Protective effect of nasal immunization of influenza virus hemagglutinin with recombinant cholera toxin B subunit as a mucosal adjuvant in mice

To develop an efficient nasal influenza vaccine, influenza A and B virus HA with rCTB as a mucosal adjuvant were administered to mice intranasally. Serum anti-HA IgG and IgA antibody responses for both HA vaccines were significantly increased in the presence of rCTB. Higher HI and neutralizing antibody titers and higher mucosal IgA antibody responses in the respiratory tract were detected when rCTB was added than without rCTB. When mice were immunized with HA vaccine with or without rCTB and challenged by intranasal administration of mouse-adapted pathogenic influenza A virus, all mice immunized with HA plus rCTB survived for seven days without any inflammatory changes in the lungs, while not all the mice immunized with HA without rCTB survived, and all of them had lung consolidations. These results demonstrate that intranasal co-administration of rCTB as a mucosal adjuvant with influenza virus HA is necessary not only for the induction of systemic and mucosal HA antibodies, but also for the protection of mice from morbidity and mortality resulting from virus infection.     

Modified pulmonary surfactant is a potent adjuvant that stimulates the mucosal IgA production in response to the influenza virus antigen

The intranasal administration of influenza hemagglutinin (HA) vaccine with Surfacten, a modified pulmonary surfactant free of antigenic c-type lectins, as a mucosal adjuvant induced the highest protective mucosal immunity in the airway. The intranasal immunization of mice with HA vaccine (0.2 microg)-Surfacten (0.2 microg) selectively induced the neutralizing anti-HA IgA, but not IgG, and conferred nearly maximal protection in the airway, without inducing a systemic response. In contrast, intranasal inoculation of vaccine with 0.2 microg of the potent mucosal adjuvant cholera toxin B* (CT-B*), prepared by adding 0.2% native CT to the B subunit of CT, induced both anti-HA IgA and IgG in the airway and in the serum. The intranasal administration of HA vaccine alone induced a limited amount of mucosal IgA against influenza virus. Although the s.c. administration of HA vaccine prominently induced serum IgG and IgA, Surfacten and CT-B* did not enhance their induction, and the concentrations of Abs leaking into the airways were insufficient to prevent viral multiplication. The intranasal administration of HA-Surfacten stimulated the expression of MHC class II, CD40, and CD86 molecules in the CD11c-positive cells isolated from the nasal mucosa, but not the expression of cells from the lungs or spleens. Lymphocytes isolated from the airway mucosa after intranasal HA-Surfacten immunization prominently induced TGF-beta1 which, compared with inoculation without Surfacten, promoted an Ag-specific mucosal IgA response. Surfacten alone, however, did not induce TGF-beta1. Our observations suggest that Surfacten, by mimicking the natural surfactant, is an effective mucosal adjuvant in the process of airway immunization.     

Modulation of glycosylation and transport of viral membrane glycoproteins by a sodium ionophore

Analysis of viral glycoprotein expression on surfaces of monensin- treated cells using a fluorescence-activated cell sorter (FACS) demonstrated that the sodium ionophore completely inhibited the appearance of the vesicular stomatitis virus (VSV) G protein on (Madin- Darby canine kidney) MDCK cell surfaces. In contrast, the expression of the influenza virus hemagglutinin (HA) glycoprotein on the surfaces of MDCK cells was observed to occur at high levels, and the time course of its appearance was not altered by the ionophore. Viral protein synthesis was not inhibited by monensin in either VSV- or influenza virus-infected cells. However, the electrophoretic mobilities of viral glycoproteins were altered, and analysis of pronase-derived glycopeptides by gel filtration indicated that the addition of sialic acid residues to the VSV G protein was impaired in monensin-treated cells. Reduced incorporation of fucose and galactose into influenza virus HA was observed in the presence of the ionophore, but the incompletely processed HA protein was cleaved, transported to the cell surface, and incorporated into budding virus particles. In contrast to the differential effects of monensin on VSV and influenza virus replication previously observed in monolayer cultures of MDCK cells, yields of both viruses were found to be significantly reduced by high concentrations of monensin in suspension cultures, indicating that cellular architecture may play a role in determining the sensitivity of virus replication to the drug. Nigericin, an ionophore that facilitates transport of potassium ions across membranes, blocked the replication of both influenza virus and VSV in MDCK cell monolayers, indicating that the ion specificity of ionophores influences their effect on the replication of enveloped viruses.     

