Regulation of antinucleoprotein IgG by systemic vaccination and its effect on influenza virus clearance

Seasonal influenza epidemics recur due to antigenic drift of envelope glycoprotein antigens and immune evasion of circulating viruses. Additionally, antigenic shift can lead to influenza pandemics. Thus, a universal vaccine that protects against multiple influenza virus strains could alleviate the continuing impact of this virus on human health. In mice, accelerated clearance of a new viral strain (cross-protection) can be elicited by prior infection (heterosubtypic immunity) or by immunization with the highly conserved internal nucleoprotein (NP). Both heterosubtypic immunity and NP-immune protection require antibody production. Here, we show that systemic immunization with NP readily accelerated clearance of a 2009 pandemic H1N1 influenza virus isolate in an antibody-dependent manner. However, human immunization with trivalent inactivated influenza virus vaccine (TIV) only rarely and modestly boosted existing levels of anti-NP IgG. Similar results were observed in mice, although the reaction could be enhanced with adjuvants, by adjusting the stoichiometry among NP and other vaccine components, and by increasing the interval between TIV prime and boost. Importantly, mouse heterosubtypic immunity that had waned over several months could be enhanced by injecting purified anti-NP IgG or by boosting with NP protein, correlating with a long-lived increase in anti-NP antibody titers. Thus, current immunization strategies poorly induce NP-immune antibody that is nonetheless capable of contributing to long-lived cross-protection. The high conservation of NP antigen and the known longevity of antibody responses suggest that the antiviral activity of anti-NP IgG may provide a critically needed component of a universal influenza vaccine.     

Evolution of the Hemagglutinin Protein of the New Pandemic H1N1 Influenza Virus: Maintaining Optimal Receptor Binding by Compensatory Substitutions

Pandemic influenza A H1N1 (pH1N1) virus emerged in 2009. In the subsequent 4 years, it acquired several genetic changes in its hemagglutinin (HA). Mutations may be expected while virus is adapting to the human host or upon evasion from adaptive immune responses. However, pH1N1 has not displayed any major antigenic changes so far. We examined the effect of the amino acid substitutions found to be most frequently occurring in the pH1N1 HA protein before 1 April 2012 on the receptor-binding properties of the virus by using recombinant soluble HA trimers. Two changes (S186P and S188T) were shown to increase the receptor-binding avidity of HA, whereas two others (A137T and A200T) decreased binding avidity. Construction of an HA protein tree revealed the worldwide emergence of several HA variants during the past few influenza seasons. Strikingly, two major variants harbor combinations of substitutions (S186P/A137T and S188T/A200T, respectively) with opposite individual effects on binding. Stepwise reconstruction of the HA proteins of these variants demonstrated that the mutations that increase receptor-binding avidity are compensated for by the acquisition of subsequent mutations. The combination of these substitutions restored the receptor-binding properties (avidity and specificity) of these HA variants to those of the parental virus. The results strongly suggest that the HA of pH1N1 was already optimally adapted to the human host upon its emergence in April 2009. Moreover, these results are in agreement with a recent model for antigenic drift, in which influenza A virus mutants with high and low receptor-binding avidity alternate.     

Hydrophobic inactivation of influenza viruses confers preservation of viral structure with enhanced immunogenicity

The use of inactivated influenza virus for the development of vaccines with broad heterosubtypic protection requires selective inactivation techniques that eliminate viral infectivity while preserving structural integrity. Here we tested if a hydrophobic inactivation approach reported for retroviruses could be applied to the influenza virus. By this approach, the transmembrane domains of viral envelope proteins are selectively targeted by the hydrophobic photoactivatable compound 1,5-iodonaphthyl-azide (INA). This probe partitions into the lipid bilayer of the viral envelope and upon far UV irradiation reacts selectively with membrane-embedded domains of proteins and lipids while the protein domains that localize outside the bilayer remain unaffected. INA treatment of influenza virus blocked infection in a dose-dependent manner without disrupting the virion or affecting neuraminidase activity. Moreover, the virus maintained the full activity in inducing pH-dependent lipid mixing, but pH-dependent redistribution of viral envelope proteins into the target cell membrane was completely blocked. These results indicate that INA selectively blocks fusion of the virus with the target cell membrane at the pore formation and expansion step. Using a murine model of influenza virus infection, INA-inactivated influenza virus induced potent anti-influenza virus serum antibody and T-cell responses, similar to live virus immunization, and protected against heterosubtypic challenge. INA treatment of influenza A virus produced a virus that is noninfectious, intact, and fully maintains the functional activity associated with the ectodomains of its two major envelope proteins, neuraminidase and hemagglutinin. When used as a vaccine given intranasally (i.n.), INA-inactivated influenza virus induced immune responses similar to live virus infection.     

