Zhu-Nan Li

Antigenic structure of the haemagglutinin of human influenza A/H2N2 virus

The antigenic structure of influenza A/H2N2 virus haemagglutinin (HA) was analysed using 19 monoclonal antibodies (MAbs) against the HA of A/Kayano/57. The antibodies were classified into three groups: group I had both haemagglutination inhibition and neutralization activities, group II had neutralization activity but no haemagglutination inhibition activity and group III had neither activity. Analysis of escape mutants selected by each of the group I and II antibodies identified six distinct antigenic sites: four (I-A to I-D) were recognized by group I MAbs and two (II-A and II-B) were recognized by group II MAbs. Sequence analysis of the HA genes of the escape mutants demonstrated that sites I-A, I-B and I-C form a contiguous antigenic area that contains the regions corresponding to antigenic sites A, B and D on the H3 molecule and that sites I-D and II-B are the equivalents of sites E and C, respectively, suggesting that the antigenic structure of the H2 molecule is largely similar to that of the H3 molecule. However, the H2 molecule differed from the H3 molecule in having a highly conserved antigenic site (II-A) in the stem domain. It was also found that most of the escape mutants selected by antibodies to sites I-A, I-B and I-C acquired a new glycosylation site at position 160, 187 or 131, respectively, which indicates that A/H2N2 viruses have the potential to gain at least one additional oligosaccharide on the tip of the HA, although this has never occurred during 11 years of its circulation in humans.

Effect of addition of new oligosaccharide chains to the globular head of influenza A/H2N2 virus haemagglutinin on the intracellular transport and biological activities of the molecule.

The haemagglutinin (HA) of influenza A/H2N2 virus possesses six antigenic sites (I-A to I-D, II-A and II-B), and sites I-A, I-B and I-C are located in the regions corresponding to sites A, B and D on the H3 HA. We demonstrated previously that most escape mutants selected by mAbs to site I-A, I-B or I-C had acquired a new oligosaccharide at position 160, 187 or 131, respectively, but this has never occurred during circulation of A/H2N2 virus in humans. Here, to examine whether the H2 HA has the potential to gain two new oligosaccharides on its tip, 31 double escape mutants were isolated by using a single escape mutant with an oligosaccharide at position 160, 187 or 131 as a parental virus and a mAb to an antigenic site different from that to which the mAb used for selection of the parental virus was directed as a selecting antibody, but there were no mutants with two new oligosaccharides. Glycosylation-site HA mutants containing one to three oligosaccharides at positions 160, 187 and 131 were also constructed and their intracellular transport and biological activities were analysed. The results showed that all of the mutant HAs were transported to the cell surface but exhibited a decrease in both receptor-binding and cell-fusing activities. Thus, influenza A/H2N2 virus may have failed to increase the number of oligosaccharides on the HA because, if this happens, the biological activities of the HA are reduced, decreasing the ability of the virus to replicate in humans.

Chimeric Influenza Virus Hemagglutinin Proteins Containing Large Domains of the Bacillus anthracis Protective Antigen: Protein Characterization, Incorporation into Infectious Influenza Viruses, and Antigenicity

ABSTRACT Large polypeptides of the Bacillus anthracis protective antigen (PA) were inserted into an influenza A virus hemagglutinin glycoprotein (HA), and the chimeric proteins were functionally characterized and incorporated into infectious influenza viruses. PA domain 1′, the region responsible for binding to the other toxin components, the lethal factor and edema factor, and domain 4, the receptor binding domain (RBD), were inserted at the C-terminal flank of the HA signal peptide and incorporated into the HA1 subunit of HA. The chimeric proteins, designated as LEF/HA (90 amino acid insertion) and RBD/HA (140 amino acid insertion), were initially analyzed following expression using recombinant vaccinia viruses. Both chimeric proteins were shown to display functional phenotypes similar to that of the wild-type HA. They transport to the cell surface, can be cleaved into the HA1 and HA2 subunits by trypsin to activate membrane fusion potential, are able to undergo the low-pH-induced conformational changes required for fusion, and are capable of inducing the fusion process. We were also able to generate recombinant influenza viruses containing the chimeric RBD/HA and LEF/HA genes, and the inserted PA domains were maintained in the HA gene segments following several passages in MDCK cells or embryonated chicken eggs. Furthermore, DNA immunization of mice with plasmids that express the chimeric RBD/HA and LEF/HA proteins, and the recombinant viruses containing them, induced antibody responses against both the HA and PA components of the protein. These approaches may provide useful tools for vaccines against anthrax and other diseases.