Epithelial cell polarization is a determinant in the infectious outcome of immunoglobulin A-mediated entry by Epstein-Barr virus.

Diseases of the nasopharyngeal epithelium due to Epstein-Barr virus (EBV) infection typically occur in chronic virus carriers with preexisting virus-specific antibodies. In vitro studies have shown that EBV-specific immunoglobulin A (IgA) promotes infection of human epithelial cells, otherwise refractory to EBV, via the polymeric immunoglobulin receptor (pIgR). To determine if EBV similarly exploits IgA transport mechanisms in vivo, we examined the fate of IgA-EBV complexes in the blood of mice, where pIgR-mediated transcytosis of IgA immune complexes through hepatocytes eliminates exogenous antigens from the circulation. By PCR analysis we showed hepatobiliary transport of IgA-EBV in viremic mice, but without detectable hepatocellular infection by immunostaining. Because efficient transport of EBV immune complexes might avert an infectious outcome, we modulated the transcytotic pathway in polarized Madin-Darby canine kidney (MDCK) cells transfected with pIgR to determine the effect on viral antigen expression. Like hepatocytes in vivo, MDCK cells in polarized monolayers translocated IgA-EBV from the basal cell face into apical medium without evidence for infection. However, when exposed to IgA-EBV as unpolarized single-cell suspensions, MDCK cells expressed EBV immediate-early and early antigens. These results suggest that pIgR-mediated transcytosis of pIgA-EBV through epithelium facilitates endogenous spread of EBV in long-term virus carriers, with infection being confined to cells with altered polarity from prior cytopathology.     

Comparison of Antiviral Activity between IgA and IgG Specific to Influenza Virus Hemagglutinin: Increased Potential of IgA for 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.     

A viral vaccine vector that expresses foreign genes in lymph nodes and protects against mucosal challenge.

A candidate live-virus vaccine strain of Venezuelan equine encephalitis virus (VEE) was configured as a replication-competent vector for in vivo expression of heterologous immunogens. Three features of VEE recommend it for use as a vaccine vector. (i) Most human and animal populations are not already immune to VEE, so preexisting immunity to the vector would not limit expression of the heterologous antigen. (ii) VEE replicates first in local lymphoid tissue, a site favoring the induction of an effective immune response. (iii) Parenteral immunization of rodents and humans with live, attenuated VEE vaccines protects against mucosal challenge, suggesting that VEE vaccine vectors might be used successfully to protect against mucosal pathogens. Upon subcutaneous (s.c.) inoculation into the footpad of mice, a VEE vector containing the complete influenza virus hemagglutinin (HA) gene expressed HA in the draining lymph node and induced anti-HA immunoglobulin G (IgG) and IgA serum antibodies, the levels of which could be increased by s.c. booster inoculation. When immunized mice were challenged intranasally with a virulent strain of influenza virus, replication of challenge virus in their lungs was restricted, and they were completely protected from signs of disease. Significant reduction of influenza virus replication in the nasal epithelia of HA vector-immunized mice suggested an effective immunity at the mucosal surface. VEE vaccine vectors represent an alternative vaccination strategy when killed or subunit vaccines are ineffective or when the use of a live attenuated vaccine might be unsafe.     

