TH cells primed during influenza virus infection provide help for qualitatively distinct antibody responses to subsequent immunization.

The quality of the primary Ab-forming cell (AFC) response in cervical lymph nodes and mediastinal lymph nodes of mice to intranasal influenza virus was strongly influenced by viral replicative capacity. IgA secretors were prominent in the early AFC response to infectious virus in mediastinal lymph nodes, while IgG expression was more frequent among isotypically switched AFC in cervical lymph nodes of the same mice; this pattern was reversed in the response to inactivated virus. Influenza viruses A/Puerto Rico/8/34 (A/PR8) and A/X-31 share six of eight genome segments, differing only in hemagglutinin (H1 in A/PR8, H3 in A/X-31) and neuraminidase (N1 in A/PR8, N2 in A/X-31) genes. These viruses therefore elicit extensively cross-reactive TH populations, though their glycoproteins are serologically unrelated. Mice recovered from an A/X-31 infection thus mount a primary B cell response against A/PR8 glycoproteins, when challenged with the latter virus, though this response can call upon memory TH cells. To assess the impact of memory TH populations on a primary Ab response, we compared the AFC response to inactivated A/PR8 in naive mice and mice that had cleared an A/X-31 infection. A/X-31 immune mice mounted a more vigorous AFC response against A/PR8 H1 and N1 glycoproteins than naive animals, when immunized intranasally with inactivated A/PR8. However the distribution of isotypes among H1/N1-specific AFC in lymph nodes of A/X-31-primed mice resembled that of naive mice. Evidently, in this functional context, memory TH cells retained the ability to help Ab responses different in quality from that generated during their primary reaction.     

H5N1 influenza marker vaccine for serological differentiation between vaccinated and infected chickens

Using plasmid-based reverse genetics, we generated a molecularly altered virus, H5N1/PR8-5B19, containing modified HA and NA genes from A/Goose/Guangdong/1/96 (GS/GD/1/96). In the H5N1/PR8-5B19 virus, the HA cleavage site was modified to resemble that of low-pathogenic avian strains and a portion of the NA stalk region was replaced by the immunodominant 5B19 epitope of the S2 glycoprotein of murine hepatitis virus (MHV). H5N1/PR8-5B19 is not lethal to embryonated eggs or chickens. Chickens immunized with the H5N1/PR8-5B19 inactivated vaccine produced high levels of HI antibody and a measurable antibody response against the MHV 5B19 epitope, and were fully protected against subsequent challenge with different highly pathogenic H5N1 avian influenza viruses. H5N1/PR8-5B19 is therefore an attractive marker vaccine candidate, eliciting a strong, protective antibody response and enabling serological discrimination between vaccinated and wild-type virus-infected chickens.     

Protection against influenza virus infection in polymeric Ig receptor knockout mice immunized intranasally with adjuvant-combined vaccines

The role of secretory IgA in conferring cross-protective immunity was examined in polymeric (p)IgR knockout (KO) mice immunized intranasally with different inactivated vaccines prepared from A/PR/8/34 (H1N1), A/Yamagata/120/86 (H1N1), A/Beijing/262/95 (H1N1), and B/Ibaraki/2/85 viruses and infected with the A/PR/8/34 virus in the upper respiratory tract (RT)-restricting volume. In wild-type mice, immunization with A/PR/8/34 or its variant (A/Yamagata/120/86 and A/Beijing/262/95) vaccines conferred complete protection or partial cross-protection against infection, while the B-type virus vaccine failed to provide protection. The protection or cross-protection was accompanied by an increase in the nasal A/PR/8/34 hemagglutinin-reactive IgA concentration, which was estimated to be >30 times the serum IgA concentration and much higher than the nasal IgG concentration. In contrast, the blockade of transepithelial transport of dimeric IgA in pIgR-KO mice reduced the degree of protection or cross-protection, in parallel with the marked increase in serum IgA concentration and the decrease in nasal IgA concentration (about 20 and 0.3 times those in wild-type mice, respectively). The degree of the reduction of protection or cross-protection was moderately reversed by the low but non-negligible level of nasal IgA, transudates from the accumulated serum IgA. These results, together with the absence of the IgA-dependent cross-protection in the lower RT and the unaltered level of nasal or serum IgG in wild-type and pIgR-KO mice, confirm that the actively secreted IgA plays an important role in cross-protection against variant virus infection in the upper RT, which cannot be substituted by serum IgG.     