Antigenic and genetic evolution of equine influenza A (H3N8) virus from 1968 to 2007

Equine influenza virus is a major respiratory pathogen in horses, and outbreaks of disease often lead to substantial disruption to and economic losses for equestrian industries. The hemagglutinin (HA) protein is of key importance in the control of equine influenza because HA is the primary target of the protective immune response and the main component of currently licensed influenza vaccines. However, the influenza virus HA protein changes over time, a process called antigenic drift, and vaccine strains must be updated to remain effective. Antigenic drift is assessed primarily by the hemagglutination inhibition (HI) assay. We have generated HI assay data for equine influenza A (H3N8) viruses isolated between 1968 and 2007 and have used antigenic cartography to quantify antigenic differences among the isolates. The antigenic evolution of equine influenza viruses during this period was clustered: from 1968 to 1988, all isolates formed a single antigenic cluster, which then split into two cocirculating clusters in 1989, and then a third cocirculating cluster appeared in 2003. Viruses from all three clusters were isolated in 2007. In one of the three clusters, we show evidence of antigenic drift away from the vaccine strain over time. We determined that a single amino acid substitution was likely responsible for the antigenic differences among clusters.     

Mucosal and systemic immune responses to a human immunodeficiency virus type 1 epitope induced upon vaginal infection with a recombinant influenza A virus

The humoral and cellular immune responses in the genital mucosa likely play an important role in the prevention of sexually transmitted infections, including infection with human immunodeficiency virus type 1 (HIV-1). Here we show that vaginal infection of progesterone-treated BALB/c mice with a recombinant influenza virus bearing the immunodominant P18IIIB cytotoxic T-lymphocyte (CTL) epitope of the gp160 envelope protein from an HIV-1 IIIB isolate (P18IIIB; RIQRGPGRAFVTIGK) can induce a specific immune response in regional mucosal lymph nodes, as well as in a systemic site (the spleen). A single inoculation of mice with the recombinant influenza virus induced long-lasting (at least 5 months) antigen-specific CTL memory detectable as a rapid recall of effector CTLs upon vaginal infection with recombinant vaccinia virus expressing HIV-1 IIIB envelope gene products. Long-term antigen-specific CTL memory was also induced and maintained in distant mucosal tissues when mice were intranasally immunized with the recombinant influenza virus. These results indicate that mucosal immunization and, in particular, local vaginal immunization with recombinant influenza virus can provide strong, durable immune responses in the female genital tract of mice.     

Computational Identification of Antigenicity-Associated Sites in the Hemagglutinin Protein of A/H1N1 Seasonal Influenza Virus

The antigenic variability of influenza viruses has always made influenza vaccine development challenging. The punctuated nature of antigenic drift of influenza virus suggests that a relatively small number of genetic changes or combinations of genetic changes may drive changes in antigenic phenotype. The present study aimed to identify antigenicity-associated sites in the hemagglutinin protein of A/H1N1 seasonal influenza virus using computational approaches. Random Forest Regression (RFR) and Support Vector Regression based on Recursive Feature Elimination (SVR-RFE) were applied to H1N1 seasonal influenza viruses and used to analyze the associations between amino acid changes in the HA1 polypeptide and antigenic variation based on hemagglutination-inhibition (HI) assay data. Twenty-three and twenty antigenicity-associated sites were identified by RFR and SVR-RFE, respectively, by considering the joint effects of amino acid residues on antigenic drift. Our proposed approaches were further validated with the H3N2 dataset. The prediction models developed in this study can quantitatively predict antigenic differences with high prediction accuracy based only on HA1 sequences. Application of the study results can increase understanding of H1N1 seasonal influenza virus antigenic evolution and accelerate the selection of vaccine strains.     

