Clinical response to pandemic H1N1 influenza virus from a fatal and mild case in ferrets.

BACKGROUND: The majority of pandemic 2009 H1N1 (A(H1N1)pdm09) influenza virus (IV) caused mild symptoms in most infected patients, however, a greater rate of severe disease was observed in healthy young adults and children without co-morbid conditions. The purpose of this work was to study in ferrets the dynamics of infection of two contemporary strains of human A(H1N1)pdm09 IV, one isolated from a patient showing mild disease and the other one from a fatal case. METHODS: Viral strains isolated from a patient showing mild disease-M (A/CastillaLaMancha/RR5661/2009) or from a fatal case-F (A/CastillaLaMancha/RR5911/2009), both without known comorbid conditions, were inoculated in two groups of ferrets and clinical and pathological conditions were analysed. RESULTS: Mild to severe clinical symptoms were observed in animals from both groups. A clinical score distribution was applied in which ferrets with mild clinical signs were distributed on a non-severe group (NS) and ferrets with severe clinical signs on a severe group (S), regardless of the virus used in the infection. Animals on S showed a significant decrease in body weight compared to animals on NS at 4 to 7 days post-infection (dpi). Clinical progress correlated with histopathological findings. Concentrations of haptoglobin (Hp) and serum amyloid A (SAA) increased on both groups after 2 dpi. Clinically severe infected ferrets showed a stronger antibody response and higher viral titres after infection (p = 0.001). CONCLUSIONS: The severity in the progress of infection was independent from the virus used for infection suggesting that the host immune response was determinant in the outcome of the infection. The diversity observed in ferrets mimicked the variability found in the human population.

Exploring the antigenic relatedness of influenza virus haemagglutinins with strain-specific polyclonal antibodies.

Alternative methods to the standard haemagglutination inhibition (HI) and neutralization tests to probe the antigenic properties of the influenza virus haemagglutinin (HA) were developed in this study. Vaccinia virus recombinants expressing reference HAs were used to immunize rabbits from which polyclonal antibodies were obtained. These antibodies were subtype specific but showed limited intra-subtype strain specificity in ELISA. The discriminatory capacity of these antibodies was, however, markedly increased after adsorption to cells infected with heterologous influenza viruses, revealing antigenic differences that were otherwise undistinguishable by standard HI and neutralization tests. Furthermore, the unadsorbed antibodies could be used to select escape mutants of the reference strain, which after sequencing unveiled amino acid changes responsible of the noted antigenic differences. These procedures therefore provide alternative methods for the antigenic characterization of influenza HA and might be useful in studies of HA antigenic evolution.

Characterization of an enhanced antigenic change in the pandemic 2009 H1N1 influenza virus haemagglutinin.

Murine hybridomas producing neutralizing mAbs specific to the pandemic influenza virus A/California/07/2009 haemagglutinin (HA) were isolated. These antibodies recognized at least two different but overlapping new epitopes that were conserved in the HA of most Spanish pandemic isolates. However, one of these isolates (A/Extremadura/RR6530/2010) lacked reactivity with the mAbs and carried two unique mutations in the HA head (S88Y and K136N) that were required simultaneously to eliminate reactivity with the murine antibodies. This unusual requirement directly illustrates the phenomenon of enhanced antigenic change proposed previously for the accumulation of simultaneous amino acid substitutions at antigenic sites of the influenza A virus HA during virus evolution (Shih et al., Proc Natl Acad Sci USA, 104 , 6283-6288, 2007). The changes found in the A/Extremadura/RR6530/2010 HA were not found in escape mutants selected in vitro with one of the mAbs, which contained instead nearby single amino acid changes in the HA head. Thus, either single or double point mutations may similarly alter epitopes of the new antigenic site identified in this work in the 2009 H1N1 pandemic virus HA. Moreover, this site is relevant for the human antibody response, as shown by competition of mAbs and human post-infection sera for virus binding. The results are discussed in the context of the HA antigenic structure and challenges posed for identification of sequence changes with possible antigenic impact during virus surveillance.

A neutralizing single-domain antibody that targets the trimer interface of the human metapneumovirus fusion protein.

