A Sendai virus-derived RNA agonist of RIG-I as a virus vaccine adjuvant.

The innate immune system is responsible for recognizing invading pathogens and initiating a protective response. In particular, the retinoic acid-inducible gene 1 protein (RIG-I) participates in the recognition of single- and double-stranded RNA viruses. RIG-I activation leads to the production of an appropriate cytokine and chemokine cocktail that stimulates an antiviral state and drives the adaptive immune system toward an efficient and specific response against the ongoing infection. One of the best-characterized natural RIG-I agonists is the defective interfering (DI) RNA produced by Sendai virus strain Cantell. This 546-nucleotide RNA is a well-known activator of the innate immune system and an extremely potent inducer of type I interferon. We designed an in vitro-transcribed RNA that retains the type I interferon stimulatory properties, and the RIG-I affinity of the Sendai virus produced DI RNA both in vitro and in vivo. This in vitro-synthesized RNA is capable of enhancing the production of anti-influenza virus hemagglutinin (HA)-specific IgG after intramuscular or intranasal coadministration with inactivated H1N1 2009 pandemic vaccine. Furthermore, our adjuvant is equally effective at increasing the efficiency of an influenza A/Puerto Rico/8/34 virus inactivated vaccine as a poly(I·C)- or a squalene-based adjuvant. Our in vitro-transcribed DI RNA represents an excellent tool for the study of RIG-I agonists as vaccine adjuvants and a starting point in the development of such a vaccine.

Hemagglutinin stalk-based universal vaccine constructs protect against group 2 influenza A viruses.

Current influenza virus vaccines contain H1N1 (phylogenetic group 1 hemagglutinin), H3N2 (phylogenetic group 2 hemagglutinin), and influenza B virus components. These vaccines induce good protection against closely matched strains by predominantly eliciting antibodies against the membrane distal globular head domain of their respective viral hemagglutinins. This domain, however, undergoes rapid antigenic drift, allowing the virus to escape neutralizing antibody responses. The membrane proximal stalk domain of the hemagglutinin is much more conserved compared to the head domain. In recent years, a growing collection of antibodies that neutralize a broad range of influenza virus strains and subtypes by binding to this domain has been isolated. Here, we demonstrate that a vaccination strategy based on the stalk domain of the H3 hemagglutinin (group 2) induces in mice broadly neutralizing anti-stalk antibodies that are highly cross-reactive to heterologous H3, H10, H14, H15, and H7 (derived from the novel Chinese H7N9 virus) hemagglutinins. Furthermore, we demonstrate that these antibodies confer broad protection against influenza viruses expressing various group 2 hemagglutinins, including an H7 subtype. Through passive transfer experiments, we show that the protection is mediated mainly by neutralizing antibodies against the stalk domain. Our data suggest that, in mice, a vaccine strategy based on the hemagglutinin stalk domain can protect against viruses expressing divergent group 2 hemagglutinins.

A novel influenza A virus mitochondrial protein that induces cell death.

While searching for alternative reading-frame peptides encoded by influenza A virus that are recognized by CD8+ T cells, we found an abundant immunogenic peptide encoded by the +1 reading frame of PB1. This peptide derives from a novel conserved 87-residue protein, PB1-F2, which has several unusual features compared with other influenza gene products in addition to its mode of translation. These include its absence from some animal (particularly swine) influenza virus isolates, variable expression in individual infected cells, rapid proteasome-dependent degradation and mitochondrial localization. Exposure of cells to a synthetic version of PB1-F2 induces apoptosis, and influenza viruses with targeted mutations that interfere with PB1-F2 expression induce less extensive apoptosis in human monocytic cells than those with intact PB1-F2. We propose that PB1-F2 functions to kill host immune cells responding to influenza virus infection.

Presentation by a major histocompatibility complex class I molecule of nucleoprotein peptide expressed in two different genes of an influenza virus transfectant.