Intraepithelial cell neutralization of HIV-1 replication by IgA

HIV is transmitted sexually through mucosal surfaces where IgA Abs are the first line of immune defense. In this study, we used paired IgA and IgG mAbs against HIV gp160 to study intraepithelial cell neutralization and inhibition of HIV replication. African green monkey kidney cells, Vero C1008, polarizable epithelial cells transfected to express the polymeric Ig receptor (pIgR), were transfected with HIV proviral DNA, and intracellular neutralization mediated by the mAbs was assessed. D47A and D19A IgA, which neutralized HIV in a conventional assay, potently inhibited intracellular HIV replication as assessed by infecting HeLa-CD4-long terminal repeat/beta-galactosidase cells (human cervical carcinoma cell line) and CEMx174 cells (human T cell line) with apical supernatant, basolateral medium, and cell lysate from transfected cells. D47A also inhibited the production of virus as assessed by direct assay of p24. In contrast, D47 and D19 IgG, sharing the same V regions, but which were not transcytosed by the pIgR, did not inhibit intracellular HIV replication, nor did D47A and D19A IgA in pIgR- cells, incapable of transcytosing IgA. Confocal immunofluorescence microscopy showed prominent colocalization of HIV protein and D47A, in agreement with the intracellular neutralization data. D10A, which did not neutralize HIV in the conventional assay, and irrelevant IgA did not show intracellular neutralization or colocalization. Control studies with two kinds of conditioned medium confirmed that HIV neutralization had indeed occurred inside the cells. Thus, during its transcytosis through epithelial cells, HIV-specific IgA can neutralize HIV replication.     

Human immune responses elicited by an intranasal inactivated H5 influenza vaccine

Intranasally administered influenza vaccines could be more effective than injected vaccines, because intranasal vaccination can induce virus-specific immunoglobulin A (IgA) antibodies in the upper respiratory tract, which is the initial site of infection. In this study, immune responses elicited by an intranasal inactivated vaccine of influenza A(H5N1) virus were evaluated in healthy individuals naive for influenza A(H5N1) virus. Three doses of intranasal inactivated whole-virion H5 influenza vaccine induced strong neutralizing nasal IgA and serum IgG antibodies. In addition, a mucoadhesive excipient, carboxy vinyl polymer, had a notable impact on the induction of nasal IgA antibody responses but not on serum IgG antibody responses. The nasal hemagglutinin (HA)-specific IgA antibody responses clearly correlated with mucosal neutralizing antibody responses, indicating that measurement of nasal HA-specific IgA titers could be used as a surrogate for the mucosal antibody response. Furthermore, increased numbers of plasma cells and vaccine antigen-specific Th cells in the peripheral blood were observed after vaccination, suggesting that peripheral blood biomarkers may also be used to evaluate the intranasal vaccine-induced immune response. However, peripheral blood immune cell responses correlated with neutralizing antibody titers in serum samples but not in nasal wash samples. Thus, analysis of the peripheral blood immune response could be a surrogate for the systemic immune response to intranasal vaccination but not for the mucosal immune response. The current study suggests the clinical potential of intranasal inactivated vaccines against influenza A(H5N1) viruses and highlights the need to develop novel means to evaluate intranasal vaccine-induced mucosal immune responses.     

Virus-neutralizing antibodies of immunoglobulin G (IgG) but not of IgM or IgA isotypes can cure influenza virus pneumonia in SCID mice.

The ability of monoclonal antibodies (MAbs) to passively cure an influenza virus pneumonia in the absence of endogenous T- and B-cell responses was investigated by treating C.B-17 mice, homozygous for the severe combined immunodeficiency (SCID) mutation, with individual monoclonal antiviral antibodies 1 day after pulmonary infection with influenza virus PR8 [A/PR/8/34 (H1N1)]. Less than 10% of untreated SCID mice survived the infection. By contrast, 100% of infected SCID mice that had been treated with a single intraperitoneal inoculation of at least 175 micrograms of a pool of virus-neutralizing (VN+) antihemagglutinin (anti-HA) MAbs survived, even if antibody treatment was delayed up to 7 days after infection. The use of individual MAbs showed that recovery could be achieved by VN+ anti-HA MAbs of the immunoglobulin G1 (IgG1), IgG2a, IgG2b, and IgG3 isotypes but not by VN+ anti-HA MAbs of the IgA and IgM isotypes, even if the latter were used in a chronic treatment protocol to compensate for their shorter half-lives in vivo. Both IgA and IgM, although ineffective therapeutically, protected against infection when given prophylactically, i.e., before exposure to virus. An Fc gamma-specific effector mechanism was not an absolute requirement for antibody-mediated recovery, as F(ab')2 preparations of IgGs could cure the disease, although with lesser efficacy, than intact IgG. An anti-M2 MAb of the IgG1 isotype, which was VN- but bound well to infected cells and inhibited virus growth in vitro, failed to cure. These observations are consistent with the idea that MAbs of the IgG isotype cure the disease by neutralizing all progeny virus until all productively infected host cells have died. VN+ MAbs of the IgA and IgM isotypes may be ineffective therapeutically because they do not have sufficient access to all tissue sites in which virus is produced during influenza virus pneumonia.     

