, mice that are depleted of B cells (i.e., µMT mice) are not protected against heterosubtypic challenge with lethal doses of influenza A viruses [47,505152. Passive transfer of immune serum prior to challenge with lethal influenza A virus also confers protection from heterosubtypic infection in some models [40, 48, 58, 69]. ...
A mouse model was used to determine if protective immunity to influenza A virus infection differs between the sexes. The median lethal dose of H1N1 or H3N2 was lower for naïve females than males. After a sublethal, primary infection with H1N1 or H3N2, females and males showed a similar transient morbidity, but females generated more neutralizing and total anti-influenza A virus antibodies. Immunized males and females showed similar protection against secondary challenge with a homologous virus, but males experienced greater morbidity and had higher lung viral titers after infection with a lethal dose of heterologous virus. Females develop stronger humoral immune responses and greater cross protection against heterosubtypic virus challenge.
... In contrast to H5N1 and H7N9 viruses, H9N2 is low pathogenic, causing mild disease among birds and human (Feng et al., 2013;Guan et al., 1999). Circulation and coexistence of these viruses highlight the need to develop a hetero-subtypic vaccine that can provide cross-subtype protection in association with CD8 + cytotoxic T lymphocytes (CTL) and CD4 + responses (Even-Or et al., 2013;Ben-Yedidia and Arnon, 2007;Grebe et al., 2008;Nguyen et al., 2001;Tumpey et al., 2001;Shaw and Palese, 2013). Induction of cross-reactive CTL responses that recognize conserved epitopes of viral antigens shared by different influenza subtypes have been investigated previously by targeting hemagglutinin (HA) or the more conserved nucleoprotein (NP) and matrix protein (M1) (Even-Or et al., 2013;Luo et al., 2012;Qiu et al., 2006;Watanabe et al., 2008). ...
... HA peptide vaccine is a potent inducer of CTL, and it confers sig-nificant protective immunity against influenza infection. Complete heterosubtypic immunity, induced by CD4 + T helper cells, involves B cell-and T-cell-dependent virus-specific antibody responses mediated by the secretion of cytokines (Nguyen et al., 2001;Tumpey et al., 2001). ...
Influenza viruses continue to emerge and re-emerge, posing new threats for public health. Control and treatment of influenza depends mainly on vaccination and chemoprophylaxis with approved antiviral drugs. Identification of specific epitopes derived from influenza viruses has significantly advanced the development of epitope-based vaccines. Here, we explore the idea of using HLA binding data to design an epitope-based vaccine that can elicit heterosubtypic T-cell responses against circulating H7N9, H5N1, and H9N2 subtypes. The hemokinin-1 (HK-1) peptide sequence was used to induce immune responses against the influenza viruses. Five conserved high score cytotoxic T lymphocyte (CTL) epitopes restricted to HLA-A*0201-binding peptides within the hemagglutinin (HA) protein of the viruses were chosen, and two HA CTL/HK-1 chimera protein models designed. Using in silico analysis, which involves interferon epitope scanning, protein structure prediction, antigenic epitope determination, and model quality evaluation, chimeric proteins were designed. The applicability of one of these proteins as a heterosubtypic epitope-based vaccine candidate was analyzed.
... Therefore, the currently used inactivated influenza vaccines, which rely on the induction of serum neutralizing antibodies, are not effective against viruses whose HA antigenicities are different from those of the vaccine strains [5]. On the other hand, infection with influenza A virus usually affords some protection against reinfection with viruses having different subtypes [6]. It has been believed that this heterosubtypic protection is mainly mediated by memory cytotoxic T lymphocytes (CTL) recognizing conserved epitopes of viral internal proteins presented with MHC class I on the surfaces of infected cells [7,8]. ...
... On the other hand, it was reported that heterosubtypic immunity was induced by intranasal immunization of mice with formalin-inactivated influenza A viruses, whereas subcutaneous immunization only protected mice from homologous viruses [6,19,20]. Interestingly, this cross-protection was dependent on B cell, but not on CTL activity [19]. However, in vitro neutralizing activity of antibodies was not detected in the sera and respiratory secretions of immunized mice. ...
Influenza A virus subtypes are classified on the basis of the antigenicity of their envelope glycoproteins, hemagglutinin (HA; H1-H17) and neuraminidase. Since HA-specific neutralizing antibodies are predominantly specific for a single HA subtype, the contribution of antibodies to the heterosubtypic immunity is not fully understood. In this study, mice were immunized intranasally or subcutaneously with viruses having the H1, H3, H5, H7, H9, or H13 HA subtype, and cross-reactivities of induced IgG and IgA antibodies to recombinant HAs of the H1-H16 subtypes were analyzed. We found that both subcutaneous and intranasal immunizations induced antibody responses to multiple HAs of different subtypes, whereas IgA was not detected remarkably in mice immunized subcutaneously. Using serum, nasal wash, and trachea-lung wash samples of H9 virus-immunized mice, neutralizing activities of cross-reactive antibodies were then evaluated by plaque-reduction assays. As expected, no heterosubtypic neutralizing activity was detected by a standard neutralization test in which viruses were mixed with antibodies prior to inoculation into cultured cells. Interestingly, however, a remarkable reduction of plaque formation and extracellular release of the H12 virus, which was bound by the H9-induced cross-reactive antibodies, was observed when infected cells were subsequently cultured with the samples containing HA-specific cross-reactive IgA. This heterosubtypic plaque reduction was interfered when the samples were pretreated with anti-mouse IgA polyclonal serum. These results suggest that the majority of HA-specific cross-reactive IgG and IgA antibodies produced by immunization do not block cellular entry of viruses, but cross-reactive IgA may have the potential to inhibit viral egress from infected cells and thus to play a role in heterosubtypic immunity against influenza A viruses.
... In contrast to the homosubtypic immunity, M2SR demonstrates heterosubtypic protection in the absence of HAI antibodies [15,16], suggesting that the immunity elicited by M2SR is unlikely to be mediated by neutralizing antibodies to the HA head. The studies in the literature on the mechanism of heterosubtypic immunity to wildtype influenza have yielded divergent results, implicating either humoral or T-cell-mediated mechanisms of protection [30][31][32][33][34][35]. The differences may relate to the combination of prime and challenge viruses used and the degree of sequence variation in protective T cell epitopes, such as the NP [36]. ...
Seasonal influenza and the threat of global pandemics present a continuing threat to public health. However, conventional inactivated influenza vaccines (IAVs) provide little cross-protective immunity and suboptimal efficacy, even against well-matched strains. Furthermore, the protection against matched strains has been shown to be of a short duration in both mouse models and humans. M2SR (M2-deficient single-replication influenza virus) is a single-replication vaccine that has been shown to provide effective cross-protection against heterosubtypic influenza viruses in both mouse and ferret models. In the present study, we investigated the duration and mechanism of heterosubtypic protection induced by M2SR in a mouse model. We previously showed that M2SR generated from influenza A/Puerto Rico/8/34 (H1N1) significantly protected C57BL/6 mice against lethal challenge with both influenza A/Puerto Rico/8/34 (H1N1, homosubtypic) and influenza A/Aichi/2/1968 (H3N2, heterosubtypic), whereas the inactivated influenza vaccine provided no heterosubtypic protection. The homosubtypic protection induced by M2SR was robust and lasted for greater than 1 year, whereas that provided by the inactivated vaccine lasted for less than 6 months. The heterosubtypic protection induced by M2SR was of a somewhat shorter duration than the homosubtypic protection, with protection being evident 9 months after vaccination. However, heterosubtypic protection was not observed at 14 months post vaccination. M2SR has been shown to induce strong systemic and mucosal antibody and T cell responses. We investigated the relative importance of these immune mechanisms in heterosubtypic protection, using mice that were deficient in B cells or mice that were depleted of T cells immediately before challenge. Somewhat surprisingly, the heterosubtypic protection was completely dependent on B cells in this model, whereas the depletion of T cells had no significant effect on survival after a lethal heterosubtypic challenge. While antibody-dependent cellular cytotoxicity (ADCC) has been demonstrated to be important in the response to some influenza vaccines, a lack of Fc receptors did not affect the survival of M2SR-vaccinated mice following a lethal challenge. We examined the influenza proteins targeted by the heterosubtypic antibody response. Shortly after the H1N1 M2SR vaccination, high titers of cross-reactive antibodies to heterosubtypic H3N2 nucleoprotein (NP) and lower titers to the stalk region of the hemagglutinin (HA2) and neuraminidase (NA) proteins were observed. The high antibody titers to heterosubtypic NP persisted one year after vaccination, whereas the antibody titers to the heterosubtypic HA2 and NA proteins were very low, or below the limit of detection, at this time. These results show that the intranasal M2SR vaccine elicits durable protective immune responses against homotypic and heterosubtypic influenza infection not seen with intramuscular inactivated vaccines. Both the homo- and heterosubtypic protection induced by the single-replication vaccine are dependent on B cells in this model. While the homosubtypic protection is mediated by antibodies to the head region of HA, our data suggest that the heterosubtypic protection for M2SR is due to cross-reactive antibodies elicited against the NP, HA2, and NA antigens that are not targeted by current seasonal influenza vaccines.
... The efficacy of current influenza vaccines is highly sensitive to antigenic changes, due to the prevalent effector mechanism of serum antibodies that block virus attachment to cells by targeting epitopes located on or in proximity to the human receptor binding site, which are subject to frequent mutations [9][10][11]. The generation of antibodies that cross-react against multiple strains is a complex event that is not yet fully elucidated but which likely involves multiple immunological mechanisms, i.e., humoral and cell mediated immunity controlled by route and mode of immunization [12][13][14][15][16][17][18][19][20]. ...
Vaccination is the most effective approach to reduce the substantial morbidity and mortality caused by influenza infection. Vaccine efficacy is highly sensitive to antigenic changes causing differences between circulating and vaccine viruses. Adjuvants such as MF59 increase antibody-mediated cross-reactive immunity and therefore may provide broader seasonal protection. A recent clinical trial showed that an MF59-adjuvanted vaccine was more efficacious than a nonadjuvanted comparator in subjects < 2 years of age, although not in those ≥ 2 years, during influenza seasons in which the predominant circulating virus was an A/H3N2 strain that was antigenically different from the vaccine virus. This finding suggested that the increased efficacy of the adjuvanted vaccine in younger subjects may be mediated by strain cross-reactive antibodies. A subset of the trial population, representing subjects with distinct age and/or immunological history, was tested for antibody responses to the vaccine A/H3N2 strain as well as A/H3N2 drifted strains antigenically matching the viruses circulating during the trial seasons. The neutralizing tests showed that, compared with nonadjuvanted vaccine, the adjuvanted vaccine improved not only the neutralizing antibody response to the vaccine strain but also the cross-reactive antibody response to the drifted strains in subjects with lower preexisting antibody titers, regardless of their age or vaccine history. The results demonstrated an immunological benefit and suggested a potential efficacy benefit by adjuvanted vaccine in subjects with lower preexisting antibody responses.
... In addition, researchers have used antibodies to deplete immune cells, such as CD4, CD8, or NK cells, to study the contribution of these cells to the protection against influenza infection in vaccinated mice [23,24]. Preclinical studies using knockout or deficient mouse models have also elucidated the roles of immune cells, including B cells, T cells, NK cells, and monocytes [25][26][27][28][29][30][31][32][33][34], as well as the IFN signaling pathway [35,36] (Table 1). Transgenic mice, such as B6-Mx1 −/− carrying mutant Mx1 alleles, B6-Mx1 r/r carrying a functional A2G Mx1 allele, and SPRET/Ei, which carry another Mx1 wild-type allele, have been applied to study the importance of the MX1 gene in influenza virus resistance [37,38]. ...
Influenza remains one of the most significant public health threats due to its ability to cause high morbidity and mortality worldwide. Although understanding of influenza viruses has greatly increased in recent years, shortcomings remain. Additionally, the continuous mutation of influenza viruses through genetic reassortment and selection of variants that escape host immune responses can render current influenza vaccines ineffective at controlling seasonal epidemics and potential pandemics. Thus, there is a knowledge gap in the understanding of influenza viruses and a corresponding need to develop novel universal vaccines and therapeutic treatments. Investigation of viral pathogenesis, transmission mechanisms, and efficacy of influenza vaccine candidates requires animal models that can recapitulate the disease. Furthermore, the choice of animal model for each research question is crucial in order for researchers to acquire a better knowledge of influenza viruses. Herein, we reviewed the advantages and limitations of each animal model—including mice, ferrets, guinea pigs, swine, felines, canines, and non-human primates—for elucidating influenza viral pathogenesis and transmission and for evaluating therapeutic agents and vaccine efficacy.
... 42 Studies performed in mice have demonstrated the predominant protective role played by sIgA, 43,44 even in case of absence of T cells. 45 Specifically, the passive intranasal transfer of anti-influenza A IgA from the respiratory tract of mice immunized with live influenza virus has been seen to provide protection in naive mice. 43 Accordingly, this protection was suppressed by the intranasal instillation of anti-IgA, 46 whereas it was not affected by treatment with anti-IgM or anti-IgG antibodies. ...