Evaluation of the efficacy of a pre-pandemic H5N1 vaccine (MG1109) in mouse and ferret models

The threat of a highly pathogenic avian influenza (HPAI) H5N1 virus causing the next pandemic remains a major concern. In this study, we evaluated the immunogenicity and efficacy of an inactivated whole-virus H5N1 pre-pandemic vaccine (MG1109) formulated by Green Cross Co., Ltd containing the hemagglutinin (HA) and neuraminidase (NA) genes of the clade 1 A/Vietnam/1194/04 virus in the backbone of A/Puerto Rico/8/34 (RgVietNam/04xPR8/34). Administration of the MG1109 vaccine (2-doses) in mice and ferrets elicited high HI and SN titers in a dose-dependent manner against the homologous (RgVietNam/04xPR8/34) and various heterologous H5N1 strains, (RgKor/W149/06xPR8/34, RgCambodia/04xPR8/34, RgGuangxi/05xPR8/34), including a heterosubtypic H5N2 (A/Aquatic bird/orea/W81/05) virus. However, efficient cross-reactivity was not observed against heterosubtypic H9N2 (A/Ck/Korea/H0802/08) and H1N1 (PR/8/34) viruses. Mice immunized with 1.9 μg HA/dose of MG1109 were completely protected from lethal challenge with heterologous wild-type HPAI H5N1 A/EM/Korea/W149/06 (clade 2.2) and mouse-adapted H5N2 viruses. Furthermore, ferrets administered at least 3.8 μg HA/dose efficiently suppressed virus growth in the upper respiratory tract and lungs. Vaccinated mice and ferrets also demonstrated attenuation of clinical disease signs and limited virus spread to other organs. Thus, this vaccine provided immunogenic responses in mouse and ferret models even against challenge with heterologous HPAI H5N1 and H5N2 viruses. Since the specific strain of HPAI H5N1 virus that would potentially cause the next outbreak is unknown, pre-pandemic vaccine preparation that could provide cross-protection against various H5 strains could be a useful approach in the selection of promising candidate vaccines in the future.     

Improved antigen yield in pandemic H1N1 (2009) candidate vaccine viruses with chimeric hemagglutinin molecules

The candidate pandemic H1N1 vaccine virus NIBRG-121 was derived by reverse genetics and comprises the hemagglutinin (HA) and neuraminidase (NA) genes from A/California/7/2009 (CAL) on an A/Puerto Rico/8/34 (PR8) backbone. NIBRG-121 was found to grow poorly in eggs, compared to seasonal H1N1 candidate vaccine viruses. Based on our previous study with H5N1 candidate vaccine viruses, we generated two new viruses with chimeric PR8/CAL HA genes. Here we show that these new viruses have considerably improved growth in eggs and are therefore better candidate vaccine viruses for use in production of pandemic H1N1 (2009) vaccine.     

Molecular Basis of Live-Attenuated Influenza Virus

Human influenza is a seasonal disease associated with significant morbidity and mortality. The most effective means for controlling infection and thereby reducing morbidity and mortality is vaccination with a three inactivated influenza virus strains mixture, or by intranasal administration of a group of three different live attenuated influenza vaccine strains. Comparing to the inactivated vaccine, the attenuated live viruses allow better elicitation of a long-lasting and broader immune (humoral and cellular) response that represents a naturally occurring transient infection. The cold-adapted (ca) influenza A/AA/6/60 (H2N2) (AA ca) virus is the backbone for the live attenuated trivalent seasonal influenza vaccine licensed in the United States. Similarly, the influenza A components of live-attenuated vaccines used in Russia have been prepared as reassortants of the cold-adapted (ca) H2N2 viruses, A/Leningrad/134/17/57-ca (Len/17) and A/Leningrad/134/47/57-ca (Len/47) along with virulent epidemic strains. However, the mechanism of temperature-sensitive attenuation is largely elusive. To understand how modification at genetic level of influenza virus would result in attenuation of human influenza virus A/PR/8/34 (H1N1,A/PR8), we investigated the involvement of key mutations in the PB1 and/or PB2 genes in attenuation of influenza virus in vitro and in vivo. We have demonstrated that a few of residues in PB1 and PB2 are critical for the phenotypes of live attenuated, temperature sensitive influenza viruses by minigenome assay and real-time PCR. The information of these mutation loci could be used for elucidation of mechanism of temperature-sensitive attenuation and as a new strategy for influenza vaccine development.     