Human intestinal epithelial cells express a novel receptor for IgA

Binding and transport of polymeric Igs (pIgA and IgM) across epithelia is mediated by the polymeric Ig receptor (pIgR), which is expressed on the basolateral surface of secretory epithelial cells. Although an Fc receptor for IgA (FcalphaR) has been identified on myeloid cells and some cultured mesangial cells, the expression of an FcalphaR on epithelial cells has not been described. In this study, binding of IgA to a human epithelial line, HT-29/19A, with features of differentiated colonic epithelial cells, was examined. Radiolabeled monomeric IgA (mIgA) showed a dose-dependent, saturable, and cation-independent binding to confluent monolayers of HT-29/19A cells. Excess of unlabeled mIgA, but not IgG or IgM, competed for the mIgA binding, indicating that the binding was IgA isotype-specific and was not mediated by the pIgR. The lack of competition by asialoorosomucoid and the lack of requirement for divalent cations excluded the possibility that IgA binding to HT-29/19A cells was due to the asialoglycoprotein receptor or beta-1, 4-galactosyltransferase, previously described on HT-29 cells. Moreover, the FcalphaR (CD89) protein and message were undetectable in HT-29/19A cells. FACS analysis of IgA binding demonstrated two discrete populations of HT-29/19 cells, which bound different amounts of mIgA. IgA binding to other colon carcinoma cell lines was also demonstrated by FACS analysis, suggesting that an IgA receptor, distinct from the pIgR, asialoglycoprotein receptor, galactosyltransferase, and CD89 is constitutively expressed on cultured human enterocytes. The function of this novel IgA receptor in mucosal immunity remains to be elucidated.     

Active synthesis of hemagglutinin-specific immunoglobulin A by lung cells of mice that were immunized intragastrically with inactivated influenza virus vaccine

Intragastric inoculation with whole-virion vaccine of inactivated influenza virus resulted in production of hemagglutinin (HA)-specific immunoglobulin A (IgA) and IgG both in lung lavage fluids and in serum samples of mice. HA-specific IgA was the predominant isotypic antibody secreted in the lung lavage fluids (average IgA/IgG ratio, 13:1), whereas HA-specific IgG was the major antibody class in serum (average IgA/IgG ratio, 0.3:1). These responses were similar to the antibody responses stimulated by intranasal infection with live influenza virus. In vitro cultures of lymphoid cells from lungs and Peyer's patches, but not from spleens, in the presence of homologous antigen, from mice vaccinated intragastrically synthesized mostly HA-specific IgA. Mice immunized parenterally with inactivated influenza virus produced only IgG in lung lavage fluids and sera. Cultures of lymphoid cells from their spleens, but not their lungs, synthesized HA-specific IgG upon antigenic stimulation in vitro; neither synthesized IgA. These in vitro cell culture results, as well as the inverse relationship of IgA/IgG ratios in lung lavage fluids and sera, demonstrated that the IgA antibody in lung lavage fluids was actively synthesized locally in the lungs of intragastrically immunized mice. This finding was consistent with the migratory distribution of antigen-primed lymphoid cells from Peyer's patches to distant lymphoid tissue such as lung. Intragastric vaccination conferred protection against intranasal challenge with a lethal dose of virulent virus.     