Secretory IgAs (sIgA) constitute the principal isotype of antibodies present in nasal and mucosal secretions. They are secreted by plasma cells adjacent to the mucosal epithelial cells, the site where infection occurs, and are the main humoral mediator of mucosal immunity. Mucosally delivered vaccines, such as live attenuated influenza vaccine (LAIV), are able to mimic natural infection without causing disease or virus transmission and mainly elicit a local immune response. The measurement of sIgA concentrations in nasal swab/wash and saliva samples is therefore a valuable tool for evaluating their role in the effectiveness of such vaccines. Here, we describe two standardized assays (enzyme‐linked immunosorbent assay and microneutralization) available for the quantification of sIgA and discuss the advantages and limitations of their use.
... Cross-protection during the secondary infection is a well-known phenomenon in mice that survive infection with pathogenic influenza viruses. In previous studies, the mice that survived pathogenic influenza virus infection and experienced substantial weight loss during primary infection were shown to have heterosubtypic immunity through the induction of cross-reactive T cell and B cell responses [32][33][34][35][36]. The heterosubtypic protection was observed in the mice that survived primary infection even in a condition with T cell depletion during the secondary infection with an antigenically different virus [37]. ...
Ginseng products used as herb nutritional supplements are orally consumed and fermented to ginsenoside compounds by the intestinal microbes. In this study, we investigated antiviral protective effects of fermented ginseng extracts against different strains of influenza viruses in genetically diverse mouse models. Intranasal coinoculation of mice with fermented ginseng extract and influenza virus improved survival rates and conferred protection against H1N1, H3N2, H5N1, and H7N9 strains, with the efficacy dependent on the dose of ginseng samples. Antiviral protection by fermented ginseng extract was observed in different genetic backgrounds of mice and in the deficient conditions of key adaptive immune components (CD4, CD8, B cell, MHCII). The mice that survived primary virus inoculation with fermented ginseng extract developed immunity against the secondary infection with homologous and heterosubtypic viruses. In vitro cell culture experiments showed moderate virus neutralizing activity by fermented ginseng extract, probably by inhibiting hemagglutination and neuraminidase activity. This study suggests that fermented ginseng extracts might provide a means to treat influenza disease regardless of virus strains.
... Development of broadly cross-reactive anti-influenza vaccine responses is challenging given the heterogeneity of influenza viruses, especially with respect to HA protein. Recently, several studies have suggested that antibody mediated cross-reactive immunity against the influenza virus HA protein strongly correlates with long-lasting cross-protection against influenza strains that differ from the primary infection or vaccination strain [15][16][17]. Induction of significant amounts of broadly cross-reactive and protective antibodies against influenza viruses is an important goal for vaccine design, as current vaccination strategies do not induce high titers of cross-reactive protective antibodies. One recent approach has been to target the conserved stalk domain of HA. ...
Annual immunization against influenza virus is a large international public health effort. Accumulating evidence suggests that antibody mediated cross-reactive immunity against influenza hemagglutinin (HA) strongly correlates with long-lasting cross-protection against influenza virus strains that differ from the primary infection or vaccination strain. However, the optimal strategies for achieving highly cross-reactive antibodies to the influenza virus HA have not yet to be defined. In the current study, using Luminex-based mPlex-Flu assay, developed by our laboratory, to quantitatively measure influenza specific IgG antibody mediated cross-reactivity, we found that prime-boost-boost vaccination of ferrets with rHA proteins admixed with adjuvant elicited higher magnitude and broader cross-reactive antibody responses than that induced by actual influenza viral infection, and this cross-reactive response likely correlated with increased anti-stalk reactive antibodies. We observed a similar phenomenon in mice receiving three sequential vaccinations with rHA proteins from either A/California/07/2009 (H1N1) or A/Hong Kong/1/1968 (H3N2) viruses admixed with Addavax, an MF59-like adjuvant. Using this same mouse vaccination model, we determined that Addavax plays a more significant role in the initial priming event than in subsequent boosts. We also characterized the generation of cross-reactive antibody secreting cells (ASCs) and memory B cells (MBCs) when comparing vaccination to viral infection. We have also found that adjuvant plays a critical role in the generation of long-lived ASCs and MBCs cross-reactive to influenza viruses as a result of vaccination with rHA of influenza virus, and the observed increase in stalk-reactive antibodies likely contributes to this IgG mediated broad cross-reactivity.
... It remains to be seen how these promising results with M2SR will translate to humans. Studies in the literature on heterosubtypic immunity to live virus have given divergent results, implicating both humoral and cell-mediated mechanisms of protection [27,[33][34][35][36]. M2SR induces strong systemic and mucosal antibody responses. ...
Despite the annual public health burden of seasonal influenza and the continuing threat of a global pandemic posed by the emergence of highly pathogenic/pandemic strains, conventional influenza vaccines do not provide universal protection, and exhibit suboptimal efficacy rates, even when they are well matched to circulating strains. To address the need for a highly effective universal influenza vaccine, we have developed a novel M2-deficient single replication vaccine virus (M2SR) that induces strong cross-protective immunity against multiple influenza strains in mice. M2SR is able to infect cells and expresses all viral proteins except M2, but is unable to generate progeny virus. M2SR generated from influenza A/Puerto Rico/8/34 (H1N1) protected mice against lethal challenge with influenza A/Puerto Rico/8/34 (H1N1, homosubtypic) and influenza A/Aichi/2/1968 (H3N2, heterosubtypic). The vaccine induced strong systemic and mucosal antibody responses of both IgA and IgG classes. Strong virus-specific T cell responses were also induced. Following heterologous challenge, significant numbers of IFN-γ-producing CD8 T cells, with effector or effector/memory phenotypes and specific for conserved viral epitopes, were observed in the lungs of vaccinated mice. A substantial proportion of the CD8 T cells expressed Granzyme B, suggesting that they were capable of killing virus-infected cells. Thus, our data suggest that M2-deficient influenza viruses represent a promising new approach for developing a universal influenza vaccine.
... The use of PapMV nanoparticles also increased the IFN-c mediated immune response against highly conserved influenza proteins within different subtypes, such as NP and M1 [35,36]. Also, it has been shown previously that antibodies directed towards an internal protein of the virus particle can contribute to protection from influenza challenge via the involvement of complement and antibody dependent cellular mechanisms [37][38][39][40]. In this study, we measured increased protection against a distant heterosubtypic strain (WSN/33) resulting from the use of the adjuvant. ...
... This process was, at least in part, dependent on alveolar macrophages [94]. However, there are also studies showing that CD4 + T cells [29,95] and/or B cells [96,97] contribute to heterosubtypic immunity. As indicated above, the immune response to influenza virus is multifactorial [82] and Table 1 Published literature on pre-existing T cell immunity in humans. ...
Since inactivated influenza vaccines mainly confer protective immunity by inducing strain-specific antibodies to the viral hemagglutinin, these vaccines only afford protection against infection with antigenically matching influenza virus strains. Due to the continuous emergence of antigenic drift variants of seasonal influenza viruses and the inevitable future emergence of pandemic influenza viruses, there is considerable interest in the development of influenza vaccines that induce broader protective immunity. It has long been recognized that influenza virus-specific CD8(+) T cells directed to epitopes located in the relatively conserved internal proteins can cross-react with various subtypes of influenza A virus. This implies that these CD8(+) T cells, induced by prior influenza virus infections or vaccinations, could afford heterosubtypic immunity. Furthermore, influenza virus-specific CD4(+) T cells have been shown to be important in protection from infection, either via direct cytotoxic effects or indirectly by providing help to B cells and CD8(+) T cells. In the present paper, we review the induction of virus-specific T cell responses by influenza virus infection and the role of virus-specific CD4(+) and CD8(+) T cells in viral clearance and conferring protection from subsequent infections with homologous or heterologous influenza virus strains. Furthermore, we discuss vector-based vaccination strategies that aim at the induction of a cross-reactive virus-specific T cell response.
Copyright © 2014. Published by Elsevier Ltd.
... Group 1 consists of 3 clades (H1, H2, H5 and H6; H8, H9 and H12; H11, H13 and H16) whereas group 2 comprises 2 clades (H3, H4 and H14; H7, H10 and H15) [1,30]. Heterosubtypic immunity has mainly been attributed to cytotoxic T-cells specific for internal proteins [31], but neutralizing antibodies also play an important role in protection [32,33]. To date, a number of broad reacting intra-subtype-, intra-clade-, intra-group-and inter-group-specific neutralizing monoclonal antibodies have been identified [34][35][36][37]. ...
Current avian influenza surveillance in poultry primarily targets subtypes of interest for the veterinary sector (H5, H7). However, as virological and serological evidence suggest, surveillance of additional subtypes is important for public health as well as for the poultry industry. Therefore, we developed a protein microarray enabling simultaneous identification of antibodies directed against different HA-types of influenza A viruses in chickens. The assay successfully discriminated negative from experimentally and naturally infected, seropositive chickens. Sensitivity and specificity depended on the cut-off level used but ranged from 84.4% to 100% and 100%, respectively, for a cut off level of ≥1∶40, showing minimal cross reactivity. As this testing platform is also validated for the use in humans, it constitutes a surveillance tool that can be applied in human-animal interface studies.
... Diversi studi hanno dimostrato l'importante ruolo svolto dai CTL cross-reattivi nella protezione da virus eterosubtipici [Yewdell JW et al. 1985;Taylor PM & Askonas BA 1986;Liang S et al. 1994;Epstein SL et al. 1998;Nguyen HH et al. 1999]. Inoltre, studi condotti sui linfociti B hanno dimostrato che anche gli anticorpi non neutralizzanti cross-protettivi sono coinvolti nell'immunità verso virus eterosubtipici [Nguyen HH et al. 2001;Tumpey TM et al. 2001]. In particolare, è stato osservato che i topi ai quali sono stati eliminati i linfociti B in circolo in seguito al trattamento con un anticorpo specifico per questa sottopopolazione linfocitaria non sono in grado di sviluppare un'immunità eterosubtipica nei confronti di un'infezione letale con il virus dell'Influenza A. Gli anticorpi cross-reattivi nei topi immunizzati sono, dunque, in grado di limitare l'infezione prodotta da un differente sottotipo antigenico. ...
... Viruses. Mouse-adapted A/PR/8/34 (PR8) (H1N1) virus was prepared as lung homogenates of intranasally infected mice were used for challenge as previously described (43). A/Aquatic bird/Korea/W81/2005 (H5N2), isolated from a wild bird in Korea in 2006 (kindly provided by Young-Ki Choi, Chungbuk University, Chungbuk, South Korea), was adapted by multiple passages (15 times) in BALB/c mice. ...
Unlabelled:
Influenza vaccines aimed at inducing antibody (Ab) responses against viral surface hemagglutinin (HA) and neuraminidase (NA) provide sterile immunity to infection with the same subtypes. Vaccines targeting viral conserved determinants shared by the influenza A viruses (IAV) offer heterosubtypic immunity (HSI), a broad protection against different subtypes. We proposed that vaccines targeting both HA and the conserved ectodomain of matrix protein 2 (M2e) would provide protection against infection with the same subtype and also HSI against other subtypes. We report here that single intranasal immunization with a recombinant adenovirus (rAd) vector encoding both HA of H5 virus and M2e (rAdH5/M2e) induced significant HA- and M2e-specific Ab responses, along with protection against heterosubtypic challenge in mice. The protection is superior compared to that induced by rAd vector encoding either HA (rAdH5), or M2e (rAdM2e). While protection against homotypic H5 virus is primarily mediated by virus-neutralizing Abs, the cross-protection is associated with Abs directed to conserved stalk HA and M2e that seem to have an additive effect. Consistently, adoptive transfer of antisera induced by rAdH5/M2e provided the best protection against heterosubtypic challenge compared to that provided by antisera derived from mice immunized with rAdH5 or rAdM2e. These results support the development of rAd-vectored vaccines encoding both H5 and M2e as universal vaccines against different IAV subtypes.
Importance:
Current licensed influenza vaccines provide protection limited to the infection with same virus strains; therefore, the composition of influenza vaccines has to be revised every year. We have developed a new universal influenza vaccine that is highly efficient in induction of long-lasting cross-protection against different influenza virus strains. The cross-protection is associated with a high level of vaccine-induced antibodies against the conserved stalk domain of influenza virus hemagglutinin and the ectodomain of matrix protein. The vaccine could be used to stimulate cross-protective antibodies for the prevention and treatment of influenza with immediate effect for individuals who fail to respond to or receive the vaccine in due time. The vaccine offers a new tool to control influenza outbreaks, including pandemics.
... Since HA-specific IgG antibodies induced by subcutaneous or intramuscular injection with inactivated influenza vaccines are principally subtype-specific, protective effects are limited to viruses whose antigenicity is closely related to those of the vaccine strains [18,19]. However, previous studies experimentally demonstrated that B-cell-dependent heterosubtypic immunity was induced by intranasal immunization of mice with formalininactivated viruses, whereas systemic immunization only protected mice from viruses with homologous HA subtypes [20][21][22]. We recently reported that both subcutaneous and intranasal immunization of mice with inactivated viruses induced antibodies that bound to HAs of multiple subtypes, but IgA antibodies showed greater ability than IgG antibodies to reduce plaque formation of viruses with heterologous subtypes [23], suggesting different antiviral potentials for IgA and IgG antibodies in heterosubtypic immunity. ...