IMPORTANCE: Human metapneumovirus (hMPV) is an important respiratory pathogen for which no licensed antivirals or vaccines exist. Single-domain antibodies represent promising antiviral biologics that can be easily produced and formatted. We describe the isolation and detailed characterization of two hMPV-neutralizing single-domain antibodies that are directed against the fusion protein F. One of these single-domain antibodies broadly neutralizes hMPV A and B strains, can prevent proteolytic maturation of F, and binds to an epitope in the F trimer interface. This suggests that hMPV pre-F undergoes trimer opening or "breathing" on infectious virions, exposing a vulnerable site for neutralizing antibodies. Finally, we show that this single-domain antibody, fused to a human IgG1 Fc, can protect cotton rats against hMPV replication, an important finding for potential future clinical applications.

Exogenous, TAP-independent lysosomal presentation of a respiratory syncytial virus CTL epitope.

Respiratory syncytial virus causes lower respiratory tract infections in infancy and old age, affecting also immunocompromised patients. The viral fusion protein is an important vaccine candidate eliciting antibody and cell-mediated immune responses. CD8(+) cytotoxic T lymphocytes (CTLs) are known to have a role in both lung pathology and viral clearance. In BALB/c mice, the fusion protein epitope F249-258 is presented to CTLs by the murine major histocompatibility complex (MHC) class I molecule K(d). In cells infected with recombinant vaccinia viruses encoding the fusion protein, F249-258 is presented by MHC class I molecules through pathways that are independent of the transporters associated with antigen processing (TAP). We have now found that F249-258 can be generated from non-infectious virus from an exogenous source. Antigen processing follows a lysosomal pathway that appears to require autophagy. As a practical consequence, inactivated virus suffices for in vivo priming of virus-specific CTLs.

Insertion of the two cleavage sites of the respiratory syncytial virus fusion protein in Sendai virus fusion protein leads to enhanced cell-cell fusion and a decreased dependency on the HN attachment protein for activity.

Cell entry by paramyxoviruses requires fusion of the viral envelope with the target cell membrane. Fusion is mediated by the viral fusion (F) glycoprotein and usually requires the aid of the attachment glycoprotein (G, H or HN, depending on the virus). Human respiratory syncytial virus F protein (F(RSV)) is able to mediate membrane fusion in the absence of the attachment G protein and is unique in possessing two multibasic furin cleavage sites, separated by a region of 27 amino acids (pep27). Cleavage at both sites is required for cell-cell fusion. We have investigated the significance of the two cleavage sites and pep27 in the context of Sendai virus F protein (F(SeV)), which possesses a single monobasic cleavage site and requires both coexpression of the HN attachment protein and trypsin in order to fuse cells. Inclusion of both F(RSV) cleavage sites in F(SeV) resulted in a dramatic increase in cell-cell fusion activity in the presence of HN. Furthermore, chimeric F(SeV) mutants containing both F(RSV) cleavage sites demonstrated cell-cell fusion in the absence of HN. The presence of two multibasic cleavage sites may therefore represent a strategy to regulate activation of a paramyxovirus F protein for cell-cell fusion in the absence of an attachment protein.

Structural properties of the human respiratory syncytial virus P protein: evidence for an elongated homotetrameric molecule that is the smallest orthologue within the family of paramyxovirus polymerase cofactors.

The oligomeric state and the hydrodynamic properties of human respiratory syncytial virus (HRSV) phosphoprotein (P), a known cofactor of the viral RNA-dependent RNA polymerase (L), and a trypsin-resistant fragment (X) that includes its oligomerization domain were analyzed by sedimentation equilibrium and velocity using analytical ultracentrifugation. The results obtained demonstrate that both P and fragment X are homotetrameric with elongated shapes, consistent with electron micrographs of the purified P protein in which thin rod-like molecules of approximately 12.5 +/- 1.0 nm in length were observed. A new chymotrypsin resistant fragment (Y*) included in fragment X has been identified and purified by gel filtration chromatography. Fragment Y* may represent a minimal version of the P oligomerization domain. Thermal denaturation curves based on circular dichroism data of P protein showed a complex behavior. In contrast, melting data generated for fragments X and particularly fragment Y* showed more homogeneous transitions indicative of simpler structures. A three-dimensional model of X and Y* fragments was built based on the atomic structure of the P oligomerization domain of the related Sendai virus, which is in good agreement with the experimental data. This model will be an useful tool to make rational mutations and test the role of specific amino acids in the oligomerization and functional properties of the HRSV P protein.