Monoclonal anti-hemagglutinin antibodies detect irreversible antigenic alterations that coincide with the acid activation of influenza virus A/PR/834-mediated hemolysis.

Exposure of influenza virus to an acidic environment, which is known to be required for viral fusion and hemolysis, has recently been shown to induce a conformational change in the hemagglutinin molecule. In the present study, we examined the effects of acid incubation on the antigenicity, biological activity, and morphology of influenza virus A/PR/8/34 (H1N1). Incubation of PR8 virus at pH 5 in the absence of erythrocytes resulted in a rapid and irreversible loss of viral hemolytic activity and infectivity. Apart from a less distinct appearance of the viral surface projections and slight damage to the envelope structure, acid incubation did not result in gross morphological changes in the viral architecture. The acid-induced change could be detected in the form of greatly increased or decreased binding of many monoclonal antibodies directed to each of the four major antigenic regions of the hemagglutinin. Triggering of viral hemolytic activity and antigenic alterations was similarly pH dependent. In addition, the different pH dependencies of egg-grown and trypsin-treated MDCK-grown viruses coincided with an analogous pH dependence of the antigenic alterations that were observed with these viruses. These observations are compatible with the idea that some of the anti-hemagglutinin antibodies detect conformational changes in the hemagglutinin which are required for the initiation of fusion and hemolysis.     

Bioinformatics models for predicting antigenic variants of influenza A/H3N2 virus

MOTIVATION: Continual and accumulated mutations in hemagglutinin (HA) protein of influenza A virus generate novel antigenic strains that cause annual epidemics. RESULTS: We propose a model by incorporating scoring and regression methods to predict antigenic variants. Based on collected sequences of influenza A/H3N2 viruses isolated between 1971 and 2002, our model can be used to accurately predict the antigenic variants in 1999-2004 (agreement rate = 91.67%). Twenty amino acid positions identified in our model contribute significantly to antigenic difference and are potential immunodominant positions.     

The fine structure of the influenza virus envelope and the concept of transmembrane asymmetry of lateral domains in biomembranes

The molecular architectures of enveloped viruses provide a demonstrative example of perfectly arranged macromolecular complexes, which are formed via highly specific interactions of all structural components. Virus morphogenesis is a multistep process that depends on the concerted actions of many viral and cell components, as well as a fitted organization of main viral constituents. The virus envelope is composed of a mixture of lipid raft and nonraft domains. The domains are recruited from the host cell membrane as discrete well-ordered lipid-protein units during virus assembly. The raft-like nature of the influenza virus A envelope was visualized using a novel approach of cold solubilization of detergent-resistant membranes from intact influenza virus A virions with a mixture of NP40 and octyl glucopyranoside, two nonionic detergents drastically differing in their raft-solubilizing activities. The virus envelope is apparently an ensemble of flexibly joint platforms, which are composed of surface glycoproteins (hemagglutinin and neuraminidase), the matrix M1 protein, and lipids. The modern concept of the transmembrane asymmetry of lateral domains in biological membranes was used to explain the solubilization mechanism revealed. Based on the principles of this concept, the M1 protein shell was assumed to provide a structure-forming framework to support asymmetrical rafts in the virus envelope.     

Novel transmembrane topology of the hepatitis B virus envelope proteins.

The small (S), middle (M) and large (L) envelope proteins of the hepatitis B virus (HBV) are initially synthesized as multispanning membrane proteins of the endoplasmic reticulum membrane. We now demonstrate that all envelope proteins synthesized in transfected cells or in a cell-free system adopt more than one transmembrane orientation. The L protein disposes its N-terminal preS domain both to the cytoplasmic and the luminal side of the membrane. This unusual topology does not depend on interaction with the viral nucleocapsid, but is preserved in secreted empty envelope particles. Pulse-chase analysis suggests a novel process of post-translational translocation leading to the non-uniform topology. Analysis of L deletion mutants indicates that the block to co-translational translocation can be attributed to a specific sequence within preS, suggesting that translocation of L may be regulated. Additional topological heterogeneity is displayed in the S region of the envelope proteins and in the S protein itself, as assayed in a cell-free system. S proteins integrated into microsomal membranes exhibit both a luminal and a cytoplasmic orientation of the internal hydrophilic region carrying the major antigenic determinants. This may explain the unusual partial glycosylation of the HBV envelope proteins.     