Both IgA and IgG antibodies are known to play important roles in protection against influenza virus infection. While IgG is the major isotype induced systemically, IgA is predominant in mucosal tissues, including the upper respiratory tract. Although IgA antibodies are believed to have unique advantages in mucosal immunity, information on direct comparisons of the in vitro antiviral activities of IgA and IgG antibodies recognizing the same epitope is limited. In this study, we demonstrate differences in antiviral activities between these isotypes using monoclonal IgA and IgG antibodies obtained from hybridomas of the same origin. Polymeric IgA-producing hybridoma cells were successfully subcloned from those originally producing monoclonal antibody S139/1, a hemaggulutinin (HA)-specific IgG that was generated against an influenza A virus strain of the H3 subtype but had cross-neutralizing activities against the H1, H2, H13, and H16 subtypes. These monoclonal S139/1 IgA and IgG antibodies were assumed to recognize the same epitope and thus used to compare their antiviral activities. We found that both S139/1 IgA and IgG antibodies strongly bound to the homologous H3 virus in an enzyme-linked immunosorbent assay, and there were no significant differences in their hemagglutination-inhibiting and neutralizing activities against the H3 virus. In contrast, S139/1 IgA showed remarkably higher cross-binding to and antiviral activities against H1, H2, and H13 viruses than S139/1 IgG. It was also noted that S139/1 IgA, but not IgG, drastically suppressed the extracellular release of the viruses from infected cells. Electron microscopy revealed that S139/1 IgA deposited newly produced viral particles on the cell surface, most likely by tethering the particles. These results suggest that anti-HA IgA has greater potential to prevent influenza A virus infection than IgG antibodies, likely due to increased avidity conferred by its multivalency, and that this advantage may be particularly important for heterosubtypic immunity.
... No neutralization activity in plasma against H7N7 was induced in vaccinated macaques, but IFN-c production by T lymphocytes against NL2586 was detected. These results suggested that antibody responses with neutralization activity were more important than T lymphocyte responses in protection against HPAIV infection, though T lymphocyte responses against H5N1 HPAIVs might have helped the elimination of VN3040 and HOK1 viruses in vaccinated macaques [35]. In summary, we demonstrated that a whole particle vaccine of Vac-3 induced protective immune responses against two clades of H5N1 HPAIVs and a pandemic (H1N1) 2009 strain in cynomolgus macaques. ...
H5N1 highly pathogenic avian influenza virus (HPAIV) infection has been reported in poultry and humans with expanding clade designations. Therefore, a vaccine that induces immunity against a broad spectrum of H5N1 viruses is preferable for pandemic preparedness. We established a second H5N1 vaccine candidate, A/duck/Hokkaido/Vac-3/2007 (Vac-3), in our virus library and examined the efficacy of inactivated whole particles of this strain against two clades of H5N1 HPAIV strains that caused severe morbidity in cynomolgus macaques. Virus propagation in vaccinated macaques infected with either of the H5N1 HPAIV strains was prevented compared with that in unvaccinated macaques. This vaccine also prevented propagation of a pandemic (H1N1) 2009 virus in macaques. In the vaccinated macaques, neutralization activity, which was mainly shown by anti-hemagglutinin antibody, against H5N1 HPAIVs in plasma was detected, but that against H1N1 virus was not detected. However, neuraminidase inhibition activity in plasma and T-lymphocyte responses in lymph nodes against H1N1 virus were detected. Therefore, cross-clade and heterosubtypic protective immunity in macaques consisted of humoral and cellular immunity induced by vaccination with Vac-3.
... Intact lungs of mice (n = 3) from each group were prepared to assess cytokine levels at days 0 and 7 p.i.. Whole lungs were homogenized using a conventional method [36]. Homogenates were centrifuged at 300 g for 10 min., and supernatants were collected. ...
Influenza virus undergoes constant antigenic evolution, and therefore influenza vaccines must be reformulated each year. Time is necessary to produce a vaccine that is antigenically matched to a pandemic strain. A goal of many research works is to produce universal vaccines that can induce protective immunity to influenza A viruses of various subtypes. Despite intensive studies, the precise mechanisms of heterosubtypic immunity (HSI) remain ambiguous.
In this study, mice were vaccinated with recombinant virus vaccine (rL H5), in which the hemagglutinin (HA) gene of influenza A/H5N1 virus was inserted into the LaSota Newcastle disease virus (NDV) vaccine strain. Following a challenge with influenza A/H1N1 virus, survival rates and lung index of mice were observed. The antibodies to influenza virus were detected using hemagglutination inhibition (HI). The lung viral loads, lung cytokine levels and the percentages of both IFN-gamma+CD4+ and IFN-gamma+CD8+ T cells in spleen were detected using real-time RT-PCR, ELISA and flow cytometry respectively.
In comparison with the group of mice given phosphate-buffered saline (PBS), the mice vaccinated with rL H5 showed reductions in lung index and viral replication in the lungs after a challenge with influenza A/H1N1 virus. The antibody titer in group 3 (H1N1-H1N1) was significantly higher than that in other groups which only low levels of antibody were detected. IFN-gamma levels increased in both group 1 (rL H5-H1N1) and group 2 (rL H5 + IL-2-H1N1). And the IFN-gamma level of group 2 was significantly higher than that of group 1. The percentages of both IFN-gamma+CD4+ and IFN-gamma+CD8+ T cells in group 1 (rL H5-H1N1) and group 2 (rL H5 + IL-2-H1N1) increased significantly, as measured by flow cytometry.
After the mice were vaccinated with rL H5, cross-protective immune response was induced, which was against heterosubtypic influenza A/H1N1 virus. To some extent, cross-protective immune response can be enhanced by IL-2 as an adjuvant. Cellular immune responses may play an important role in HSI against influenza virus.
... In addition to their ability to ingest Ab-coated particles by opsonophagocytosis, macrophages also could potentially destroy infected cells by ADCC (52). Interestingly, B cells but not cytotoxic lymphocytes were recently found to be required for heterosubtypic immunity to influenza virus infection (53). Our findings have important implications for antiviral vaccination strategies and stress the need for the targeting of Ab responses at mucosal sites that preferentially stimulate Fc receptor-mediated host mechanisms. ...
Fc receptors for IgG expressed on macrophages and NK cells are important mediators of opsonophagocytosis and Ab-dependent cell-mediated cytotoxicity. Phagocyte-mediated opsonophagocytosis is pivotal for protection against bacteria, but its importance in recovery from infection with intracellular pathogens is unclear. We have now investigated the role of opsonophagocytosis in protection against lethal influenza virus infection by using FcR γ−/− mice. Absence of the FcR γ-chain did not affect the expression of IFN-γ and IL-10 in the lungs and spleens after intranasal immunization with an influenza subunit vaccine. Titers of serum and respiratory Abs of the IgM, IgG1, IgG2a, and IgA isotypes in FcR γ−/− mice were similar to levels seen in FcR γ+/+ mice. Nevertheless, FcR γ−/− mice were highly susceptible to influenza infection, even in the presence of anti-influenza Abs from immune FcR γ+/+ mice. NK cells were not necessary for the observed Ab-mediated viral clearance, but macrophages were found to be capable of actively ingesting opsonized virus particles. We conclude that Fc receptor-mediated phagocytosis plays a pivotal role in clearance of respiratory virus infections.
... IgA was shown to be more cross-reactive than IgG against heterologous influenza viruses and passive transfer of IgA to non-immune mice conferred protection (Tamura et al., 1991). Although cytotoxic T-lymphocyte activity has been shown to be stimulated in heterosubtypic primed mice (Nguyen et al., 1999), protection against heterosubtypic challenge in mice was largely dependent on the presence of B-cells and CD4þ T-helper cells, specifically those with a Th1 phenotype (Moran et al., 1999; Nguyen et al., 1999 Nguyen et al., , 2001). CD4þ T cells primed against conserved internal influenza proteins may be responsible for the rapid development of cross-reacting antibodies following a heterosubtypic challenge (Scherle and Gerhard, 1986). ...
Influenza is a zoonotic viral disease that represents a health and economic threat to both humans and animals worldwide. Swine influenza (SI) was first recognized clinically in pigs in the Midwestern U.S., in 1918, coinciding with the human influenza pandemic known as the Spanish flu. Since that time SI has remained of importance to the swine industry throughout the world. In this review, the epidemiology of swine influenza virus (SIV) infection in North American pigs is described in detail. The first 80 years of SI remained relatively static, whereas the last decade has become dynamic with the establishment of many emerging subtypes. With the increasing number of novel subtypes and genetic variants, the control of SI has become increasingly difficult and innovative strategies to combat this economically important zoonotic disease are critical. Therefore, protective immune responses against influenza virus infections as well as new paradigms of vaccine development in pigs are discussed in the review. It is expected that the dynamic evolutionary changes of SIVs in North American pigs will continue, making currently available prophylactic approaches of limited use to control the spread and economic losses associated with this important swine pathogen.
... Such immunity is thought to be mediated mainly by cross-reactive cytotoxic T lymphocytes (CTLs) directed against the highly conserved internal proteins but not the surface glycoproteins [64][65][66][67][68][69]. However, different reports have shown that crossprotective antibodies against HA can also be conferred by immunization [63,[70][71][72][73][74][75][76] and natural infection [77][78][79]. ...
Influenza A surface glycoproteins, hemagglutinin (HA) and neuraminidase (NA), are the major targets of neutralizing antibodies which necessitate their inclusion in influenza vaccines. The frequent antigenic drift and shift of these proteins are responsible for the annual epidemics and occasional pandemics of influenza. Therefore, vaccines must be reformulated annually to include the HA and NA proteins of the viral strains predicted for the upcoming flu season. There are inherent limitations in the annual vaccine preparation process since testing for effectiveness, approval by regulatory agencies and distribution of these vaccines can take approximately 6 months. These drawbacks are most pronounced in situations of pandemic influenza such as the recent swine-origin 2009 H1N1 pandemic, thus there is a critical need for the development of new antiviral and vaccine strategies. One promising new area of research is targeting the conserved regions of influenza surface glycoproteins. Recent studies have demonstrated that vaccines based on highly conserved sequences or antibodies raised against conserved epitopes can protect animals against diverse influenza A virus subtypes. In this review, we focus on the challenges associated with the two main surface glycoproteins, HA and NA, while emphasizing recent advances in bioinformatics tools and their contribution to the design of new diagnostics, vaccines and antivirals.
... In addition to their ability to ingest Ab-coated particles by opsonophagocytosis, macrophages also could potentially destroy infected cells by ADCC (52). Interestingly, B cells but not cytotoxic lymphocytes were recently found to be required for heterosubtypic immunity to influenza virus infection (53). Our findings have important implications for antiviral vaccination strategies and stress the need for the targeting of Ab responses at mucosal sites that preferentially stimulate Fc receptor-mediated host mechanisms. ...
... B cells play a role in the heterosubtypic immunity to influenza infection. 27 Inactivated whole-virus vaccines also produce a good crossclade cellular immune response, 28 which may well explain CD4 + T cells have indeed been shown to recognize H5N1 viral antigens in human: peptides from matrix 1, nucleocapsid, neuraminidase, and H5 HA proteins. [29][30][31] A key variable in such an approach is the duration of crossprotective immunity, a variable that we measured in this study. ...
Background:
Highly pathogenic H5N1 influenza viruses reemerged in humans in 2003 and have caused fatal human infections in Asia and Africa as well as ongoing outbreaks in poultry. These viruses have evolved substantially and are now so antigenically varied that a single vaccine antigen may not protect against all circulating strains. Nevertheless, studies have shown that substantial cross-reactivity can be achieved with H5N1 vaccines. These studies have not, however, addressed the issue of duration of such cross-reactive protection.
Objectives:
To directly address this using the ferret model, we used two recommended World Health Organization H5N1 vaccine seed strains - A/Vietnam/1203/04 (clade 1) and A/duck/Hunan/795/02 (clade 2.1) - seven single, double, or triple mutant viruses based on A/Vietnam/1203/04, and the ancestral viruses A and D, selected from sequences at nodes of the hemagglutinin and neuraminidase gene phylogenies to represent antigenically diverse progeny H5N1 subclades as vaccine antigens.
Results:
All inactivated whole-virus vaccines provided full protection against morbidity and mortality in ferrets challenged with the highly pathogenic H5N1 strain A/Vietnam/1203/04 5 months and 1 year after immunization.
Conclusion:
If an H5N1 pandemic was to arise, and with the hypothesis that one can extrapolate the results from three doses of a whole-virion vaccine in ferrets to the available split vaccines for use in humans, the population could be efficiently immunized with currently available H5N1 vaccines, while the homologous vaccine is under production.
... Furthermore, mucosal S-IgA is known as a noninflammatory Ab (21). Although heterosubtypic protection from influenza is not only mediated by cytotoxic T cells (31), mucosal S-IgA and respiratory B cells induced by nasal vaccination and natural infection (7,32) are crucial in protecting against IAV infection. In addition, humoral and protective immunity associated with antihemagglutinin specific IgA elicited by primary infection or vaccination is important for the prevention of secondary infection with different virus subtypes (41). ...