High level expression of soluble glycoproteins in the allantoic fluid of embryonated chicken eggs using a Sendai virus minigenome system.

BACKGROUND: Embryonated chicken eggs have been used since the mid-20th century to grow a wide range of animal viruses to high titers. However, eggs have found so far only limited use in the production of recombinant proteins. We now describe a system, based on a Sendai virus minigenome, to produce large amounts of heterologous viral glycoproteins in the allantoic cavity of embryonated eggs. RESULTS: Soluble forms of human respiratory syncytial virus (HRSV) and human metapneumovirus (HMPV) fusion (F) proteins, devoid of their transmembrane and cytoplasmic domains, were produced in allantoic fluids using the Sendai minigenome system. The first step was rescuing in cell cultures Sendai virus minigenomes encoding the proteins of interest, with the help of wild type Sendai virus. The second step was propagating such recombinant defective viruses, together with the helper virus, in the allantoic cavity of chicken embryonated eggs, and passage to optimize protein production. When compared with the production of the same proteins in the culture supernatant of cells infected with vaccinia recombinants, the yield in the allantoic fluid was 5-10 fold higher. Mutant forms of these soluble proteins were easily constructed by site-directed mutagenesis and expressed in eggs using the same approach. CONCLUSION: The simplicity and economy of the Sendai minigenome system, together with the high yield achieved in the allantoic fluid of eggs, makes it an attractive method to express soluble glycoproteins aimed for structural studies.

Characterization in vitro and in vivo of a pandemic H1N1 influenza virus from a fatal case.

Pandemic 2009 H1N1 (pH1N1) influenza viruses caused mild symptoms in most infected patients. However, a greater rate of severe disease was observed in healthy young adults and children without co-morbid conditions. Here we tested whether influenza strains displaying differential virulence could be present among circulating pH1N1 viruses. The biological properties and the genotype of viruses isolated from a patient showing mild disease (M) or from a fatal case (F), both without known co-morbid conditions were compared in vitro and in vivo. The F virus presented faster growth kinetics and stronger induction of cytokines than M virus in human alveolar lung epithelial cells. In the murine model in vivo, the F virus showed a stronger morbidity and mortality than M virus. Remarkably, a higher proportion of mice presenting infectious virus in the hearts, was found in F virus-infected animals. Altogether, the data indicate that strains of pH1N1 virus with enhanced pathogenicity circulated during the 2009 pandemic. In addition, examination of chemokine receptor 5 (CCR5) genotype, recently reported as involved in severe influenza virus disease, revealed that the F virus-infected patient was homozygous for the deleted form of CCR5 receptor (CCR5Δ32).

Cultures of HEp-2 cells persistently infected by human respiratory syncytial virus differ in chemokine expression and resistance to apoptosis as compared to lytic infections of the same cell type.

HEp-2 cells that survived a lytic infection with Human Respiratory Syncytial Virus (HRSV) were grown to obtain a persistently infected culture that produced relatively high amounts of virus (10(6)-10(7) pfu/ml) for more than twenty passages. The cells in this culture were heterogeneous with regard to the expression of viral antigens, ranging from high to undetectable levels. However, all cell clones derived from the persistent culture did not produce infectious virus or viral antigens and grew more slowly than the original uninfected HEp-2 cells. When these "cured" cell clones were infected with wild-type HRSV, delayed virus production and reduction in the number and size of syncytia were observed compared to lytically infected HEp-2 cells. Most significantly, differences in gene expression between persistently and lytically infected cultures were also observed, including genes that encode for cytokines, chemokines and other gene products that either promote cell survival or inhibit apoptosis. These results highlight the significantly different responses of the same cell type to HRSV infection depending on the outcome of such infection, i.e., lytic versus persistent.

Relevance of viral context and diversity of antigen-processing routes for respiratory syncytial virus cytotoxic T-lymphocyte epitopes.