Envelope protein(s) derived from influenza virus

Eckert, Edward A. (University of Michigan, Ann Arbor). Envelope protein(s) derived from influenza virus. J. Bacteriol. 91:1907-1910. 1966.-Lipids were extracted from influenza virus, strain PR8, with methanol-chloroform, and the protein residue was dissolved in 67% glacial acetic acid. Hemagglutinating activity and complement-fixing reactivity were markedly reduced or lost during lipid extraction, and then increased after acetic acid treatment and subsequent dialysis. Evidence is presented that the envelope protein(s) responsible for these activities is dissociated in acetic acid and reassociated at neutral pH.     

Plant-produced recombinant influenza vaccine based on virus-like HBc particles carrying an extracellular domain of M2 protein

Conventional influenza vaccines are based on a virus obtained in chicken embryos or its components. The high variability of the surface proteins of influenza virus, hemagglutinin and neuraminidase, requires strain-specific vaccines matching the antigenic specificity of newly emerging virus strains to be developed. A recombinant vaccine based on a highly conservative influenza virus protein M2 fused to a nanosized carrier particle can be an attractive alternative to traditional vaccines. We have constructed a recombinant viral vector based on potato X virus that provides for expression in the Nicotiana benthamiana plants of a hybrid protein M2eHBc consisting of an extracellular domain of influenza virus M2 protein (M2e) fused to hepatitis B core antigen (HBc). This vector was introduced into plant cells by infiltrating leaves with agrobacteria carrying the viral vector. The hybrid protein M2eHBc was synthesized in the infected N. benthamiana plants in an amount reaching 1–2% of the total soluble protein and formed virus-like particles with the M2e peptide presented on the surface. Methods of isolation and purification of M2eHBc particles from plant producers were elaborated. Experiments on mice have shown a high immunogenicity of the plant-produced M2eHBc particles and their protective effect against lethal influenza challenge. The developed transient expression system can be used for production of M2e-based candidate influenza vaccine in plants.     

Glycosylation and an amino acid insertion in the head of hemagglutinin independently affect the antigenic properties of H5N1 avian influenza viruses

Antigenic drift forces us to frequently update influenza vaccines; however, the genetic basis for antigenic variation remains largely unknown. In this study, we used clade 7.2 H5 viruses as models to explore the molecular determinants of influenza virus antigenic variation. We generated eight monoclonal antibodies(MAbs) targeted to the hemagglutinin(HA) protein of the index virus A/chicken/Shanxi/2/2006 and found that two representative antigenically drifted clade 7.2 viruses did not react with six of the eight MAbs. The E131 N mutation and insertion of leucine at position 134 in the HA protein of the antigenically drifted strains eliminated the reactivity of the virus with the MAbs. We also found that the amino acid N131 in the H5 HA protein is glycosylated. Our results provide experimental evidence that glycosylation and an amino acid insertion or deletion in HA influence antigenic variation.     

A cross-neutralizing antibody between HIV-1 and influenza virus

Incessant antigenic evolution enables the persistence and spread of influenza virus in the human population. As the principal target of the immune response, the hemagglutinin (HA) surface antigen on influenza viruses continuously acquires and replaces N-linked glycosylation sites to shield immunogenic protein epitopes using host-derived glycans. Anti-glycan antibodies, such as 2G12, target the HIV-1 envelope protein (Env), which is even more extensively glycosylated and contains under-processed oligomannose-type clusters on its dense glycan shield. Here, we illustrate that 2G12 can also neutralize human seasonal influenza A H3N2 viruses that have evolved to present similar oligomannose-type clusters on their HAs from around 20 years after the 1968 pandemic. Using structural biology and mass spectrometric approaches, we find that two N-glycosylation sites close to the receptor binding site (RBS) on influenza hemagglutinin represent the oligomannose cluster recognized by 2G12. One of these glycan sites is highly conserved in all human H3N2 strains and the other emerged during virus evolution. These two N-glycosylation sites have also become crucial for fitness of recent H3N2 strains. These findings shed light on the evolution of the glycan shield on influenza virus and suggest 2G12-like antibodies can potentially act as broad neutralizers to target human enveloped viruses.     