We previously reported that the macrolide antibiotic clarithromycin (CAM) enhanced the mucosal immune response in pediatric influenza, particularly in children treated with the antiviral neuraminidase inhibitor oseltamivir (OSV) with low production of mucosal antiviral secretory IgA (S-IgA). The aims of the present study were to confirm the effects of CAM on S-IgA immune responses, by using influenza A virus (IAV) H1N1-infected mice treated with or without OSV, and to determine the molecular mechanisms responsible for the induction of mucosal IgA class switching recombination in IAV-infected CAM-treated mice. The anti-IAV S-IgA responses and expression levels of IgA class switching recombination-associated molecules were examined in bronchus-lymphoid tissues and spleens of infected mice. We also assessed neutralization activities of S-IgA against IAV. Data show that CAM enhanced anti-IAV S-IgA induction in the airway of infected mice and restored the attenuated antiviral S-IgA levels in OSV-treated mice to the levels in the vehicle-treated mice. The expression levels of B-cell-activating factor of the tumor necrosis factor family (BAFF) molecule on mucosal dendritic cells as well as those of activation-induced cytidine deaminase and Iμ-Cα transcripts on B cells were enhanced by CAM, compared with the levels without CAM treatment, but CAM had no effect on the expression of the BAFF receptor on B cells. Enhancement by CAM of neutralization activities of airway S-IgA against IAV in vitro and reinfected mice was observed. This study identifies that CAM enhances S-IgA production and neutralizing activities through the induction of IgA class switching recombination and upregulation of BAFF molecules in mucosal dendritic cells in IAV-infected mice.
... The immunologic basis underlying heterosubtypic immunity has been the topic of numerous studies [23]. Experiments in multiple knock-out and transgenic mouse models have shown that virus-specific CD4+ T cells (T helper cells), CD8+ Cytotoxic T cells (CTL), mucosal antibodies (IgA) and B cells can contribute to heterosubtypic immunity [28–33]. Especially cell-mediated immune responses directed to conserved proteins of influenza A viruses are believed to play an important role. ...
The current pandemic caused by the new influenza A(H1N1) virus of swine origin and the current pandemic threat caused by the highly pathogenic avian influenza A viruses of the H5N1 subtype have renewed the interest in the development of vaccines that can induce broad protective immunity. Preferably, vaccines not only provide protection against the homologous strains, but also against heterologous strains, even of another subtype. Here we describe viral targets and the arms of the immune response involved in protection against influenza virus infections such as antibodies directed against the hemagglutinin, neuraminidase and the M2 protein and cellular immune responses directed against the internal viral proteins.
... Male Imprinting Control Region 8-to 10-week-old mice, purchased from Orient, Sungnam, Korea, were used in all experiments. The influenza virus strain used in this study is PR8, kindly provided by Dr. Huan H. Nguyen, International Vaccine Institute, Seoul, Korea, and was prepared as previously reported (15). For total respiratory tract infection, the mice were anesthetized with ketamine/xylazine and infected with 15 PFU in 40 μL PBS via the intranasal route. ...
The influenza A virus is one of the main causes of respiratory infection. Although influenza virus infection alone can result in pneumonia, secondary bacterial infection combined with the virus is the major cause of morbidity and mortality. Interestingly, while influenza infection increases susceptibility to some bacteria, including Streptococcus pneumoniae, Staphylococcus aureus (S. aureus), and Haemophilus influenzae, other bacteria such as Escherichia coli (E. coli) and Klebsiella pneumoniae are not associated with influenza infection. The reason for this discrepancy is not known. In this study, it was found that prior influenza virus infection inhibits murine alveolar macrophage phagocytosis of S. aureus but not of E. coli. Here, the mechanism for this inhibition is elucidated: prior influenza virus infection strongly increases interferon gamma (IFN-γ) production. Furthermore, it was shown that IFN-γ differentially affects alveolar macrophage phagocytosis of S. aureus and E. coli. The findings of the present study explain how influenza virus infection increases susceptibility to some bacteria, such as S. aureus, but not others, and provides evidence that IFN-γ might be a promising target for protecting the human population from secondary bacterial infection by influenza.
... At the same time, several studies in mouse models suggest that also humoral immunity, especially in its IgG component, when directed against conserved epitopes contributes to heterosubtypic protection (Nguyen et al., 2001;Quan et al., 2008a,b;Takada et al., 2003;Tumpey et al., 2001). As an example, a heterosubtypic immune response was successfully elicited in infected b2-microglobulin knockout mice or in mice lacking the secretory IgA Jchain, evidencing that neither MHC-I-restricted T-cells nor secretory IgA are absolutely required for cross-protection (Epstein et al., 1997). ...
The role of humoral response in the effective control of infection by influenza viruses is well known, but the protection is usually limited to the infecting or vaccinating isolate and to few related strains. Recent studies have evidenced the existence of B-cell epitopes broadly conserved among different influenza subtypes recognized by monoclonal antibodies endowed with unprecedented broad activity. In this review, all major monoclonal antibodies directed against different influenza virus proteins are reported and their potential in the design of new anti-influenza prophylactic or therapeutic strategies is discussed.
Acute viral infections are characterized by rapid increases in viral load, leading to cellular damage and the resulting induction of complex innate and adaptive antiviral immune responses that cause local and systemic inflammation. Successful antiviral immunity requires the activation of many immune cells, including T cells, natural killer cells, and macrophages. B cells play a unique part through their production of antibodies that can both neutralize and clear viral particles before virus entry into a cell. Protective antibodies are produced even before the first exposure of a pathogen, through the regulated secretion of so-called natural antibodies that are generated even in the complete absence of prior microbial exposure. An early wave of rapidly secreted antibodies from extrafollicular (EF) responses draws on the preexisting naive or memory repertoire of B cells to induce a strong protective response that in kinetics tightly follows the clearance of acute infections, such as with influenza virus. Finally, the generation of germinal centers (GCs) provides long-term protection through production of long-lived plasma cells and memory B cells, which shape and broaden the B cell repertoire for more effective responses following repeat exposures. In this study, we review B cell responses to acute viral infections, primarily influenza virus, from the earliest nonspecific B-1 cell to early, antigen-specific EF responses and finally to GC responses. Throughout, we address known factors that lead to distinct B cell response outcomes and discuss how their functions effect viral clearance, highlighting the critical contributions of each response type to the induction of highly protective antiviral humoral immunity.
It has been known that influenza A virus infection induces a cross-protective immunity against infection by viruses with different subtypes of viral envelope proteins, hemagglutinin (HA) and neuraminidase (NA). This heterosubtypic immunity is generally mediated by cytotoxic T lymphocytes (CTL) reactive to specific epitopes in the viral internal proteins, such as nucleoprotein and matrix protein. By contrast, immunization with inactivated virus antigens has been thought to be unable to generate heterosubtypic immunity, since inactivated antigens do not usually induce CTL responses. However, we show that intranasal immunization with formalin-inactivated intact virus, but not ether-split vaccines, induced a broad spectrum of heterosubtypic protective immunity in mice. The protection may be mediated by the mucosal immune response, most likely secretory IgA antibodies to the viral proteins. This approach may overcome limitations in the efficacy of inactivated influenza vaccines and confer potent immunity to humans against viruses with new pandemic potential.
Both the cellular and humoral immune systems participate actively in the resolution of first and subsequent mucosal viral infections. Specifically, cytotoxic T cells (TCs) and IgA/IgM antibodies each make important contributions during resolution of primary infection, while IgA antibodies induced by prior infection or immunization offer the most effective resistance to reinfection. IgG antibodies are less effective at accessing the lumenal surfaces but are an important component of long-term immunity, especially in the lung. TCs and helper T (Th) memory cells take time to expand and activate reinfection and make a smaller contribution than IgA antibodies to the inhibition of the viral replication during reinfection. Studies demonstrate that local IgA memory to bovine respiratory syncytial virus in the absence of detectable IgA antibodies is associated with resistance to reinfection with virus. It appears that a local IgA response to reinfection is sufficiently rapid to limit the extent of virus replication. Thus implying that the mucosal IgA antibodies participate in the resolution of primary viral infection.
This chapter reviews the main viral pathogens of the respiratory tract, the immune responses they induce, currently available vaccines, and vaccines that are in development to control them. The main viruses responsible for acute respiratory infection in people include respiratory syncytial, influenza, human parainfluenza, human metapneumo-, human rhino-, corona-, and adenoviruses. Licensed vaccines are available only for influenza virus, with vaccines against the other pathogens either in clinical trials or in preclinical stages of development. The majority of studies evaluating respiratory virus vaccines measure serum antibody responses, because, although both cellular and humoral responses contribute to the clearance of a primary infection, neutralizing antibodies are known to protect against secondary infection. Humoral responses can be readily detected after vaccination with inactivated or subunit vaccines; however, fewer individuals seroconvert after vaccination with live vaccines. Alternative immune mechanisms such as mucosal antibody responses are probably responsible for protection by live attenuated vaccines, and immune correlates of protection are under investigation.
This study was undertaken to determine the differences in genotypes of rotavirus and their incidence between patients with acute rotaviral enteritis who suffered neurologic complications and those who did not suffer neurologic complications.
Rotavirus (RV) is the most common cause of childhood diarrhea worldwide, and several vaccines have been successfully developed to reduce the burden of disease. However, lower vaccine immunogenicity and efficacy in developing countries might be related to the virus-neutralizing activity of breast milk. We examined possible differences in breast milk antibody levels (total IgA antibody, RV-specific antibodies, and RV-neutralizing antibodies) between healthy mothers living in a rural area (n = 145) and mothers living in an urban area (n = 147) of Vietnam. Total IgA concentration was significantly higher in samples from mothers in the rural region than in samples from mothers in the urban region, whereas urban mothers had significantly higher RV-specific IgA antibody titers than did rural mothers. Neutralizing antibodies against RV strain G1P[8] were undetected in nearly one-half of the breast milk samples (45-48%), whereas the majority of the remaining samples had low antibody titers (2-16). Despite these low titers, the breast milk still reduced vaccine strain titers (2 × 10(6) plaque forming units/mL) up to 80% or more, even at a milk-to-virus ratio of 1:8. An increase in neutralizing anti-G1P[8] antibody titers (P < 0.05) in rural infants over time suggests a continuous exposure to circulating RV. These results contribute to the understanding of the potential interference of breast milk with RV vaccine efficacy and immunogenicity in Vietnamese infants.
We investigated the cross-neutralising potential of serum and nasal wash samples from volunteers who were intranasally immunised once with a monovalent replication-deficient delNS1-H1N1 influenza virus vaccine (7.7 log10 TCID50/volunteer). Eight out of twelve (8/12) vaccinees responded to vaccination with a significant increase of antibody levels in serum IgG ELISA, mucosal IgA ELISA, MNA or HAI. Four responders showed delNS1-specific ELISA IgA increases and revealed excellent homosubtypic neutralising activity in serum and mucosal washings (4/4). However, 0/4 of the sera but 3/4 of the nasal washings neutralised also heterosubtypic H3N2 and H5N1 influenza viruses. Depletion experiments proved that IgA but not IgG is responsible for the cross-neutralising activity of the nasal wash sample. Our findings indicate that the induction of virus-neutralising IgA may represent a valuable correlate of cross-protection of intranasal influenza vaccines and that the delNS1 concept constitutes a promising approach to protect humans from seasonal and pandemic influenza threats.
Clinical trial registration: NCT00724997.
Influenza virus infections are a serious global health threat, particularly in light of newly emerging strains, such as the avian virus H5N1. In this study, a sample of avian influenza A virus subtype H3N8 inactivated by high hydrostatic pressure was used as a vaccine. Our goal was to study pressurized virus preparations for their ability to induce an immunogenic and protective response when using mice as an animal model. Here, Balb/c mice were treated through the intranasal route with three doses of pressurized virus. After vaccination, the mice were challenged and monitored for virus-specific antibodies (ELISA and neutralization assay), clinical symptoms and death. After immunization, there was an increase of IgG1 and IgG2a in sera and IgA in nasal lavages, which indicated that the serum antibodies were showing neutralizing ability. The viral neutralization assay demonstrated that the produced antibodies were neutralizing. After the challenge, the control group (immunized with saline) showed all measured clinical signs of disease (weight loss, ruffled fur, lethargy and huddling). The vaccinated animals did not develop any clinical signs. The results reveal that the animals were able to produce a satisfactory humoral response after vaccination and protected against the challenge. Our work reaffirms the use of hydrostatic pressure as a means for developing low-cost viral vaccines with good immune response.
Infection with one serotype of the influenza A virus confers a certain level of heterosubtypic immunity (HSI) to infection with influenza A virus of a different serotype. Effective HSI is thus predicated on prior sensitization of the host, implicating that adaptive immunity plays a vital role. It had been proposed that HSI is mediated by serotype cross-reactive cytotoxic T lymphocytes (CTL), but more recent studies suggest a principal role for antibodies (Abs) in HSI. Our results from microarray analysis of gene expression suggest a role for complement (C) in HSI. Accordingly, we found that complement protein C3 is required for induction of antigen-specific Ab responses and subsequent complete HSI. In addition, complement per se may play a role in effector mechanisms in HSI. These findings suggest that both adaptive and innate immunity are required for protective HSI.