The Antiviral CD8+ T Cell Response Is Differentially Dependent on CD4+ T Cell Help Over the Course of Persistent Infection

Although many studies have investigated the requirement for CD4+ T cell help for CD8+ T cell responses to acute viral infections that are fully resolved, less is known about the role of CD4+ T cells in maintaining ongoing CD8+ T cell responses to persistently infecting viruses. Using mouse polyoma virus (PyV), we asked whether CD4+ T cell help is required to maintain antiviral CD8+ T cell and humoral responses during acute and persistent phases of infection. Though fully intact during acute infection, the PyV-specific CD8+ T cell response declined numerically during persistent infection in MHC class II-deficient mice, leaving a small antiviral CD8+ T cell population that was maintained long term. These unhelped PyV-specific CD8+ T cells were functionally unimpaired; they retained the potential for robust expansion and cytokine production in response to Ag rechallenge. In addition, although a strong antiviral IgG response was initially elicited by MHC class II-deficient mice, these Ab titers fell, and long-lived PyV-specific Ab-secreting cells were not detected in the bone marrow. Finally, using a minimally myeloablative mixed bone marrow chimerism approach, we demonstrate that recruitment and/or maintenance of new virus-specific CD8+ T cells during persistent infection is impaired in the absence of MHC class II-restricted T cells. In summary, these studies show that CD4+ T cells differentially affect CD8+ T cell responses over the course of a persistent virus infection.

The sites for fatty acylation, phosphorylation and intermolecular disulphide bond formation of influenza C virus CM2 protein

The sites for fatty acylation, disulphide bond formation and phosphorylation of influenza C virus CM2 were investigated by site-specific mutagenesis. Cysteine 65 in the cytoplasmic tail was identified as the site for palmitoylation. Removal of one or more of three cysteine residues in the ectodomain showed that all of cysteines 1, 6 and 20 can participate in the formation of disulphide-linked dimers and/or tetramers, although cysteine 20 may play the most important role in tetramer formation. Furthermore, it was found that serine 78, located within the recognition motifs for mammary gland casein kinase and casein kinase I, is the predominant site for phosphorylation, although serine 103 is phosphorylated to a minor extent by proline-dependent protein kinase. The effects of acylation and phosphorylation on the formation of disulphide-linked oligomers were also studied. The results showed that, while palmitoylation has no role in oligomer formation, phosphorylation accelerates tetramer formation without influencing dimer formation. CM2 mutants defective in acylation, phosphorylation or disulphide bond formation were all transported to the cell surface, suggesting that none of these modifications is required for proper oligomerization. When proteins solubilized in detergent were analysed on sucrose gradients, however, the mutant lacking cysteines 1, 6 and 20 sedimented as monomers, raising the possibility that disulphide bond formation, although not essential for proper oligomerization, may stabilize the CM2 multimer. This was supported by the results of chemical cross-linking analysis, which showed that the triple-cysteine mutant can form multimers.

Infectivity Studies of Influenza Virus Hemagglutinin Receptor Binding Site Mutants in Mice

ABSTRACT The replicative properties of influenza virus hemagglutinin (HA) mutants with altered receptor binding characteristics were analyzed following intranasal inoculation of mice. Among the mutants examined was a virus containing a Y98F substitution at a conserved position in the receptor binding site that leads to a 20-fold reduction in binding. This mutant can replicate as well as wild-type (WT) virus in MDCK cells and in embryonated chicken eggs but is highly attenuated in mice, exhibiting titers in lungs more than 1,000-fold lower than those of the WT. The capacity of the Y98F mutant to induce antibody responses and the structural locations of HA reversion mutations are examined.

Immunogenicity of low-pH treated whole viral influenza vaccine.

Low pH treatment of influenza virus hemagglutinin (HA) exposes its relatively conserved stalk domain, suggesting a potential immunogen with capability to induce broader immune responses. Here, we describe characterization, immunogenicity, antigenicity, and protective immunity induced by low pH treated inactivated whole viral vaccine in comparison with the untreated vaccine. The acidic pH treated viral vaccine showed high susceptibility to proteolytic cleavage and low hemagglutination activity indicating conformational changes. Immunization of mice with low pH treated viral vaccine induced lower levels of homologous or heterologous virus-specific binding and neutralizing antibodies compared to the untreated vaccine. Also, low pH treated influenza viral antigen showed lower antigenicity compared to the untreated influenza viral antigen. Lower efficacy of cross-protection against heterosubtypic virus was observed in the low-pH treated vaccine group. The results provide evidence that there is a correlation between protective efficacy and the stability of vaccines.