CK2beta gene silencing increases cell susceptibility to influenza A virus infection resulting in accelerated virus entry and higher viral protein content

Background

Influenza A virus (IVA) exploits diverse cellular gene products to support its replication in the host. The significance of the regulatory (β) subunit of casein kinase 2 (CK2β) in various cellular mechanisms is well established, but less is known about its potential role in IVA replication. We studied the role of CK2β in IVA-infected A549 human epithelial lung cells.

Results

Activation of CK2β was observed in A549 cells during virus binding and internalization but appeared to be constrained as replication began. We used small interfering RNAs (siRNAs) targeting CK2β mRNA to silence CK2β protein expression in A549 cells without affecting expression of the CK2α subunit. CK2β gene silencing led to increased virus titers, consistent with the inhibition of CK2β during IVA replication. Notably, virus titers increased significantly when CK2β siRNA-transfected cells were inoculated at a lower multiplicity of infection. Virus titers also increased in cells treated with a specific CK2 inhibitor but decreased in cells treated with a CK2β stimulator. CK2β absence did not impair nuclear export of viral ribonucleoprotein complexes (6 h and 8 h after inoculation) or viral polymerase activity (analyzed in a minigenome system). The enhancement of virus titers by CK2β siRNA reflects increased cell susceptibility to influenza virus infection resulting in accelerated virus entry and higher viral protein content.

Conclusion

This study demonstrates the role of cellular CK2β protein in the viral biology. Our results are the first to demonstrate a functional link between siRNA-mediated inhibition of the CK2β protein and regulation of influenza A virus replication in infected cells. Overall, the data suggest that expression and activation of CK2β inhibits influenza virus replication by regulating the virus entry process and viral protein synthesis.     

An RNA Aptamer That Specifically Binds to the Glycosylated Hemagglutinin of Avian Influenza Virus and Suppresses Viral Infection in Cells

The influenza virus surface glycoprotein hemagglutinin (HA) is responsible for viral attachment to sialic acid-containing host cell receptors and it facilitates the initial stage of viral infection. In the present study, we isolated an RNA aptamer specific to the glycosylated receptor-binding domain of the HA protein (gHA1) after 12 cycles of the systematic evolution of ligands by exponential enrichment procedure (SELEX), and we then investigated if the selected aptamer suppresses viral infection in host cells. Nitrocellulose filter binding and enzyme-linked immunosorbent assay (ELISA) experiments revealed that 1 RNA aptamer, HA12-16, bound specifically to the gHA1 protein. Cell viability assay showed that the HA12-16 RNA aptamer suppressed viral infection in host cells by enhancing cell viability. Immunofluorescence microscopic analysis further demonstrated that the HA12-16 RNA aptamer suppresses viral attachment to host cells by neutralizing the receptor-binding site of influenza virus HA. These results indicate that the isolated RNA aptamer can be developed as an antiviral reagent against influenza through appropriate therapeutic formulation.     

Influenza A virus neuraminidase protein enhances cell survival through interaction with carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6) protein

The influenza virus neuraminidase (NA) protein primarily aids in the release of progeny virions from infected cells. Here, we demonstrate a novel role for NA in enhancing host cell survival by activating the Src/Akt signaling axis via an interaction with carcinoembryonic antigen-related cell adhesion molecule 6/cluster of differentiation 66c (C6). NA/C6 interaction leads to increased tyrosyl phosphorylation of Src, FAK, Akt, GSK3β, and Bcl-2, which affects cell survival, proliferation, migration, differentiation, and apoptosis. siRNA-mediated suppression of C6 resulted in a down-regulation of activated Src, FAK, and Akt, increased apoptosis, and reduced expression of viral proteins and viral titers in influenza virus-infected human lung adenocarcinoma epithelial and normal human bronchial epithelial cells. These findings indicate that influenza NA not only aids in the release of progeny virions, but also cell survival during viral replication.     