Memory CD4+ T cells combat viral infection and contribute to protective immune responses through multiple mechanisms, but how these pathways interact is unclear. We found that several pathways involving memory CD4+ T cells act together to effectively clear influenza A virus (IAV) in otherwise unprimed mice. Memory CD4+ T cell protection was enhanced through synergy with naive B cells or CD8+ T cells and maximized when both were present. However, memory CD4+ T cells protected against lower viral doses independently of other lymphocytes through production of IFN-γ. Moreover, memory CD4+ T cells selected for epitope-specific viral escape mutants via a perforin-dependent pathway. By deconstructing protective immunity mediated by memory CD4+ T cells, we demonstrated that this population simultaneously acts through multiple pathways to provide a high level of protection that ensures eradication of rapidly mutating pathogens such as IAV. This redundancy indicates the need for reductionist approaches for delineating the individual mechanisms of protection mediated by memory CD4+ T cells responding to pathogens.
Heterosubtypic immunity (HSI) is defined as protective cross-reactive immune responses to lethal infection with influenza A virus of a different serotype than the virus initially encountered, and is thought to be mediated by serotype cross-reactive cytotoxic T lymphocytes (CTL). These CTL recognize conserved epitopes of internal proteins, such as nucleoprotein (NP) or matrix (M) protein shared by influenza A virus subtypes. Despite extensive studies, the precise effector mechanism for HSI remains elusive. For example, our recent studies and those of others reported HSI in T cell-depleted, β2-microglobulin-deficient, and CD8 cell-deficient mice. The role for humoral immune responses in HSI is also unclear. Passive transfer of heterosubtypic immune serum did not provide protection against lethal heterosubtypic challenge, while B cell-deficient mice failed to develop HSI. Our recent findings and those of others now allow us to suggest a two-tiered HSI. Early after heterosubtypic challenge, a number of factors including subtype-specific CTL as well as antibody (Ab) responses and other as yet not well characterized host factors are able to minimize temporarily the virus spread, but are unable to clear the infection. In the later phase, the development of virus-neutralizing (VN) antibodies is important for virus clearance resulting in complete host recovery.
A major challenge for research on influenza vaccines is the selection of an appropriate animal model that accurately reflects the disease and the protective immune response to influenza infection in humans. Vaccines for seasonal influenza have been available for decades and there is a wealth of data available on the immune response to these vaccines in humans, with well-established correlates of protection for inactivated influenza virus vaccines. Many of the seminal studies on vaccines for epidemic influenza were conducted in human subjects. Studies in humans are performed less frequently now than they were in the past. Therefore, as the quest for improved influenza vaccines continues, it is important to consider the use of animal models for the evaluation of influenza vaccines, and a major challenge for research on influenza vaccines is the selection of an appropriate animal model that accurately reflects the disease and the protective immune response to influenza infection in humans.
The emergence of highly pathogenic H5N1 avian influenza (AI) viruses and the threat of a pandemic caused by AI viruses of this or another subtype has resulted in a resurgence of interest in influenza vaccine research. The development of vaccines for pandemic influenza presents a unique set of obstacles, not the least of which is that the demonstration of efficacy in humans is not possible. Since the correlates of protection from pandemic influenza are not known, we rely on extrapolation of lessons from seasonal influenza vaccines and on data from the evaluation of pandemic influenza vaccines in animal models to guide our decisions on vaccines for use in humans. The features and contributions of commonly used animal models for influenza vaccine research are discussed.
The influenza A viruses are dangerous pathogens with the potential to provoke devastating disease. The challenge for the medical
research community is to design preventive measures and therapeutic interventions that will limit the severe consequences
of pandemic influenza A virus infections. Vaccines have long been available, but there is considerable scope for improvement
as they target only the prevailing influenza A virus strains, do not give broad immunity, and work poorly in the elderly,
the target group that is most at risk of fatal disease. Improved vaccines will only emerge if the development strategy is
based on a firm understanding of the host immune response to the virus. Here, we summarize the research to date that details
immune mechanisms participating in the control and elimination of influenza A viruses.
BackgroundAntibody alone cannot provide optimal protection against many infectious diseases impacting global heath. In these cases,
our challenge is to develop innovative vaccines that generate protective populations of memory T cells. However, our studies
suggest that current paradigms explaining how memory CD4 T cells provide protection are inadequate. This is likely due to
both the paucity of and heterogeneity of memory CD4 T cells observed in vivo, which make analysis extremely difficult.
SummaryHere, we discuss new findings that indicate there is extensive functional heterogeneity within effector and memory CD4 T cell
populations both in vivo and in vitro. Using influenza as an example, we also discuss the merits of employing reductionist approaches to explore how unique subsets
of CD4 T cells are generated, what mechanisms of protection they use, and where they stand on the axes of differentiation
that define T cell subsets.
During the 2009 H1N1 influenza virus pandemic (pdmH1N1) outbreak, it was found that most individuals lacked antibodies against the new pdmH1N1 virus, and only the elderly showed anti-hemagglutinin (anti-HA) antibodies that were cross-reactive with the new strains. Different studies have demonstrated that prior contact with the virus can confer protection against strains with some degree of dissimilarity; however, this has not been sufficiently explored within the context of a pdmH1N1 virus infection. In this study, we have found that a first infection with the A/Brisbane/59/2007 virus strain confers heterologous protection in ferrets and mice against a subsequent pdmH1N1 (A/Mexico/4108/2009) virus infection through a cross-reactive but non-neutralizing antibody mechanism. Heterologous immunity is abrogated in B cell-deficient mice but maintained in CD8(-/-) and perforin-1(-/-) mice. We identified cross-reactive antibodies from A/Brisbane/59/2007 sera that recognize non-HA epitopes in pdmH1N1 virus. Passive serum transfer showed that cross-reactive sH1N1-induced antibodies conferred protection in naive recipient mice during pdmH1N1 virus challenge. The presence or absence of anti-HA antibodies, therefore, is not the sole indicator of the effectiveness of protective cross-reactive antibody immunity. Measurement of additional antibody repertoires targeting the non-HA antigens of influenza virus should be taken into consideration in assessing protection and immunization strategies. We propose that preexisting cross-protective non-HA antibody immunity may have had an overall protective effect during the 2009 pdmH1N1 outbreak, thereby reducing disease severity in human infections.
There is considerable interest in the development of broadly protective influenza vaccines because of the continuous emergence of antigenic drift variants of seasonal influenza viruses and the threat posed by the emergence of antigenically distinct pandemic influenza viruses. It has been recognized more than three decades ago that influenza A virus-specific cytotoxic T lymphocytes recognize epitopes located in the relatively conserved proteins like the nucleoprotein and that they cross-react with various subtypes of influenza A viruses. This implies that these CD8+ T lymphocytes may contribute to protective heterosubtypic immunity induced by antecedent influenza A virus infections. In the present paper, we review the evidence for the role of virus-specific CD8+ T lymphocytes in protective immunity against influenza virus infections and discuss vaccination strategies that aim at the induction of cross-reactive virus-specific T-cell responses.
Intramuscular injection of BALB/c mice with a DNA plasmid encoding nucleoprotein (NP) from influenza virus A/PR/8/34 (H1N1) provides cross-strain protection against lethal challenge with influenza virus A/HK/68 (H3N2). CTL specific for the H-2Kd-restricted epitope NP147–155 are present in these mice and are thought to play a role in the protection. To assess the effectiveness of NP DNA immunization in comparison with influenza virus infection in the induction of CTL responses, we monitored the frequency of CTL precursors (CTLp) in mice following i.m. injection with NP DNA or intranasal infection with influenza virus and showed that the CTLp frequency in NP DNA-immunized mice can reach levels found in mice that had been infected with influenza virus. We also measured the CTLp frequency, anti-NP Ab titers, and T cell proliferative responses in mice that were injected with titrated dosages of NP DNA and documented a correlation of the CTLp frequency and the Ab titers, but not proliferative responses, with the injection dose. Furthermore, we observed a positive correlation between the frequency of NP147–155 epitope-specific CTLp and the extent of protective immunity against cross-strain influenza challenge induced by NP DNA injection. Collectively, these results and our early observations from adoptive transfer experiments of in vitro activated lymphocytes from NP DNA-immunized mice suggest a protective function of NP-specific CTLp in mice against cross-strain influenza virus challenge.
Local administration of nucleoprotein purified from X31 (H3N2) influenza A virus primed for A virus cross-reactive cytotoxic T cells and resulted in substantial protection (75%) of mice from a lethal challenge with the heterologous mouse-adapted A/PR/8/34 (H1N1) virus. By following the course of a lethal virus challenge we found that nucleoprotein priming did not prevent virus infection but rather aided recovery. Nucleoprotein-primed mice suffered initial symptoms of infection, i.e. weight loss and surface temperature changes, but started to recover after approximately 7 days. We suggest that such heterotypic protection can be attributed to priming of A virus cross-reactive cytotoxic T cells.
Natural killer cell stimulatory factor (NKSF), or interleukin 12 (IL-12), is a 70-kD heterodimeric cytokine composed of two covalently linked chains, p40 and p35. NKSF/IL-12 has multiple effects on T and NK cells and was originally identified and purified from the supernatant fluid of Epstein-Barr virus (EBV)-transformed human B lymphoblastoid cell lines. We have produced a panel of monoclonal antibodies against both chains of NKSF/IL-12. Some of these antibodies have neutralizing activity, and several combinations of them have been used to establish sensitive radioimmunoassays detecting the free p40 chain, the free p35 chain, or the p70 heterodimer. Using these reagents, we have determined that most EBV-transformed human B lymphoblastoid cell lines constitutively produce low levels of the p70 heterodimer and an excess of the free p40 chain, whereas Burkitt lymphoma-derived, T, myeloid, and many solid tumor-derived cell lines produce neither. Production of both p40 and p70 is increased several-fold upon stimulation of the EBV-transformed cell lines with phorbol diesters. The ability of supernatant fluids from unstimulated and phorbol diester-stimulated cell lines to induce interferon gamma (IFN-gamma) production from T and NK cells, one of the effects of NKSF/IL-12, parallels the levels of production of the p70 heterodimer, known to be the biologically active form of NKSF/IL-12. Staphylococcus aureus Cowan I strain (SAC) and other stimuli induce accumulation of p40 mRNA and production of both p40 and p70 by peripheral blood mononuclear cells (PBMC). The producer cells appear to include both adherent cells and nonadherent lymphocytes, possibly B cells. The supernatant fluids from SAC-stimulated PBMC mediate the typical functions of NKSF/IL-12 (i.e., IFN-gamma induction, mitogenic effects on T/NK blasts, enhancement of NK cell cytotoxicity) at concentrations of p70 similar to those at which recombinant NKSF/IL-12 mediates the same functions. Moreover, these activities are significantly inhibited by anti-NKSF/IL-12 antibodies. The neutralizing anti-NKSF/IL-12 antibodies also inhibit 85% of the IFN-gamma production in response to SAC, an NKSF/IL-12 inducer, and approximately 50% of the IFN-gamma production in response to non-NKSF/IL-12-inducers such as IL-2, phytohemagglutinin, and anti-CD3 antibodies. These results indicate that induced or constitutively produced NKSF/IL-12 has a major role in facilitating IFN-gamma production by peripheral blood lymphocytes.(ABSTRACT TRUNCATED AT 400 WORDS)
Transgenic mice homozygous for a beta 2-microglobulin (beta 2-m) gene disruption and normal mice that had been treated with a CD8-specific mAb were infected intranasally with an H3N2 influenza A virus. Both groups of CD8T cell-deficient mice eliminated the virus from the infected respiratory tract. Potent CTL activity was detected in lung lavage populations taken from mice with intact CD8+ T cell function, with minimal levels of cytotoxicity being found for inflammatory cells obtained from the antibody-treated and beta 2-m mutant mice. We therefore conclude that cells infected with an influenza A virus can be cleared from the respiratory tract of mice lacking both functional class I major histocompatibility complex (MHC) glycoproteins and class I MHC-restricted, CD8+ effector T cells.
Influenza A virus-specific cytotoxic T lymphocytes (CTL) capable of lysing cells infected with any influenza A virus ("cross-reactive CTL") constitute a major portion of the host CTL response to influenza. The viral nucleoprotein (NP), a major internal virion structural protein, has been implicated as a possible target antigen for cross-reactive CTL. To directly examine CTL recognition of NP, a vaccinia virus recombinant containing a DNA copy of an influenza A virus NP gene was constructed. We found that murine cells infected with this virus were efficiently lysed in a major histocompatibility complex-restricted manner by cross-reactive CTL populations obtained by immunization with a variety of influenza A virus subtypes. In addition, the recombinant vaccinia virus containing the PR8 NP gene was able to both stimulate and prime for a vigorous secondary cross-reactive CTL response. Significantly, splenocytes from mice primed by inoculation with the recombinant vaccinia virus containing the PR8 NP gene could be stimulated by influenza A viruses of all three major human subtypes. Finally, unlabeled target competition experiments suggest that NP is a major, but not the sole, viral target antigen recognized by cross-reactive CTL.
Cloned lines of murine cytotoxic T lymphocytes (CTL) directed to type A influenza virus confer complete protection upon adoptive transfer to syngeneic mice lethally infected by influenza virus. The exquisite specificity exhibited by a subtype-specific cloned CTL in culture is reflected in its capacity to eliminate pulmonary virus and mediate recovery only in those mice infected by the virus subtype recognized by this cloned line in vitro. A cross-reactive CTL cloned line protects mice infected by either of two influenza virus subtypes. In mice dually infected with two virus subtypes, the subtype-specific CTL clone only reduces lung virus levels of the recognized virus subtype and cannot prevent these mice from dying. In contrast, adoptive transfer of the cross-reactive CTL clone into mice simultaneously infected with two virus subtypes results in reduction of pulmonary titers of both subtypes and promotes complete recovery. These results directly implicate CTL as an important antiviral defense mechanism in experimental influenza infection. In addition, these results indicate that both the induction and expression of antiviral effector activity by CTL in vivo is highly specific and therefore favor the concept that CTL express their antiviral effect in vivo by direct cytolysis of infected cells.