Length Requirements for Membrane Fusion of Influenza Virus Hemagglutinin Peptide Linkers to Transmembrane or Fusion Peptide Domains

ABSTRACT During membrane fusion, the influenza A virus hemagglutinin (HA) adopts an extended helical structure that contains the viral transmembrane and fusion peptide domains at the same end of the molecule. The peptide segments that link the end of this rod-like structure to the membrane-associating domains are approximately 10 amino acids in each case, and their structure at the pH of fusion is currently unknown. Here, we examine mutant HAs and influenza viruses containing such HAs to determine whether these peptide linkers are subject to specific length requirements for the proper folding of native HA and for membrane fusion function. Using pairwise deletions and insertions, we show that the region flanking the fusion peptide appears to be important for the folding of the native HA structure but that mutant proteins with small insertions can be expressed on the cell surface and are functional for membrane fusion. HA mutants with deletions of up to 10 residues and insertions of as many as 12 amino acids were generated for the peptide linker to the viral transmembrane domain, and all folded properly and were expressed on the cell surface. For these mutants, it was possible to designate length restrictions for efficient membrane fusion, as functional activity was observed only for mutants containing linkers with insertions or deletions of eight residues or less. The linker peptide mutants are discussed with respect to requirements for the folding of native HAs and length restrictions for membrane fusion activity.

Induction of Neutralizing Antibody Responses to Anthrax Protective Antigen by Using Influenza Virus Vectors: Implications for Disparate Immune System Priming Pathways

ABSTRACT Viral vectors based on influenza virus, rabies virus (RV), and vaccinia virus (VV) were used to express large polypeptide segments derived from the Bacillus anthracis protective antigen (PA). For the infectious influenza virus vector and recombinant VV constructs, the receptor binding domain (RBD or domain 4) or the lethal and edema factor binding domain (LEF or domain 1′) were engineered into functional chimeric hemagglutinin (HA) glycoproteins. In the case of the RV vector, the viral glycoprotein (G) was used as a carrier for RBD in an inactivated form of the vector. These constructs were examined by using multiple homologous and heterologous prime/boost immunization regimens in order to optimize the induction of α-PA antibody responses. Several immunization combinations were shown to induce high titers of antibody recognizing the anthrax RBD and LEF domains, as well as the full-length PA protein in mice. The heterologous prime/boost immunization regimens that involved an initial intranasal administration of a live influenza virus vector, followed by an intramuscular boost with either the killed RV vector or the VV vector, were particularly effective, inducing antigen-specific antibodies at levels severalfold higher than homologous or alternative heterologous protocols. Furthermore, sera from several groups of the immunized mice demonstrated neutralization activity in an in vitro anthrax toxin neutralization assay. In some cases, such toxin-neutralizing activity was notably high, indicating that the mechanisms by which immunity is primed by live influenza virus vectors may have beneficial properties.

The effects of preexisting immunity to influenza on responses to influenza vectors in mice.

The use of viral vectors as vaccine candidates has shown promise against a number of pathogens. However, preexisting immunity to these vectors is a concern that must be addressed when deciding which viruses are suitable for use. A number of properties, including the existence of antigenically distinct subtypes, make influenza viruses attractive candidates for use as viral vectors. Here, we evaluate the ability of influenza viral vectors containing inserts of foreign pathogens to elicit antibody and CD8(+) T cell responses against these foreign antigens in the presence of preexisting immunity to influenza virus in mice. Specifically, responses to an H3N1-based vector expressing a 90 amino acid polypeptide derived from the protective antigen (PA) of Bacillus anthracis or an H1N1-based vector containing a CD8(+) T cell epitope from the glycoprotein (GP) of lymphocytic choriomeningitis virus were evaluated following infections with either homosubtypic or heterosubtypic influenza viruses. We found that mice previously infected with influenza viruses, even those expressing HA and NA proteins of completely different subtypes, were severely compromised in their ability to mount an immune response against the inserted epitopes. This inhibition was demonstrated to be mediated by CD8(+) T cells, which recognize multiple strains of influenza viruses. These CD8(+) T cells were further shown to protect mice from a lethal challenge by a heterologous influenza subtype. The implication of these data for the use of influenza virus vectors and influenza vaccination in general are discussed.