甲型H1N1流感病毒血凝素HA1在糖基工程毕赤酵母中的表达

目的:建立用糖基工程酵母制备流感血凝素的方法 ,研究其免疫原性,为酵母表达流感疫苗提供基础。方法:通过PCR的方法扩增编码H1N1流感病毒血凝素HA1(1～330 aa)的基因片段,将HA1基因克隆到表达载体pPIC9质粒上,电转化到糖基工程酵母中,甲醇诱导表达并用镍亲和层析柱纯化重组蛋白,N-糖苷酶F(PNGF)酶切分析N-糖链,Western印迹验证纯化蛋白,免疫小鼠并测定HA1诱导抗体的滴度。结果:获得HA1基因的酵母重组表达菌株,SDS-PAGE分析可见野生型GS115表达的重组HA1相对分子质量约为100×103,而糖基工程酵母GJK01表达的HA1约为60×103,PNGF酶切后相对分子质量均降至45×103左右;经Western印迹检测,这些条带均为目的蛋白条带,野生型和糖基工程酵母表达的HA1分子大小不同是由于不同的N-糖基化修饰引起的。重组HA1免疫小鼠可产生抗HA1抗体,随着抗原剂量的增加,其产生的抗体滴度相应增加;3次免疫后,4μg HA1诱导小鼠产生的抗体滴度最高。结论:利用糖基工程酵母表达制备了低糖化的流感病毒血凝素HA1,该重组蛋白可以诱导小鼠产生HA1抗体,且产生的抗体滴度具有HA1剂量依赖性。     

Pre-Existing Immunity with High Neutralizing Activity to 2009 Pandemic H1N1 Influenza Virus in Shanghai Population

Pre-existing immunity is an important factor countering the pandemic potential of an emerging influenza virus strain. Thus, studying of pre-existing immunity to the 2009 pandemic H1N1 virus (2009 H1N1) will advance our understanding of the pathogenesis and epidemiology of this emerging pathogen. In the present study, sera were collected from 486 individuals in a hospital in Shanghai, China, before the 2009 H1N1 influenza pandemic. The serum anti-hemagglutinins (HA) antibody, hemagglutination inhibition (HI) antibody and neutralizing antibody against the 2009 H1N1 were assayed. Among this population, 84.2%, 14.61% and 26.5% subjects possessed anti-HA antibody, HI antibody and neutralizing antibody, respectively. Although neutralizing antibody only existed in those sera with detectable anti-HA antibody, there was no obvious correlation between the titers of anti-HA and neutralizing antibody. However, the titers of anti-HA and neutralizing antibody against seasonal H1N1 virus were highly correlated. In the same population, there was no correlation between titers of neutralizing antibody against 2009 H1N1 and seasonal H1N1. DNA immunization performed on mice demonstrated that antibodies to the HA of 2009 pandemic and seasonal H1N1 influenza viruses were strain-specific and had no cross-neutralizing activity. In addition, the predicted conserved epitope in the HA of 2009 H1N1 and recently circulating seasonal H1N1 virus, GLFGAIAGFIE, was not an immunologically valid B-cell epitope. The data in this report are valuable for advancing our understanding of 2009 H1N1 influenza virus infection.     

Impact of chicken-origin cells on adaptation of a low pathogenic influenza virus

Understanding the growth dynamics of influenza viruses is an essential step in virus replication and cell-adaptation. The aim of this study was to elucidate the growth kinetic of a low pathogenic avian influenza H9N2 subtype in chicken embryo fibroblast (CEF) and chicken tracheal epithelial (CTE) cells during consecutive passages. An egg-adapted H9N2 virus was seeded into both cell culture systems. The amount of infectious virus released into the cell culture supernatants at interval times post-infection were titered and plaque assayed. The results as well as cell viability results indicate that the infectivity of the influenza virus was different among these primary cells. The egg-adapted H9N2 virus featured higher infectivity in CTE than in CEF cells. After serial passages and plaque purifications of the virus, a CTE cell-adapted strain was generated which carried amino acid substitutions within the HA stem region. The strain showed faster replication kinetics in cell culture resulting in an increase in virus titer. Overall, the present study provides the impact of cell type, multiplicity of infection, cellular protease roles in virus infectivity and finally molecular characterization during H9N2 virus adaptation procedure.     