We tested two biological properties of a continuously growing mouse cytotoxic T cell line, L4, which is specific for influenza A virus and has been cloned and recloned many times. We previously reported that L4 cells are H-2 restricted and cross-reactive for all type A influenza viruses, whereas they do not recognize type B influenza viruses. They bear Thy-1 and Lyt-2 markers. In the present study, we show that L4 cytotoxic T cells protect mice against a lethal influenza infection on transfer to syngeneic recipients, and reduce virus titers in the lungs of mice challenged with a heterologous type A influenza virus. This provides further support for the active role of cytotoxic T cells in limiting virus replication in influenza infection. We could also demonstrate that the cloned cytotoxic T cells induce a delayed-type hypersensitivity skin reaction in the footpads of mice challenged with live or inactivated influenza virus. This reaction can be observed at 24 h, but has declined by 48 h. A clone of cells derived from L4 that has lost its cytotoxic potential and its ability to recognize infected cells did not induce a delayed-type hypersensitivity reaction in the presence of virus. Thus, cytotoxic T cells actively killing influenza virus-infected cells are able to induce a delayed-type hypersensitivity skin reaction to homologous and heterologous type A influenza viruses.
The nucleoprotein (NP) of influenza A virus is the dominant antigen recognized by influenza virus-specific cytotoxic T lymphocytes (CTLs), and adoptive transfer of NP-specific CTLs protects mice from influenza A virus infection. BALB/c mouse cells (H-2d) recognize a single Kd-restricted CTL epitope of NP consisting of amino acids 147 to 155. In the present study, mice were immunized with various vaccinia virus recombinant viruses to examine the effect of the induction of primary pulmonary CTLs on resistance to challenge with influenza A/Puerto Rico/8/34 virus. The minigene ESNP(147-155)-VAC construct, composed of a signal sequence from the adenovirus E3/19K glycoprotein (designated ES) and expressing the 9-amino-acid NP natural determinant (amino acids 147 to 155) preceded by an alanine residue, a similar minigene NP(Met 147-155)-VAC lacking ES, and a full-length NP-VAC recombinant of influenza virus were analyzed. The two minigene NP-VAC recombinants induced a greater primary pulmonary CTL response than the full-length NP-VAC recombinant. However, NP-specific CTLs induced by immunization with ESNP(147-155)-VAC did not decrease peak virus titer or accelerate clearance of virus in the lungs of mice challenged intranasally with A/PR/8/34. Furthermore, NP-specific CTLs induced by immunization did not protect mice challenged intranasally with a lethal dose of A/PR/8/34. Sequence analysis of the NP CTL epitope of A/PR/8/34 challenge virus obtained from lungs after 8 days of replication in ESNP(147-155)-VAC-immunized mice showed identity with that of the input virus, demonstrating that an escape mutant had not emerged during replication in vivo. Thus, in contrast to adoptively transferred CTLs, pulmonary NP-specific CTLs induced by recombinant vaccinia virus immunization do not have protective in vivo antiviral activity against influenza virus infection.
Immunity that is cross-protective between different influenza A virus subtypes (termed heterosubtypic immunity) can be demonstrated readily in some animals but only rarely in humans. Induction of heterosubtypic immunity in humans by vaccines would provide public health benefit, perhaps offering some protection against pandemics or other new influenza A strains. Therefore, we studied mechanisms mediating heterosubtypic immunity in mice. Immunization with either A/H1N1 or A/H3N2 virus protected mice against mortality following heterosubtypic challenge while providing modest reductions in lung virus titers. No cross-protection was seen with distantly related type B influenza virus. Depletion of CD4+ or CD8+ T cells or both around the time of challenge had no significant effect on survival, indicating that these cells are not required at the effector stage. beta2-microglobulin knockout mice could be protected readily against heterosubtypic challenge, confirming that class I-restricted T cells are not required. In beta2-microglobulin -/- mice, depletion of CD4+ T cells partially abrogated heterosubtypic immunity, showing that they play a role in these mice. Passive transfer of Abs to naive recipients protected against subsequent challenge with homologous but not heterosubtypic virus. Because a role for secretory Abs has been suggested, we studied dependence on the J chain, which is required for polymeric Ig receptor-mediated IgA transport. J chain knockout mice were readily protected by heterosubtypic immunity, indicating that polymeric Ig receptor-mediated transport is not required. Better understanding of heterosubtypic immunity should be valuable in analyzing new vaccines, including peptide and DNA vaccines, intended to induce broadly cross-reactive immunity.
We have previously shown that a pulmonary influenza virus infection in SCID mice can be cured by treatment with monoclonal antibodies (MAbs) specific for the viral transmembrane protein hemagglutinin (HA) but not for matrix 2. Since both types of MAbs react with infected cells but only the former neutralizes the virus, it appeared that passive MAbs cured by neutralization of progeny virus rather than reaction with infected host cells. To prove this, we selected a set of four HA-specific MAbs, all of the immunoglobulin G2a isotype, which reacted well with native HA expressed on infected cells yet differed greatly (>10,000-fold) in virus neutralization (VN) activity in vitro, apparently because of differences in antibody avidity and accessibility of the respective determinants on the HA of mature virions. Since the VN activities of these MAbs in vitro were differentially enhanced by serum components, we determined their prophylactic activities in vivo and used them as measures of their actual VN activities in vivo. The comparison of therapeutic and prophylactic activities indicated that these MAbs cured the infection to a greater extent by VN activity (which was greatly enhanced in vivo) and to a lesser extent by reaction with infected host cells. Neither complement- nor NK cell-dependent mechanisms were involved in the MAb-mediated virus clearance.
In the adaptive immune response to most viruses, both the cellular and humoral arms of the immune system play complementary roles in eliminating virus and virus-infected cells and in promoting recovery. To evaluate the relative contribution of CD4+ and CD8+ effector T lymphocytes in virus clearance and recovery, we have examined the host response to lethal type A influenza virus infection in B lymphocyte-deficient mice with a targeted disruption in the immunoglobulin mu heavy chain. Our results indicate that naive B cell-deficient mice have a 50- 100-fold greater susceptibility to lethal type A influenza virus infection than do wild type mice. However, after priming with sublethal doses of influenza, immune B cell-deficient animals show an enhanced resistance to lethal virus infection. This finding indicates that an antibody-independent immune-mediated antiviral mechanism accounts for the increased resistance to lethal virus challenge. To assess the contribution of influenza-specific CD4+ and CD8+ effector T cells in this process, defined clonal populations of influenza-specific CD4+ and CD8+ effector T cells were adoptively transferred into lethally infected B cell-deficient mice. Cloned CD8+ effectors efficiently promoted recovery from lethal infection, whereas cloned CD4+ T cells conferred only partial protection. These results suggest that memory T lymphocytes can act independently of a humoral immune response in order to confer resistance to influenza infection in immune individuals. The potential implications of these results for vaccination against human influenza infection are discussed.
We have investigated the mechanisms involved in the clearance of viral infection at the epithelium level by analyzing the activity of influenza virus-specific cytotoxic T lymphocytes (CTL) against virus-infected CMT-93 intestinal epithelial cells. Epithelial cells infected with live influenza virus effectively present viral antigens and were lysed by both homotypic and heterotypic influenza virus-specific CD8+ T cells. These results shed new light on the control of viral infection through the elimination of virus-infected epithelial cells by virus-specific CTL and demonstrate that CMT-93 cells furnish an appropriate model for in vitro evaluation of CTL activity against virus-infected epithelial cells.
Heterosubtypic immunity (HSI) is defined as cross-protection against influenza virus of a different serotype than the virus initially encountered and is thought to be mediated by influenza virus-specific cytotoxic T lymphocytes (CTL). Since gamma interferon (IFN-gamma) stimulates cytotoxic cells, including antigen-specific CTL which may control virus replication by secretion of antiviral cytokines such as tumor necrosis factor alpha and IFN-gamma, we have investigated the mechanism of HSI by analyzing the role of IFN-gamma for HSI in IFN-gamma gene-deleted (IFN-gamma(-/-)) mice. It has been reported that IFN-gamma is not required for recovery from primary infection with influenza virus but is important for HSI. Here, we conclusively show that IFN-gamma is not required for induction of secondary influenza virus-specific CTL responses in mediastinal lymph nodes and HSI to lethal influenza A virus infection. Although T helper 2 (Th2)-type cytokines were upregulated in the lungs of IFN-gamma(-/-) mice after virus challenge, either Th1- or Th2-biased responses could provide heterosubtypic protection. Furthermore, titers of serum-neutralizing and cross-reactive antibodies to conserved nucleoprotein in IFN-gamma(-/-) mice did not differ significantly from those in immunocompetent mice. These results indicate that lack of IFN-gamma does not impair cross-reactive virus-specific immune responses and HSI to lethal infection with influenza virus. Our findings provide new insight for the mechanisms of HSI and should be valuable in the development of protective mucosal vaccines against variant virus strains, such as influenza and human immunodeficiency virus.
Transgenic mice homozygous for a beta 2-microglobulin (beta 2-m) gene disruption and normal mice that had been treated with a CD8-specific mAb were infected intranasally with an H3N2 influenza A virus. Both groups of CD8T cell-deficient mice eliminated the virus from the infected respiratory tract. Potent CTL activity was detected in lung lavage populations taken from mice with intact CD8+ T cell function, with minimal levels of cytotoxicity being found for inflammatory cells obtained from the antibody-treated and beta 2-m mutant mice. We therefore conclude that cells infected with an influenza A virus can be cleared from the respiratory tract of mice lacking both functional class I major histocompatibility complex (MHC) glycoproteins and class I MHC-restricted, CD8+ effector T cells.
Cloned lines of murine cytotoxic T lymphocytes (CTL) directed to type A influenza virus confer complete protection upon adoptive transfer to syngeneic mice lethally infected by influenza virus. The exquisite specificity exhibited by a subtype-specific cloned CTL in culture is reflected in its capacity to eliminate pulmonary virus and mediate recovery only in those mice infected by the virus subtype recognized by this cloned line in vitro. A cross-reactive CTL cloned line protects mice infected by either of two influenza virus subtypes. In mice dually infected with two virus subtypes, the subtype-specific CTL clone only reduces lung virus levels of the recognized virus subtype and cannot prevent these mice from dying. In contrast, adoptive transfer of the cross-reactive CTL clone into mice simultaneously infected with two virus subtypes results in reduction of pulmonary titers of both subtypes and promotes complete recovery. These results directly implicate CTL as an important antiviral defense mechanism in experimental influenza infection. In addition, these results indicate that both the induction and expression of antiviral effector activity by CTL in vivo is highly specific and therefore favor the concept that CTL express their antiviral effect in vivo by direct cytolysis of infected cells.
Immunity that cross-reacts between influenza type A viruses of distinct subtypes is called hetero(sub)typic (Het-I). We have studied Het-I by challenging PR8-immune mice with the heterosubtypic virus X31. Het-I did not prevent infection by X31 but, at its height, strongly aided in recovery. The nature of the effector mechanisms involved was investigated by simultaneous challenge with X31 and an immunologically unrelated influenza type B virus and by depleting individual lymphocyte subsets in PR8-immune mice before challenge. The study showed the following: 1) The effector mechanisms were intimately associated with immune recognition events. 2) In the nose, depletion of CD8+ or CD4+ T cells led to partial reduction of Het-I, and simultaneous depletion of both T cell subsets abrogated Het-I almost completely. This T cell-mediated immunity was short lived and had disappeared 4 to 5 mo after induction. 3) In trachea and lung, depletion of CD8+ T cells led to a partial reduction of Het-I, whereas depletion of CD4+ T cells was without significant effect. The CD8-mediated component appeared short lived, whereas the residual immunity (in CD4/8-depleted mice) was long lived and persisted past 7 mos after induction. 4) Depletion of NK cells did not significantly reduce the strength of Het-I in either nose or lung. In conclusion, the study shows that Het-I in this system is mediated by a complex combination of immune mechanisms that differ, in part, between upper and lower respiratory tract.