Antigen processing of respiratory syncytial virus (RSV) fusion (F) protein epitopes F85-93 and F249-258 presented to cytotoxic T-lymphocytes (CTLs) by the murine major histocompatibility complex (MHC) class I molecule Kd was studied in different viral contexts. Epitope F85-93 was presented through a classical endogenous pathway dependent on the transporters associated with antigen processing (TAP) when the F protein was expressed from either RSV or recombinant vaccinia virus (rVACV). At least in cells infected with rVACV encoding either natural or cytosolic F protein, the proteasome was required for epitope processing. In cells infected with rVACV encoding the natural F protein, an additional endogenous TAP-independent presentation pathway was found for F85-93. In contrast, epitope F249-258 was presented only through TAP-independent pathways, but presentation was brefeldin A sensitive when the F protein was expressed from RSV, or mostly resistant when expressed from rVACV. Therefore, antigen-processing pathways with different mechanisms and subcellular localizations are accessible to individual epitopes presented by the same MHC class I molecule and processed from the same protein but in different viral contexts. This underscores both the diversity of pathways available and the influence of virus infection on presentation of epitopes to CTLs.

Distinct gene subsets are induced at different time points after human respiratory syncytial virus infection of A549 cells.

cDNA microarray technology was applied to time course analysis of differentially expressed genes in A549 cells following human respiratory syncytial virus (HRSV) infection. Both up- and down-regulation of cellular genes were observed in a time-dependent manner. However, gene up-regulation prevailed over gene down-regulation. Virus infectivity was required as UV-inactivated virus failed to up-regulate/down-regulate those genes. At early times post-infection (0-6 h p.i.) 85 genes were up-regulated. Some of those genes were involved in cell growth/proliferation, cellular protein metabolism and cytoskeleton organization. Among the most strongly up-regulated genes at that time were the urokinase plasminogen activator (PLAU) and its receptor (PLAUR), a pleiotropic system involved in many biological processes, including chemotaxis and inflammation. Functionally related genes encoding the alpha- and beta-chains of several integrins were also up-regulated within the first 12 h of infection. Genes up-regulated between 6 and 12 h p.i. included interferon-stimulated genes (ISGs), genes related to oxidative stress and genes of the non-canonical NF-kappaB pathway. At later times, genes involved in the immune response became predominant among the up-regulated genes, most of them being ISGs. Different up-regulation kinetics of cytokine and cytokine-signalling-related genes were also observed. These results highlight the dynamic interplay between the virus and the host cell and provide a general picture of changes in cellular gene expression along the HRSV replicative cycle.

Sequence elements of the fusion peptide of human respiratory syncytial virus fusion protein required for activity.

We have reported previously the expression and purification of an anchorless form of the human respiratory syncytial virus (HRSV) F protein (F(TM-)) representing the ectodomain of the full-length F. F(TM-) molecules are seen as unaggregated cones by electron microscopy but completion of proteolytic cleavage of the F0 monomers in the F(TM-) trimer leads to a change in shape from cones to lollipops that aggregate into rosettes. This aggregation apparently occurs by interaction of the fusion peptides of F(TM-) molecules that are exposed after cleavage. Since exposure of the fusion peptide is a key event in the process of membrane fusion, changes associated with F(TM-) cleavage may reflect those occurring in full-length F during membrane fusion. Deletions or substitutions that changed either the length, charge or hydrophobicity of the fusion peptide inhibited aggregation of F(TM-), and these mutants remained as unaggregated cones after cleavage. In contrast, more conservative changes did not inhibit the change of shape and aggregation of F(TM-). When the same changes were introduced in the fusion peptide of full-length F, only the mutations that inhibited aggregation of F(TM-) prevented membrane fusion. Thus, the conformational changes that follow completion of cleavage of the F(TM-) protein require a functional fusion peptide. These sequence constraints may restrict accumulation of sequence changes in the fusion peptide of HRSV F when compared with other hydrophobic regions of the molecule.

Structural analysis of the human respiratory syncytial virus phosphoprotein: characterization of an alpha-helical domain involved in oligomerization.