The analysis of the monoclonal immune response to influenza virus. III. The relationship between stimulation of virus-primed precursor B cells by heterologous viruses and reactivity of secreted antibodies.

Individual splenic precursor B cells from BALB/c mice primed with influenza virus PR8[A/PR/8/34 (H0N1)] were stimulated in vitro in the splenic fragment culture system by homologous or various heterologous influenza viruses. The specificity of the stimulated precursor cells was determined by analysis of the antibodies secreted by the ensuing plasma cell clone in a radioimmunoassay (RIA). Viruses of the H2N2 and H3N2 subtypes were unable to stimulate hemagglutinin (HA)- or neuraminidase (NA)-committed precursor B cells but did efficiently stimulate chicken host component (ChHC)-committed precursors. Viruses of the H1N1 and H0N1 subtypes could stimulate precursors committed to any of the three viral surface components. Analysis of the fine specificity of HA-committed B cells showed that BEL(H0N1) and CAM(H1N1) stimulated almost exclusively precursors whose clonal antibody product reacted with the stimulating virus in the RIA. On the other hand, WSE and MEL (both H0N1) quite frequently were able to stimulate precursors whose clonal antibody product did not react with the stimulating virus in the RIA. These results suggest that the stimulatory interaction of viruses with the cell-bound immunoglobulin receptors is slightly less affinity dependent than the antigen-antibody interaction in the RIA.     

The origin of a novel kind of reassortant (H1N2) of influenza A virus

Genetic analysis of three H1N2 viruses indicated that only HA genes of H1N2 viruses were similar to that of A/Guangdong/6/91(H1N1) virus (PR8-like strain), while the other seven genes of them were similar to those of H3N2 virus circulating in man in 1995. Therefore, it could be considered that the H1N2 viruses were derived from reassortment between PR8-like strain and H3N2 virus circulating in man in 1995. However, the genomes of H1N2 viruses were very similar to each other. So the H1N2 viruses isolated in 1998 were not derived from new reassortment between PR8-like strain and H3N2 virus circulating in man in 1998, but derived from the evolution of H1N2 virus found in 1995.     

The origin of a novel kind of reassortant (H1N2) of influenza A virus

Genetic analysis of three H1N2 viruses indicated that only HA genes of H1N2 viruses were similar to that of A/Guangdong/6/91(H1N1) virus (PR8-like strain), while the other seven genes of them were similar to those of H3N2 virus circulating in man in 1995. Therefore, it could be considered that the H1N2 viruses were derived from reassortment between PR8-like strain and H3N2 virus circulating in man in 1995. However, the genomes of H1N2 viruses were very similar to each other. So the H1N2 viruses isolated in 1998 were not derived from new reassortment between PR8-like strain and H3N2 virus circulating in man in 1998, but derived from the evolution of H1N2 virus found in 1995.     

Preparative isolation of basic structural proteins of the influenza virus]

The schemes for preparative electrophoretic isolation and purification of major proteins from influenza virus are described. The viral envelope protein, hemagglutinin, two of its subunits, internal M and NP proteins of influenza viruses A/FPV/Rostock (H7N1), A/PR/8/34 (H1N1) and X-31 (H3N2) were obtained in preparative amounts and characterized by amino acid and N-terminus analyses.     

Cytotoxic T cell recognition of the influenza nucleoprotein and hemagglutinin expressed in transfected mouse L cells

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.     

The M segment of the 2009 new pandemic H1N1 influenza virus is critical for its high transmission efficiency in the guinea pig model

A remarkable feature of the 2009 pandemic H1N1 influenza virus is its efficient transmissibility in humans compared to that of precursor strains from the triple-reassortant swine influenza virus lineage, which cause only sporadic infections in humans. The viral components essential for this phenotype have not been fully elucidated. In this study, we aimed to determine the viral factors critical for aerosol transmission of the 2009 pandemic virus. Single or multiple segment reassortments were made between the pandemic A/California/04/09 (H1N1) (Cal/09) virus and another H1N1 strain, A/Puerto Rico/8/34 (H1N1) (PR8). These viruses were then tested in the guinea pig model to understand which segment of Cal/09 virus conferred transmissibility to the poorly transmissible PR8 virus. We confirmed our findings by generating recombinant A/swine/Texas/1998 (H3N2) (sw/Tx/98) virus, a representative triple-reassortant swine virus, containing segments of the Cal/09 virus. The data showed that the M segment of the Cal/09 virus promoted aerosol transmissibility to recombinant viruses with PR8 and sw/Tx/98 virus backgrounds, suggesting that the M segment is a critical factor supporting the transmission of the 2009 pandemic virus.     