Natural Killer cell Stimulatory Factor (NKSF) or interleukin-12 (IL-12) is a heterodimeric cytokine of 70 kDa formed by a heavy chain of 40 kDa (p40) and a light chain of 35 kDa (p35). Although it was originally identified and purified from the supernatant of Epstein-Barr virus-transformed B cell lines, it has been shown that among peripheral blood cells is predominantly produced by monocytes, with lower production by B cells and other accessory cells. The most powerful inducers of production are bacteria, bacterial products and parasites. In addition to the biologically active p70 heterodimer, the cells producing also secrete a large excess of monomeric p40, a molecule with no demonstrable biological activity. is active on T lymphocytes and NK cells on which it induces production of lymphokines, enhancement of cytotoxic activity and mitogenic effects. induces T and NK cells to produce IFN-γ and synergizes with other IFN-γ inducers in this effect. In vitro, and probably in vivo, is required for optimal IFN-γ production. When human lymphocytes are stimulated with antigens in vitro, addition of exogenous to the culture induces differentiation of T helper type 1 (Th1) cells, whereas neutralization of endogenous with antibodies favors differentiation of Th2 cells. IFN-γ, a product of Th1 cells, enhances production by mononuclear cells, whereas IL-10 and IL-4, products of Th2 cells, efficiently inhibit it. Therefore, appears to be an important inducer of Th1 responses produced by accessory cells during early antigenic stimulation and its production is regulated by a positive feedback mechanism mediated by Th1 cells through IFN-γ and a negative one by Th2 cells through IL-10 and IL-4. The balance of IL-12 production versus IL-10 and IL-4 production early during an immune response might therefore be instrumental in determining Th1-type versus Th2-type immune responses. Because of this potential role of IL-12 during immune responses, our results demonstrating the impaired ability of HIV seropositive patients to produce in response to bacterial stimulation suggest that this defect in production might be a factor contributing to their immune depression.
There is considerable interest in developing viral vaccines intended to induce T cell immunity, especially cytotoxic CD8+ T lymphocytes, when Abs are not protective or are too narrow in viral strain specificity. We have studied protective immunity in doubly inactivated (DI) mice devoid of Abs and mature B cells. When infected with influenza B virus, these mice cleared the virus in a process dependent upon CD8+ T lymphocytes. Cytotoxic activity was detected in lung lymphocytes of DI mice after primary or secondary infection, and was abrogated by depletion of CD8+ cells in vivo. Challenge experiments showed that DI mice could be protected by immunization against reinfection 1 mo later, and protection was virus specific. Depletion of CD4+ or CD8+ T cells in vivo during the challenge period partially abrogated, and depletion of both subsets completely abrogated, the protection. This indicates that both CD4+ and CD8+ T cells are required effectors in the optimal control of virus replication. Thus, when Abs fail to protect against varying challenge viruses, as is the case with variant strains of influenza and HIV, there is hope that T cells might be able to act alone.
Immune spleen cells enhanced for influenza-specific cytotoxic activity after exposure to virus-infected stimulator cells in vitro effect recovery when transferred to nude and immunocompetent mice with influenza pneumonia (5). This protective effect correlated with the virus-specific cytotoxic activity of the transferred lymphocytes and is removed by treatment with anti-0 serum and complement. The experiments presented here indicate that spleen cells taken directly from mice undergoing a primary or secondary infection are less protective than immune spleen cells that are restimulated in vitro before transfer. This decreased ability to clear pulmonary virus and effect survival correlated with their relatively lower levels of influenza-specific cytotoxicity. Protection did not correlate with the level of natural killer cell activity of transferred cells. The results also indicate the immune spleen cells that are protective are influenza A subtype cross-reactive and are H-2-restricted; H-2d immune spleen cells effected recovery of H-2d but not H-2k challenged mice.
THERE is remarkably little information about the possible importance of
cell-mediated immune responses in the protection of hosts during
influenza virus infection. It has recently been found that specific
cytotoxic T cells (Tc) can be recovered from the spleens of
mice previously inoculated intranasally or intravenously with live
virus1-4 and peak activity occurs about 6 d after virus
inoculation. Furthermore, Tc are found in the lungs and the
lymph nodes draining the lungs of infected mice in conditions which
suggest that these cells might be important in host recovery from this
infection5. Adoptive transfer of primary or secondary immune
spleen cells to mice inoculated intranasally with a lethal dose of A/WSN
virus caused a significant reduction of infectious virus levels in the
lungs and prevented death6. The active cells in the
transferred population were T lymphocytes. This report shows that the
protective effects conferred by the transferred T cells in these
conditions are largely, if not entirely, due to Tc.
Cytotoxic T cells are present in the lungs and the bronchoalveolar washings of mice infected intravenously (i.v.) or intranasally (i.n.) with live influenza A/WSN virus. After i.v. injection, cytotoxic T cell activity in both spleens and lungs reaches a peak at 6 days when the level of infectious virus recovered from the lungs falls sharply and the mice do not die. If a lethal dose of virus is given intranasally, very high levels of virus appear rapidly in the lungs, and the development of lung consolidation follows slightly behind the appearance of cytotoxic T cells there. When a non-lethal dose of virus is given intranasally, lower levels of virus are found in the lung and the appearance of cytotoxic T cells is delayed. These results suggest that the cytotoxic T cells play a protective role if the level of virus in the lungs does not reach very high levels. After injection of antithymocyte serum, the subsequent level of cytotoxic T cell activity in the lungs was greatly reduced, suggesting that the T cells recovered in lungs had at an earlier stage been circulating cells. However, splenectomized mice develop high levels of cytotoxic T cell activity, after intranasal infection of mice, indicating that the spleen did not contribute substantially to the T cells recovered in the lungs.
Intranasal exposure of athymic (nu/nu) BALB/c mice to influenza virus leads to a persistent infection of the respiratory tract from which the mice die, usually within 3 to 4 wk with symptoms of general cachexia. However, if these nude mice were injected 1 day after infection, with approximately 10(6) cells from individual virus-specific MHC class II-restricted Th cell clones, they showed greatly reduced mortality and the titers of infectious virus in their lungs were reduced, often to undetectable levels. By coinfecting mice with pairs of antigenically distinct viruses and subsequently determining the extent of clearance of each type of virus, it could be shown first that the clearance mechanism was immunologically specific but did not display the typical crossreaction of class I-restricted cytotoxic T (Tc) cells. In addition, neither primary nor memory Tc responses could be detected in these mice. Second, Th cell clones promoted clearance solely of those viruses that contained the specific Th cell determinant, i.e., Th cell-nonreactive bystander viruses were not cleared. These findings were compatible with virus clearance being effected either directly after recognition of infected class II-positive cells by the transferred Th cells or indirectly via promotion of a glycoprotein-specific antibody response. The latter seems to be the case because transfer of Th cells into infected T and B cell-deficient SCID mice did not result in virus clearance, although transfer of an anti-hemagglutinin antibody cocktail did. Thus, a virus-specific Tc cell response is not a requirement for recovery from a pulmonary influenza virus infection.
A mutant mouse strain without CD8 (Lyt-2 and Lyt-3) expression on the cell surface has been generated by disrupting the Lyt-2 gene using embryonic stem cell technology. In these mice, CD8+ T lymphocytes are not present in peripheral lymphoid organs, but the CD4+ T lymphocyte population seems to be unaltered. Cytotoxic response of T lymphocytes from these mice against alloantigens and viral antigens is dramatically decreased. Proliferative response against alloantigens and in vivo help to B lymphocytes, however, are not affected. These data suggest that CD8 is necessary for the maturation and positive selection of class I MHC restricted cytotoxic T lymphocytes but is not required on any of the intermediate thymocyte populations (CD8+CD4-TcR- or CD4+CD8+TcRlow) during the development of functional class II MHC restricted helper T cells.
Of the various classes of antibodies that B lymphocytes can produce, class M (IgM) is the first to be expressed on the membrane of the developing cells. Pre-B cells, the precursors of B-lymphocytes, produce the heavy chain of IgM (mu chain), but not light chains. Recent data suggest that pre-B cells express mu chains on the membrane together with the 'surrogate' light chains lambda 5 and V pre B (refs 2-7). This complex could control pre-B-cell differentiation, in particular the rearrangement of the light-chain genes. We have now assessed the importance of the membrane form of the mu chain in B-cell development by generating mice lacking this chain. We disrupted one of the membrane exons of the gene encoding the mu-chain constant region by gene targeting in mouse embryonic stem cells. From these cells we derived mice heterozygous or homozygous for the mutation. B-cell development in the heterozygous mice seemed to be normal, but in homozygous animals B cells were absent, their development already being arrested at the stage of pre-B-cell maturation.
Influenza nucleoprotein (NP) serves as a target antigen on abortively infected cells for cytotoxic T cells (Tc) cross-reactive for all type A influenza viruses, and it can also prime mice for such Tc. It is important to test the protective ability of NP-specific Tc clones in vivo in a productive influenza infection. In this report, we show that Tc clones of this antigenic specificity protect mice against a lethal influenza infection on transfer to syngeneic recipients, and also that they reduce virus titres in the lungs and trachea of mice challenged with homologous or heterologous type A influenza viruses. Simultaneous injection of IL-2 to maintain the viability of the Tc clones is not essential, but has made the clonal transfer experiments highly reproducible.
Influenza nucleoprotein (NP) is an important target antigen for influenza A virus cross-reactive cytotoxic T cells (Tc). Here we examine the NP epitope recognized by cloned and polyclonal BALB/c Tc and the genetics of this recognition pattern. We can define NP residues 147–161 as the epitope seen in conjunction with K
d
, the only H-2d class I responder allele for NP restriction. H-2
d
/H-2
b
F1 mice (C57BL × DBA/2) primed by influenza infection lyse only H-2d target cells treated with peptide 147–161 while H-2b targets are recognized only after treatment with NP residues 365–379 (previously found to be recognized by Db restricted Tc cells). Tc cell recognition of NP peptide 147–161 is entirely dictated by expression of K
d
and not by other B10 or OH background genes of congenic mice. Restriction of a unique NP sequence by each responder class I major histocompatibility complex (MHC) allele suggests that antigen and class I MHC interact for Tc recognition.
The lymphokine IFN-gamma has been shown in vitro to stimulate IgG2a secretion and inhibit IgG1 and IgE secretion by LPS-activated B lymphocytes. To determine whether IFN-gamma has a similar isotype regulatory role in vivo, we studied the abilities of rIFN-gamma and a mAb to IFN-gamma to modify the isotypes of Ig secreted in mice injected with a goat antibody to mouse IgD, which by itself induces large increases in levels of serum IgG1 and IgE and a relatively small increase in serum IgG2a. Multiple injections of IFN-gamma substantially inhibited production of IgG1 and IgE, and stimulated production of IgG2a in affinity purified goat antibody specific for mouse IgD-treated mice; anti-IFN-gamma antibody blocked the effects of IFN-gamma and in fact enhanced IgG1 and IgE secretion and inhibited the IgG2a response in these mice. The role of IFN-gamma in the selection of isotypes of Ig produced in response to injection of mice with the bacterium Brucella abortus (BA) was also studied, because killed, fixed BA are known to stimulate IFN secretion and a predominantly IgG2a antibody response. Anti-IFN-gamma antibody strongly suppressed IgG2a secretion and stimulated IgG1, but not IgE, secretion in BA-immunized mice. BA suppressed IgG1 and IgE secretion and enhanced IgG2a secretion in affinity purified goat antibody specific for mouse IgD-injected mice; treatment of these mice with anti-IFN-gamma antibody reversed the effects of BA on IgG1 and IgG2a secretion, but not the suppressive effect of BA on IgE secretion. These observations demonstrate that IFN-gamma has an important and perhaps unique physiologic role in the stimulation of IgG2a secretion and in the suppression of secretion of IgG1, whereas bacterial antigens can suppress IgE secretion by other mechanisms in addition to IFN-gamma secretion.
B cell stimulatory factor 1 (BSF-1) (IL-4) was shown to synergize with phorbol esters or with monoclonal anti-TCR antibody in stimulation of the development of CTL from small resting murine T cells. IL-2 also synergized with PMA in such differentiation but was less effective than BSF-1. The combination of these two lymphokines with PMA had the most potent effect on the development of CTL. BSF-1 plus PMA stimulated a significant increase in the intracellular content of N-benzyloxycarbonyl-L-lysine thiobenzylester esterase, a granule-associated biochemical marker, whereas IL-2 plus PMA was only marginally effective. Depletion of L3T4+ cells did not result in the abrogation of these effects. Lyt-2+ T cells that were incubated for 72 h with BSF-1 plus PMA accumulated N-benzyloxycarbonyl-L-lysine thiobenzylester esterase and secreted this intragranular marker after interaction with immobilized anti-T cell receptor mAb. These BSF-1/PMA-stimulated Lyt-2+, L3T4- T cells were also able to kill FcR positive target cells in a retargeting assay with a mAb to murine T3 Ag, providing evidence that BSF-1 plus PMA acted directly on precursors of cytotoxic T cells.
The growth and differentiation of cytolytic T lymphocytes (CTL) is regulated by soluble growth hormones, of which interleukin-2 (IL-2) is considered to be of prime importance. Here we report that the lymphokine B-cell stimulatory factor (BSF-1 or interleukin-4) also has profound effects on the generation of these functionally active T cells. In particular, BSF-1 acts as a potent helper factor for the generation of CTL in primary mixed leukocyte culture (MLC) and induces cytolytic activity in in vitro primed, MLC memory populations. Direct comparison of purified recombinant BSF-1 and IL-2 reveals BSF-1 to be the more potent CTL helper factor in primary MLC. Interestingly, the two lymphokines differed in that IL-2, but not BSF-1, induced a lytic population in cultures of unprimed cells without an overt antigenic stimulus. Collectively, our data provide a direct demonstration of a heretofore undefined mechanism by which CTL activation and amplification can occur.