Human respiratory syncytial virus (HRSV) phosphoprotein (P), an essential cofactor of the viral polymerase, is much shorter (241 aa) than and has no sequence similarity to P of other paramyxoviruses. Nevertheless, bioinformatic analysis of HRSV P sequence revealed a modular organization, reminiscent of other paramyxovirus Ps, with a central structured domain (aa 100-200), flanked by two intrinsically disordered regions (1-99 and 201-241). To test the predicted structure experimentally, HRSV P was purified from cell extracts infected with recombinant vaccinia virus or HRSV. The estimated molecular mass of P by gel filtration (approximately 500 kDa) greatly exceeded the theoretical mass of a homotetramer, proposed as the oligomeric form of native P. Nevertheless, the profile of cross-linked products obtained with purified P resembled that reported by others with P purified from bacteria or mammalian cells. Thus, the shape of HRSV P probably influences its elution from the gel filtration column, as reported for other paramyxovirus Ps. Digestion of purified HRSV P with different proteases identified a trypsin-resistant fragment (X) that reacted with a previously characterized monoclonal antibody (021/2P). N-terminal sequencing and mass spectrometry analysis placed the X fragment boundaries (Glu-104 and Arg-163) within the predicted structured domain of P. Cross-linking and circular dichroism analyses indicated that fragment X was oligomeric, with a high alpha-helical content, properties resembling those of the multimerization domain of Sendai and rinderpest virus P. These results denote structural features shared by HRSV and other paramyxovirus Ps and should assist in elucidation of the HRSV P structure.

Comparison of affinity chromatography and adsorption to vaccinia virus recombinant infected cells for depletion of antibodies directed against respiratory syncytial virus glycoproteins present in a human immunoglobulin preparation.

Antibodies directed against human respiratory syncytial virus (HRSV) glycoproteins were depleted from a commercial immunoglobulin preparation (RespiGam) by two different methods. The first method consisted of repeated adsorption of RespiGam to Sepharose beads with covalently bound soluble forms of the two major viral glycoproteins (F or G). The second method consisted of adsorption of immunoglobulins to live cells expressing F or G glycoproteins on their surfaces after infection with vaccinia virus recombinants. While the first method removed efficiently antibodies that reacted with F and/or G glycoproteins by ELISA, it was inefficient in the elimination of anti-HRSV neutralizing antibodies. In contrast, the second method removed efficiently anti-HRSV antibodies that both reacted by ELISA and neutralized virus infectivity. These results confirm that human neutralizing antibodies are directed exclusively against HRSV F and G glycoproteins, and, they raise the possibility that F and G glycoproteins inserted into cell membranes differ antigenically from their soluble forms linked covalently to Sepharose beads.

Epitope mapping of human respiratory syncytial virus 22K transcription antitermination factor: role of N-terminal sequences in protein folding.

The reactivity of a panel of 12 monoclonal antibodies raised against the human respiratory syncytial virus 22 kDa (22K) protein was tested by Western blotting with a set of 22K deletion mutants. The results obtained identified sequences in the C-terminal half of the 22K polypeptide required for integrity of most antibody epitopes, except for epitope 112, which was lost in mutants with short N-terminal deletions. This antibody, in contrast to the others, failed to immunoprecipitate the native 22K protein, indicating that the N terminus of this protein is buried in the native molecule and exposed only under the denaturing conditions of Western blotting. In addition, N-terminal deletions that abolished reactivity with monoclonal antibody 112 also inhibited phosphorylation of the 22K protein previously identified at Ser-58 and Ser-61, suggesting that the N terminus is important in regulating the 22K protein phosphorylation status, most likely as a result of its requirement for protein folding.

Comparison of antibodies directed against human respiratory syncytial virus antigens present in two commercial preparations of human immunoglobulins with different neutralizing activities.