Intranasal immunization with inactivated influenza virus enhances immune responses to coadministered simian-human immunodeficiency virus-like particle antigens

Intranasal immunization with inactivated influenza virus vaccine can provide protective immunity, whereas many other antigens are less effective when used for mucosal immunization. To determine whether influenza virus could enhance immune responses to an antigen coadministered to a mucosal surface, we studied the intranasal immunization of mice with a mixture of simian-human immunodeficiency virus (SHIV) virus-like particles (VLPs) and inactivated influenza virus. Compared to mice immunized with SHIV VLPs alone, mice coimmunized with SHIV VLPs and inactivated influenza virus showed significant increases in serum immunoglobulin G (IgG) and mucosal IgA antibodies specific to the human immunodeficiency virus envelope protein, neutralizing activities, numbers of gamma interferon- and interleukin 4-secreting lymphocytes, and cytotoxic-T-lymphocyte activities. The levels of enhancement of immune response by coimmunization with inactivated influenza virus were equivalent to those induced by inclusion of immunostimulatory CpG oligodeoxynucleotides (CpG DNA). We also observed that SHIV VLPs bind to influenza virus virions, forming mixed aggregates. These results indicate that inactivated influenza virus can play a role as a mucosal adjuvant to coadministered antigens.     

Pathogenic significance of alpha-N-acetylgalactosaminidase activity found in the hemagglutinin of influenza virus

Serum vitamin D3-binding protein (Gc protein) is the precursor for the principal macrophage activating factor (MAF). The precursor activity of serum Gc protein was reduced in all influenza virus-infected patients. These patient sera contained alpha-N-acetylgalactosaminidase (Nagalase) that deglycosylates Gc protein. Deglycosylated Gc protein cannot be converted to MAF, thus it loses the MAF precursor activity, leading to immunosuppression. An influenza virus stock contained a large amount of Nagalase activity. A sucrose gradient centrifugation analysis of the virus stock showed that the profile of Nagalase activity corresponds to that of hemagglutinating activity. When these gradient fractions were treated with 0.01% trypsin for 30 min, the Nagalase activity of each fraction increased significantly, suggesting that the Nagalase activity resides on an outer envelope protein of the influenza virion and is enhanced by the proteolytic process. After disruption of influenza virions with sodium deoxycholate, fractionation of the envelope proteins with mannose-specific lectin affinity column along with electrophoretic analysis of the Nagalase peak fraction revealed that Nagalase is the intrinsic component of the hemagglutinin (HA). Cloned HA protein exhibited Nagalase activity only if treated with trypsin. Since both fusion capacity and Nagalase activity of HA protein are expressed by proteolytic cleavage, Nagalase activity appears to be an enzymatic basis for the fusion process. Thus, Nagalase plays dual roles in regulating both infectivity and immunosuppression.     

Antigenic and genetic evolution of swine influenza A (H3N2) viruses in Europe

In the early 1970s, a human influenza A/Port Chalmers/1/73 (H3N2)-like virus colonized the European swine population. Analyses of swine influenza A (H3N2) viruses isolated in The Netherlands and Belgium revealed that in the early 1990s, antigenic drift had occurred, away from A/Port Chalmers/1/73, the strain commonly used in influenza vaccines for pigs. Here we show that Italian swine influenza A (H3N2) viruses displayed antigenic and genetic changes similar to those observed in Northern European viruses in the same period. We used antigenic cartography methods for quantitative analyses of the antigenic evolution of European swine H3N2 viruses and observed a clustered virus evolution as seen for human viruses. Although the antigenic drift of swine and human H3N2 viruses has followed distinct evolutionary paths, potential cluster-differentiating amino acid substitutions in the influenza virus surface protein hemagglutinin (HA) were in part the same. The antigenic evolution of swine viruses occurred at a rate approximately six times slower than the rate in human viruses, even though the rates of genetic evolution of the HA at the nucleotide and amino acid level were similar for human and swine H3N2 viruses. Continuous monitoring of antigenic changes is recommended to give a first indication as to whether vaccine strains may need updating. Our data suggest that humoral immunity in the population plays a smaller role in the evolutionary selection processes of swine H3N2 viruses than in human H3N2 viruses.     