L cells expressing either the A/NT/60/68 nucleoprotein or the A/PR/8/34 (H1) hemagglutinin by DNA mediated gene transfer were used to investigate recognition by influenza A specific cytotoxic T lymphocytes (CTL). A subpopulation of CTL that recognized the H1 hemagglutinin was detected in mice primed with either A/PR/8/34 (H1N1) or A/JAP/305/57 (H2N2) influenza viruses. However, neither CTL from mice primed with A/NT/60/68 (H3N2) nor the recombinant virus X31 (H3N2) showed any activity on L cells expressing H1. These results showed that the majority of fully crossreactive CTL do not recognize the hemagglutinin molecule. A comparison between nucleoprotein and hemagglutinin transfected L cells reveals the nucleoprotein as the major target for CTL that are crossreactive on the three pandemic strains of human influenza A virus.
Subunit and intact influenza A virus vaccines have been compared with infectious virus in a mouse model for their ability to induce memory for cross-reactive cytotoxic T cell responses and to protect mice from challenge with different subtypes of influenza A virus. There is an overall correlation between secondary cytotoxic T cell responses and cross-protection. The most long-lasting and successful cross-protection was observed after intranasal infection with influenza virus A/X31 (H3 N2) that replicates efficiently in mice and induces high levels of memory for cross-reactive cytotoxic T cell responses. Short-lasting cross-protection and low levels of T cell-mediated cytotoxicity were associated with infection by A/USSR (H1 N1) virus, that replicates to lower titers in mice, or after multiple injections of inactivated whole virus vaccine. No cross-protection to challenge with heterologous influenza virus was detectable after 1-2 injections of HANA influenza subunit vaccine which failed to prime hosts for cytotoxic T cell responses. These findings may have important implications for vaccination strategy. If cytotoxic T cells play a role in the protection of humans from influenza, live attenuated vaccines should be considered instead of the currently recommended inactivated virus or subunit vaccines.
Effective host defense against bacterial infection is dependent upon the vigorous recruitment and activation of neutrophils and macrophages. We hypothesized that IL-10 is produced in the setting of bacterial pneumonia, and this cytokine may attenuate host defense by inhibiting the expression of important activating and chemotactic cytokines. CD-1 mice were challenged with either 30 microliters of saline or saline containing 10(3) CFUs of Klebsiella pneumoniae intratracheally (i.t.) and lungs were harvested at 8, 24, and 48 h. The i.t. inoculation with K. pneumoniae resulted in a 13-, 14-, and 8-fold increase in lung homogenate TNF, macrophage inflammatory protein-2 (MIP-2), and macrophage inflammatory protein-1 alpha (MIP-1 alpha) levels, respectively, as compared with control animals. In addition, we observed an increase in IL-10 mRNA and protein levels in lung homogenates, maximal at 48 h postinoculation. To establish the biologic relevance of IL-10 in Klebsiella pneumonia, we passively immunized CD-1 mice with 0.5 ml of rabbit anti-murine IL-10 serum or preimmune serum i.p. 2 h before i.t. administration of K. pneumoniae. Treatment of animals with anti-IL-10 serum resulted in increased levels of TNF, MIP-2, and MIP-1 alpha, respectively, within lung homogenates at 24 and 48 h, as compared with preimmune-treated animals. Furthermore, neutralization of IL-10 resulted in a significant decrease in K. pneumoniae CFU in both lung homogenates and plasma harvested at 48 h, as well as a significant increase in survival in these animals. Our studies indicate that 1) IL-10 is produced during Klebsiella pneumonia; and 2) inhibition of IL-10 bioactivity in vivo results in enhanced bacterial clearance, increased expression of proinflammatory cytokines, and prolonged survival.
Mice transgenic for p2-micrQglobulin deletion (β2 M-/-) were immunized intranasally with either a recombinant vaccinia virus that expressed both nucleoprotein and interleukin-2
or by infection with H3N2 influenza virus; 3–4 weeks later they were challenged with H1N1 influenza virus. The immunized β2 M-/- mice had increased survival and enhanced clearance of virus relative to nonimmune controls. This protection correlated
with the development of class II major histocompatibility complex-restricted pulmonary cytotoxic T lymphocyte activity and
nasal IgA anti-nucleoprotein antibody. Heterotypic immunity can therefore be generated by a mechanism that does not involve
class I major histocompatibility complex-restricted T cells.
Immunity that cross-reacts between influenza type A viruses of distinct subtypes is called hetero(sub)typic (Het-I). We have studied Het-I by challenging PR8-immune mice with the heterosubtypic virus X31. Het-I did not prevent infection by X31 but, at its height, strongly aided in recovery. The nature of the effector mechanisms involved was investigated by simultaneous challenge with X31 and an immunologically unrelated influenza type B virus and by depleting individual lymphocyte subsets in PR8-immune mice before challenge. The study showed the following: 1) The effector mechanisms were intimately associated with immune recognition events. 2) In the nose, depletion of CD8+ or CD4+ T cells led to partial reduction of Het-I, and simultaneous depletion of both T cell subsets abrogated Het-I almost completely. This T cell-mediated immunity was short lived and had disappeared 4 to 5 mo after induction. 3) In trachea and lung, depletion of CD8+ T cells led to a partial reduction of Het-I, whereas depletion of CD4+ T cells was without significant effect. The CD8-mediated component appeared short lived, whereas the residual immunity (in CD4/8-depleted mice) was long lived and persisted past 7 mos after induction. 4) Depletion of NK cells did not significantly reduce the strength of Het-I in either nose or lung. In conclusion, the study shows that Het-I in this system is mediated by a complex combination of immune mechanisms that differ, in part, between upper and lower respiratory tract.
The role of B lymphocytes and their Ig product in the development and maintenance of virus-specific CD4+ T cells has been analyzed in mice homozygous for disruption of the Ig mu gene (mu MT). These mice lack mature B220+ B cells and do not secrete Ig, but generate normal CD8+ cytotoxic T lymphocyte responses and have no difficulty clearing the HKx31 influenza A virus from the infected respiratory tract. Sequential limiting dilution analysis of virus-specific CD4+ T cells established that the frequencies of IL-2-producing T helper cell precursors in the draining lymph nodes and/or spleen from 7 days to 6 mo after infection were essentially similar in mu MT and C57BL/6 (B6) mice. Ag presentation and processing mechanisms involving Ig or B cells are apparently not required to generate virus-specific T helper cell precursors, and Ag-Ig complexes on follicular dendritic cells are not essential for the persistence of virus-specific CD4+ T cell memory. The main difference was that the spleens of the mu MT mice were much smaller than those of the B6 controls, and greater numbers of CD4+ T cells were found consistently in the regional mediastinal lymph nodes. This could be the result of abnormal expression of the lymph node homing receptor (CD62L) on the mu MT CD4+ T cells. However, the profiles of CD62L expression over the long term were comparable for both total and virus-specific CD4+ T cells from the two groups. The diminished role of the mu MT spleen is thus more likely to reflect the absence of germinal centers and/or Ig rather than a disruption of CD62L-mediated T cell trafficking.
Replication-deficient adenovirus (Ad) vectors are effective to specifically target the respiratory epithelium for either corrective gene therapy such as cystic fibrosis or for mucosal immunization. As a consequence of transducing the lower respiratory tract with an E1/E3 deleted Ad5 vector, host responses have been characterized by the duration of transgene expression and by the induction of CTL responses. However, limited emphasis has been devoted to understanding the contribution of CD4+ T cell responses to the Ad vector. Both CD4+ and CD8+ T cells migrate into the lung following sequential intratracheal Ad5 transgene instillations. Isolated CD3+ T lymphocytes from the lungs were predominantly of the Th2 type, and after cell sorting, the IL-4-producing T cells were largely CD4+, while IFN-gamma expression was associated with both CD4+ and CD8+ T cells. Ab responses to the Ad5 vector and to the expressed transgene beta-galactosidase (beta gal) revealed elevated bronchial and serum IgA and IgG Abs with low neutralization titers. Analysis of serum IgG subclass responses showed IgG1 and IgG2b with lower IgG2a Abs to Ad5 and IgG2a and IgG2b Ab responses to beta gal. Ad5-specifc CD4+ T cells produced both Th1 (IFN-gamma and IL-2)- and Th2 (IL-4, IL-5, IL-6)-type cytokines, while beta gal-specific CD4+ T cells secreted IFN-gamma and IL-6. This study provides direct evidence for the concomitant induction of Th2- with Th1-type responses in both the pulmonary systemic and mucosal immune compartments to the Ad5 vector as well as a Th1-dominant response to the transgene.
Recovery from influenza virus infection has long been known to require an intact T-cell compartment. More recent studies revealed that CD8 and CD4 T cells can promote recovery through independent mechanisms. The CD4 T-cell-dependent recovery process appears to operate primarily through promotion of the T-dependent antibody response as B-cell-deficient microMT mice cannot recover from infection if they have been depleted of CD8 T cells. The potential therapeutic activity of the B-cell response was further studied by transfer of antibodies into infected SCID mice. At the dose of 200 micrograms/mouse, most antibodies (of IgG2a isotype) to the viral transmembrane protein HA cured the infection, while those to the transmembrane proteins NA and M2 suppressed virus titers in the lung but failed to clear the infection. The ability of passive antibody to resolve the infection was closely related to its prophylactic activity, suggesting that neutralization of progeny virus (VN) played an important role in the process of virus clearance in vivo, while reaction of antibodies with infected host cells contributed to but was insufficient, on its own, for cure. HA-specific antibodies of IgM and IgA isotypes were therapeutically ineffective against pulmonary infection, presumably because of a preferential delivery into the upper respiratory tract, while IgG exhibited highest activity against pulmonary and minimal activity against nasal infection. B cells appear to be of similar importance for recovery from primary infection as CD8 T cells.
The primary CD8+ T-cell response protected most B-cell-deficient muMT mice against intranasal infection with the HKx31 influenza A virus. Prior exposure did not prevent reinfection upon homologous challenge, and the recall CD8+ T-cell response cleared the virus from the lung within 7 days. Depleting the CD8+ T cells substantially reduced the capacity of these primed mice to deal with the infection, in spite of evidence for established CD4+ T-cell memory. Thus, the control of this relatively mild influenza virus by both primary and secondary CD4+ T-cell responses is relatively inefficient in the absence of B cells and CD8+ T cells.
Recovery from influenza virus infection is dependent on T cell functions which can be provided either by CD8 or CD4 T cells. To identity the functions involved in recovery promoted by CD4 T cells, we have studied the course of the infection in B-cell deficient micro MT mice which had been depleted of CD8 T cells by antibody treatment. Upon infection with PR8 [A/PR/8/34(H1N1)], such B- and CD8 T cell-deficient mice mounted strong CD4 T cell responses that were comparable in size and cytokine secretion to those seen in intact mice. Yet, these B- and CD8 T cell-deficient mice could not clear the infection, in contrast to (CD8-depleted) mice containing both B- and CD4 T cells. These findings indicate that the promotion of the T-dependent antibody response is an indispensable component in the CD4 T cell-dependent recovery process.
Antibodies (Abs) can contribute to the cure of a viral infection, in principle, in two ways by: (1) binding to infected cells and thereby reducing the production of progeny virus [here termed cell-targeting (CT) activity] and (2) reacting with released progeny virus and thereby inhibiting the spread of the infection [termed virus neutralizing (VN) activity]. We have previously shown that a pulmonary influenza virus infection in severe combined immunodeficient mice could be cured by treatment of these mice with hemagglutinin (HA)-specific monoclonal Abs (mAbs) that mediated both of the above activities. Although the therapeutic activity of these mAbs correlated with their VN activity, it remained unclear how much their CT activity contributed to the Ab-mediated recovery process. To clarify this point, we tested the therapeutic efficacy of two mAbs of IgG2a isotype that mediated CT but no VN activity: one specific for the viral neuraminidase and the other for matrix protein 2. Both mAbs reduced pulmonary virus titers by 100- to 1000-fold but they failed to clear the infection, even when administered in combination and at therapeutically saturating concentrations. The results suggest that CT activity contributes significantly also to the therapeutic activity of HA-specific mAbs and further support the notion that VN-activity is required for Ab-mediated virus clearance.
Heterosubtypic immunity, defined as cross-reactive immune responses to influenza virus of a different serotype than the virus initially encountered, was investigated in association with virus-specific cytotoxic T lymphocyte (CTL) responses induced in systemic and mucosa-associated lymph nodes after immunization via different routes. Mice immunized by the pulmonary route with live nonpathogenic influenza virus, strain Udorn (H3N2), survived challenge with mouse-adapted pathogenic influenza virus, strain PR/8/34 (H1N1). These mice developed strong heterosubtypic CTL responses in spleen, cervical lymph nodes (CLN), and mediastinal lymph nodes (MLN). Alternately, only 20% of mice immunized intravenously, intraperitoneally, or intranasally survived the challenge; all of these developed CTL responses in spleen and CLN, but not in MLN. Direct correlation between short-term and long-term memory heterosubtypic CTL responses induced in MLN and host recovery after lethal infection indicates that these CTL responses may play an important role in heterosubtypic immunity. Furthermore, induction and maintenance of memory CTL in regional mucosa-associated lymphoid tissues are highly dependent on mucosal immunization. The results implicate the mechanism of heterosubtypic immunity and should be an important consideration in the development of protective mucosal vaccines against variant strains of influenza and HIV.
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.
89
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.
Natural killer cell stimulatory factor (NKSF) or interleukin-12 is a key regulator of immune response and inflammation
- G Trinchieri
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