Fusion-Related Host Proteins Are Actively Regulated by NA during Influenza Infection as Revealed by Quantitative Proteomics Analysis

Three recombinant influenza A viruses with different neuraminidases (NAs) in the background of A/PR/8/34 (PR8), named rPR8-H5N1NA, rPR8-H9N2NA, and rPR8-H1N1NA, derived from H5N1, H9N2, H1N1 (swine) viruses, respectively, were constructed. We performed a quantitative proteomics analysis to investigate differential protein expression in Madin-Darby canine kidney (MDCK) cells infected with recombinant and wild-type influenza viruses to determine whether NA replacement would alter host cell gene expression. Using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-TOF MS) and two-dimensional gel electrophoresis (2-DE), we identified 12 up-regulated and 49 down-regulated protein spots, including cytoskeletal proteins, molecular biosynthesis proteins, ubiquitin-proteasome pathway proteins, and heat shock proteins. The most significant changes in infected cells were observed for molecular biosynthesis proteins. We found more differentially expressed protein spots in cells infected with rPR8-H5N1NA or rPR8-H9N2NA viruses than cells infected with wild-type virus. Many of those proteins are postulated to be involved in cell-cell fusion, but the full mechanism remains to be explored. Meanwhile, our data demonstrate that the wild-type virus has evolutionary advantages over recombinant viruses.     

The evaluation of the reactogenicity and immunological activity of an inactivated 3-component influenza vaccine with an elevated hemagglutinin concentration in the inoculation dosage

In this work the reactogenic properties and antigenic potency of inactivated trivalent influenza vaccine, obtained by elution and centrifugation and containing up to 9-11 micrograms of hemagglutinin for influenza viruses A(H1N1) and A(H3N2) and up to 14 micrograms for influenza virus B, were studied. The reactogenicity of the preparation was found to correspond to the regulations. The immunogenic potency characteristics of individual batches of this trivaccine were higher than the immunogenicity of divaccines, but did not meet the requirements of technical specifications.     

Neuraminidase inhibitor-resistant recombinant A/Vietnam/1203/04 (H5N1) influenza viruses retain their replication efficiency and pathogenicity in vitro and in vivo

Effective antiviral drugs are essential for early control of an influenza pandemic. It is therefore crucial to evaluate the possible threat posed by neuraminidase (NA) inhibitor-resistant influenza viruses with pandemic potential. Four NA mutations (E119G, H274Y, R292K, and N294S) that have been reported to confer resistance to NA inhibitors were each introduced into recombinant A/Vietnam/1203/04 (VN1203) H5N1 influenza virus. For comparison, the same mutations were introduced into recombinant A/Puerto Rico/8/34 (PR8) H1N1 influenza virus. The E119G and R292K mutations significantly compromised viral growth in vitro, but the H274Y and N294S mutations were stably maintained in VN1203 and PR8 viruses. In both backgrounds, the H274Y and N294S mutations conferred resistance to oseltamivir carboxylate (50% inhibitory concentration [IC(50)] increases, >250-fold and >20-fold, respectively), and the N294S mutation reduced susceptibility to zanamivir (IC(50) increase, >3.0-fold). Although the H274Y and N294S mutations did not compromise the replication efficiency of VN1203 or PR8 viruses in vitro, these mutations slightly reduced the lethality of PR8 virus in mice. However, the VN1203 virus carrying either the H274Y or N294S mutation exhibited lethality similar to that of the wild-type VN1203 virus. The different enzyme kinetic parameters (V(max) and K(m)) of avian-like VN1203 NA and human-like PR8 NA suggest that resistance-associated NA mutations can cause different levels of functional loss in NA glycoproteins of the same subtype. Our results suggest that NA inhibitor-resistant H5N1 variants may retain the high pathogenicity of the wild-type virus in mammalian species. Patients receiving NA inhibitors for H5N1 influenza virus infection should be closely monitored for the emergence of resistant variants.     