Antigenic characterization of flavivirus structural proteins separated by isoelectric focusing.

Isoelectrofocusing of nonionic-detergent-disrupted flaviviruses separated the envelope glycoprotein of 53,000 to 58,000 daltons and the nucleocapsid protein of 14,000 daltons. The envelope protein and nucleocapsid protein were isolated at isoelectric points of pI 7.8 and 10.3, respectively. The antigenic determinants of St. Louis encephalitis, Japanese encephalitis, and dengue virus envelope and nucleocapsid proteins were examined by solid-phase competition radioimmunoassay. By the appropriate selection of antiserum and competing proteins, it was possible to distinguish type-specific, complex-reactive and flavivirus group-reactive antigenic determinants. The envelope glycoproteins of St. Louis encephalitis, Japanese encephalitis, and dengue viruses were found to contain each of these three classes of antigenic determinants. Most of the determinants on the envelope protein were type specific, some were complex reactive, and a small fraction were flavivirus group reactive. The nucleocapsid protein contained only flavivirus group-reactive antigenic determinants.     

Opposite polarity of virus budding and of viral envelope glycoprotein distribution in epithelial cells derived from different tissues

We compared the surface envelope glycoprotein distribution and the budding polarity of four RNA viruses in Fischer rat thyroid (FRT) cells and in CaCo-2 cells derived from a human colon carcinoma. Whereas both FRT and CaCo-2 cells sort similarly influenza hemagglutinin and vesicular stomatitis virus (VSV) G protein, respectively, to apical and basolateral membrane domains, they differ in their handling of two togaviruses, Sindbis and Semliki Forest virus (SFV). By conventional EM Sindbis virus and SFV were shown to bud apically in FRT cells and basolaterally in CaCo-2 cells. Consistent with this finding, the distribution of the p62/E2 envelope glycoprotein of SFV, assayed by immunoelectronmicroscopy and by domain-selective surface biotinylation was predominantly apical on FRT cells and basolateral on CaCo-2 cells. We conclude that a given virus and its envelope glycoprotein can be delivered to opposite membrane domains in epithelial cells derived from different tissues. The tissue specificity in the polarity of virus budding and viral envelope glycoprotein distribution indicate that the sorting machinery varies considerably between different epithelial cell types.     

Structural features of T-cell recognition: applications to vaccine design

T lymphocytes, in contrast to antibodies, appear to recognize primarily a limited number of antigenic sites on any given antigenic protein. We find that a single site can so dominate the T-cell repertoire that the presence or absence of a response to one immunodominant site can make the difference between a high responder and a low responder, even though low responders respond to other sites almost as well as high responders. Besides interaction with major histocompatibility complex (MHC) molecules, the mode by which the antigen is processed into fragments for T-cell recognition also determines which sites are seen. The products of natural processing of the protein appear to be larger than the synthetic peptides and contain structures which hinder binding to certain MHC molecules or to the T-cell receptor. A third factor in immunodominance is the intrinsic structure of the antigenic site. We have shown that amphipathic helices have a higher than random chance of being immunodominant, and have developed a computer program to locate such structures in protein amino acid sequences. We prospectively predicted sites in the malaria circumsporozoite protein and found that the four most widely recognized sites in an endemic area of West Africa were all predicted. Similarly, we identified two helper T-cell sites from the HIV (AIDS virus) envelope, and have now shown that immunization with these elicits enhanced antibody responses to the whole envelope when injected into monkeys. These sites are also recognized by human T cells from volunteers who had been immunized with a recombinant vaccinia virus expressing the HIV envelope. Also, because cytotoxic T lymphocytes (CTLS) may play a critical role in defence against AIDS, we have used a recombinant vaccinia virus and transfectants expressing the HIV envelope gene to induce specific CTLS against the HIV envelope. Using synthetic peptides, we were able to identify the first CTL recognition site in the AIDS virus. These results may contribute to the rational design of vaccines.