Cleavage of influenza a virus hemagglutinin in human respiratory epithelium is cell associated and sensitive to exogenous antiproteases

Proteolytic cleavage of the hemagglutinin (HA) of human influenza viruses A/Aichi/2/68 (H3N2) and A/WSN/34 (H1N1) from HA0 to HA1/HA2 was studied in primary human adenoid epithelial cells (HAEC). HAEC contain a mixture of ciliated and nonciliated secretory cells and mimic the epithelium membrane of the human respiratory tract. Pulse-chase labeling with [(35)S]methionine and Western blot analysis with anti-HA antibodies of cellular and virion polypeptides showed that HAEC cleaved newly synthesized HA0 to HA1/HA2 ("cleavage from within") and significant amounts of cleaved HA accumulated within cells. It was also shown that HAEC was able to cleave HA0 of incoming virions ("cleavage from without"), whereas the HA0 of nonabsorbed virions free in extracellular fluid were not cleaved, supporting the conclusion that HA0 cleavage in HAEC is cell associated. Low-molecular-weight inhibitors of serine proteases, aprotinin and leupeptin, when added to influenza virus-infected HAEC suppressed HA0 cleavage and reduced the amount of cleaved HA1/HA2 both in cells and in progeny virions and thus diminished the infectivity of the virus. In contrast, the addition of fetal bovine serum, containing a number of high-molecular-weight antiproteases that compete for proteases in the extracellular environment, did not inhibit influenza virus growth in HAEC. These data suggest that in human respiratory epithelium the cleavage of influenza virus HA containing a single arginine in the proteolytic site (i) is a cell-associated process accomplished by serine-type protease(s) and (ii) is sensitive to low-molecular-weight exogenous inhibitors of serine proteases.     

Exposure to ozone modulates human airway protease/antiprotease balance contributing to increased influenza A infection

Exposure to oxidant air pollution is associated with increased respiratory morbidities and susceptibility to infections. Ozone is a commonly encountered oxidant air pollutant, yet its effects on influenza infections in humans are not known. The greater Mexico City area was the primary site for the spring 2009 influenza A H1N1 pandemic, which also coincided with high levels of environmental ozone. Proteolytic cleavage of the viral membrane protein hemagglutinin (HA) is essential for influenza virus infectivity. Recent studies suggest that HA cleavage might be cell-associated and facilitated by the type II transmembrane serine proteases (TTSPs) human airway trypsin-like protease (HAT) and transmembrane protease, serine 2 (TMPRSS2), whose activities are regulated by antiproteases, such as secretory leukocyte protease inhibitor (SLPI). Based on these observations, we sought to determine how acute exposure to ozone may modulate cellular protease/antiprotease expression and function, and to define their roles in a viral infection. We utilized our in vitro model of differentiated human nasal epithelial cells (NECs) to determine the effects of ozone on influenza cleavage, entry, and replication. We show that ozone exposure disrupts the protease/antiprotease balance within the airway liquid. We also determined that functional forms of HAT, TMPRSS2, and SLPI are secreted from human airway epithelium, and acute exposure to ozone inversely alters their expression levels. We also show that addition of antioxidants significantly reduces virus replication through the induction of SLPI. In addition, we determined that ozone-induced cleavage of the viral HA protein is not cell-associated and that secreted endogenous proteases are sufficient to activate HA leading to a significant increase in viral replication. Our data indicate that pre-exposure to ozone disrupts the protease/antiprotease balance found in the human airway, leading to increased influenza susceptibility.     

In vitro antibody response to influenza virus. I. T cell dependence of secondary response to hemagglutinin.

A good secondary IgG response to the hemagglutinin (HA) of influenza virus has been obtained in vitro in Marbrook-type cultures of influenza-primed mouse spleen cell suspensions stimulated with inactivated influenza virus. Anti-HA antibody was quantitated by a solid phase radioimmunoassay (RIA) by using purified HA as substrate. The T dependence of this secondary response was shown by depletion of T cells and reconstitution with a source of primed or unprimed T cells. The help given by T cells primed to the homologous virus was many times greater than that given by unprimed T cells, although the latter was significant. The system described will allow investigation of the specificity requirements of helper T cells engaged in the anti-HA response.