Antibodies directed against human respiratory syncytial virus (HRSV) from two commercial preparations of human immunoglobulins (Igs) were compared. One of the Ig preparations (RespiGam) was obtained from blood samples selected for high titres of anti-HRSV neutralizing antibodies. The other preparation (Flebogamma) was obtained from unselected blood donations. RespiGam and Flebogamma had very similar anti-HRSV ELISA titres, but RespiGam neutralized virus infectivity 8-10 times more efficiently than Flebogamma. The same behaviour was observed when purified antibodies from RespiGam and Flebogamma, specific for either the fusion (F) or the attachment (G) glycoprotein, were compared. To gain further information about differences in neutralization between these two Ig preparations, antibodies recognizing certain F and G protein fragments or peptides were purified and their neutralizing activities were compared. In general, antibodies purified from RespiGam showed higher neutralizing activity that those purified from Flebogamma, but those differences were higher with antibodies specific for certain protein segments than for others. Some of the protein regions recognized by human neutralizing antibodies were mapped outside antigenic sites identified previously with panels of murine monoclonal antibodies. These results offer the possibility of searching for new neutralizing antibodies that could be used to study the molecular basis of neutralization and to prevent HRSV infections.

The soluble form of human respiratory syncytial virus attachment protein differs from the membrane-bound form in its oligomeric state but is still capable of binding to cell surface proteoglycans.

The soluble (Gs) and membrane-bound (Gm) forms of human respiratory syncytial virus (HRSV) attachment protein were purified by immunoaffinity chromatography from cultures of HEp-2 cells infected with vaccinia virus recombinants expressing either protein. Sucrose gradient centrifugation indicated that Gs, which is secreted into the culture medium, remains monomeric, whereas Gm is an oligomer, probably a homotetramer. Nevertheless, Gs was capable of binding to the surface of cells in vitro, as assessed by a flow cytometry-based binding assay. The attachment of Gs to cells was inhibited by previous heparinase treatment of living cells, and Gs did not bind to CHO cell mutants defective in proteoglycan biosynthesis. Thus, Gs, as previously reported for the G protein of intact virions, binds to glycosaminoglycans presented at the cell surface as proteoglycans. Deletion of a previously reported heparin binding domain from Gs protein substantially inhibited its ability to bind to cells, but the remaining level of binding was still sensitive to heparinase treatment, suggesting that other regions of the Gs molecule may contribute to attachment to proteoglycans. The significance of these results for HRSV infection is discussed.

Prevalence of anti-human respiratory syncytial virus antibodies over three consecutive years in a healthy adult population.

The titers of anti-human respiratory syncytial virus (HRSV) antibodies have been measured by enzyme-linked immunosorbent assay (ELISA) in a cohort of healthy adult volunteers over 3 consecutive years. Significant increases in the level of these antibodies were detected in a small percentage of individuals (3-3.3%), when paired serum samples taken from two consecutive seasons were compared. In these cases, a significant increase was generally noted in the level of antibodies directed against either the F or the G glycoproteins, and in the level of neutralizing antibodies. However, no correlation was found between the level of binding and neutralizing antibodies, when samples representative of the population under study were analyzed. These results indicate some level of infection by HRSV in the adult population. The implications of these results for the epidemiology of HRSV are discussed.

Shifting immunodominance pattern of two cytotoxic T-lymphocyte epitopes in the F glycoprotein of the Long strain of respiratory syncytial virus.

Human respiratory syncytial virus (RSV) is a major cause of respiratory infection in children and in the elderly. The RSV fusion (F) glycoprotein has long been recognized as a vaccine candidate as it elicits cytotoxic T-lymphocyte (CTL) and antibody responses. Two murine H-2K(d)-restricted CTL epitopes (F85-93 and F92-106) are known in the F protein of the A2 strain of RSV. F-specific CTL lines using BCH4 fibroblasts that are persistently infected with the Long strain of human RSV as stimulators were generated, and it was found that in this strain only the F85-93 epitope is conserved. Motif based epitope prediction programs and an F2 chain deleted F protein encoded in a recombinant vaccinia virus enabled identification of a new epitope in the Long strain, F249-258, which is presented by K(d) as a 9-mer (TYMLTNSEL) or a 10-mer (TYMLTNSELL) peptide. The results suggest that the 10-mer might be a naturally processed endogenous K(d) ligand. The CD8(+) T-lymphocyte responses to epitopes F85-93 and F249-258 present in the F protein of RSV Long were found to be strongly skewed to F85-93 in in vitro multispecific CTL lines and in vivo during a secondary response to a recombinant vaccinia virus that expresses the entire F protein. However, no hierarchy in CD8(+) T-lymphocyte responses to F85-93 and F249-258 epitopes was observed in vivo during a primary response.