Growth determinants for H5N1 influenza vaccine seed viruses in MDCK cells

H5N1 influenza A viruses are exacting a growing human toll, with more than 240 fatal cases to date. In the event of an influenza pandemic caused by these viruses, embryonated chicken eggs, which are the approved substrate for human inactivated-vaccine production, will likely be in short supply because chickens will be killed by these viruses or culled to limit the worldwide spread of the infection. The Madin-Darby canine kidney (MDCK) cell line is a promising alternative candidate substrate because it supports efficient growth of influenza viruses compared to other cell lines. Here, we addressed the molecular determinants for growth of an H5N1 vaccine seed virus in MDCK cells, revealing the critical responsibility of the Tyr residue at position 360 of PB2, the considerable requirement for functional balance between hemagglutinin (HA) and neuraminidase (NA), and the partial responsibility of the Glu residue at position 55 of NS1. Based on these findings, we produced a PR8/H5N1 reassortant, optimized for this cell line, that derives all of its genes for its internal proteins from the PR8(UW) strain except for the NS gene, which derives from the PR8(Cambridge) strain; its N1 NA gene, which has a long stalk and derives from an early H5N1 strain; and its HA gene, which has an avirulent-type cleavage site sequence and is derived from a circulating H5N1 virus. Our findings demonstrate the importance and feasibility of a cell culture-based approach to producing seed viruses for inactivated H5N1 vaccines that grow robustly and in a timely, cost-efficient manner as an alternative to egg-based vaccine production.     

Induction of heterosubtypic immunity to influenza virus by intranasal immunization

Recovery from live influenza virus infection is known to induce heterosubtypic immunity. In contrast, immunity induced by inactivated vaccines is predominantly subtype specific. In this study, we investigated the heterosubtypic protective immunity induced by inactivated influenza virus. Intranasal immunization of mice with inactivated influenza virus A/PR8 (H1N1) provided complete protection against the homologous virus and a drift virus within the same subtype, A/WSN (H1N1), but not against the heterosubtypic virus A/Philippines (H3N2). However, coadministration of inactivated virus with cholera toxin as an adjuvant conferred complete heterosubtypic protection, without observed illness, even under conditions of CD4⁺ or CD8⁺ T-cell depletion. Analysis of immune correlates prior to challenge and postchallenge indicated that humoral immune responses with cross-neutralizing activity in lungs and in sera play a major role in conferring protective immunity against heterosubtypic challenge. This study has significant implications for developing broadly cross-reactive vaccines against newly emerging pathogenic influenza viruses.     

Nonspecificity of the phenomenon of influenza virus phagocytosis by murine macrophages

Peritoneal macrophage cultures from intact mice and those immune to influenza virus A/PR/8/34 (HON1) were infected with homologous virus or influenza virus A/England/42/72 (H3N2) whereupon virus was isolated from chick embryos. It was established that in intact macrophages, both viruses duplicated similarly. Macrophages immune to virus HON1 equally disintegrated both in homologous virus and heterologous influenza virus H3N2.     

Limiting dilution analysis of the specificity of influenza-immune cytotoxic T cells

The frequency of memory T cells in the spleens of mice primed with the A/Puerto Rico/8/34/1 (H1N1) (PR8) influenza A virus was determined using limiting dilution protocols. The mean frequency of memory cytotoxic T lymphocytes (CTL) in spleen populations from mice primed with PR8 and restimulated in vitro with the same virus ranged, in six experiments, from 1 in 1600 to 1 in 4800. In the same experiments, the frequencies of CTL capable of lysing targets infected with the heterologous A/Hong Kong/×31/68 (H3N2) (HK) virus ranged from 1 in 1700 to 1 in 4700 nucleated spleen cells. Thus, at least 80% of PR8 (H1N1) influenza-specific cytotoxic T cells are lytic for both HK (H3N2)- and PR8-infected target cells. Further analysis of the specificity of a series of monoclonal influenza-specific CTL was achieved by expanding limit dilution cultures and then testing lytic capacity for targets infected with a range of influenza A viruses. This approach confirmed that the great majority of PR8-primed influenza-specific CTL are cross-reactive for a variety of influenza A subtypes. These experiments demonstrate the feasibility of quantitating different influenza-immune CTL specificities at a stage very close to removal of cells from the animal.