Major histocompatibility (MHC) class I glycoproteins are specialized to present to CD8+ T cells, peptides that originate from proteins synthesized within the cytoplasm. Conventional killed vaccines are unable to get into the cell cytoplasm and therefore fail to expand the CD8+ T cell population. We have created a novel influenza transfectant virus, R10, which carries an immunogenic peptide from the nucleoprotein (NP) of PR8 influenza virus in its hemagglutinin (HA) and another similar peptide in its HK influenza virus NP. The two peptides are both presented by H-2Db and bind with approximately equal affinity. They can compete with one another for binding to H-2Db. Yet in cells infected with R10, both peptides are presented efficiently enough to expand the respective cytotoxic T lymphocyte (CTL) precursors in vivo and to serve as targets for CTL lysis in vitro. It has been proposed that proteins bearing signal sequences may be processed by a transporter-independent pathway. To investigate this, we infected the transporter-deficient cell line RMA-S with the R10 virus to see if the NP peptide expressed by the HA would be presented. The result shows that even the presence of a signal peptide in the HA does not overcome the lack of a transporter function, suggesting that the presentation of both peptides is dependent on functional transporter proteins. Our data also suggest the feasibility of creating by genetic engineering, recombinant vaccines expressing multiple epitopes that can effectively stimulate a cellular immune response.

Variation of influenza A, B, and C viruses.

Influenza is caused by highly variable RNA viruses belonging to the orthomyxovirus group. These viruses are capable of constantly changing the genes coding for their surface proteins as well as for their nonsurface proteins. The mechanisms responsible for these changes in type A influenza viruses include recombination (reassortment) of genes among strains, deletions and insertions in genes, and, frequently, point mutations. In addition, old strains may reappear in the population. Influenza viruses of types B and C appear to vary to a lesser degree. The mechanisms responsible for changes in these viruses are not well characterized.

Genetic composition of a high-yielding influenza A virus recombinant: a vaccine strain against "Swine" influenza.

Analysis of the RNA migration pattern of a high-yielding influenza virus recombinant, X-53, used in vaccine production, reveals that only the two genes coding for hemagglutinin and neuraminidase antigens were derived from the "swine" influenza virus parent. A/New Jersey/11/76, while six were acquired from the A/PR/8/34 (HON1) parent, donor of the high yield characteristic.

Evolution of human influenza A viruses over 50 years: rapid, uniform rate of change in NS gene.

Variation in influenza A viruses was examined by comparison of nucleotide sequences of the NS gene (890 bases) of 15 human viruses isolated over 53 years (1933 to 1985). Changes in the genes accumulate with time, and an evolutionary tree based on the maximum parsimony method can be constructed. The evolutionary rate is approximately 2 X 10(-3) substitution per site per year in the NS genes, which is about 10(6) times the evolutionary rate of germline genes in mammals. This uniform and rapid rate of evolution in the NS gene is a good molecular clock and is compatible with the hypothesis that positive selection is operating on the hemagglutinin (or perhaps some other viral genes) to preserve random mutations in the NS gene.

Measurement of suppressor transfer RNA activity.

Transfer RNA (tRNA) suppression of nonsense mutations in prokaryotic systems has been widely used to study the structure and function of different prokaryotic genes. Through genetic engineering techniques, it is now possible to introduce suppressor (Su+) tRNA molecules into mammalian cells. A quantitative assay of the suppressor tRNA activity in these mammalian cells is described; it is based on the amount of tRNA-mediated readthrough of a terminating codon in the influenza virus NS1 gene after the cells are infected with virus. Suppressor activity in L cells continuously expressing Su+ (tRNAtyr) was 3.5 percent and that in CV-1 cells infected with an SV40- Su+ (tRNAtyr) recombinant was 22.5 percent.

Influenza A viruses with truncated NS1 as modified live virus vaccines: pilot studies of safety and efficacy in horses.

REASONS FOR PERFORMING STUDY: Three previously described NS1 mutant equine influenza viruses encoding carboxy-terminally truncated NS1 proteins are impaired in their ability to inhibit type I IFN production in vitro and are replication attenuated, and thus are candidates for use as a modified live influenza virus vaccine in the horse. HYPOTHESIS: One or more of these mutant viruses is safe when administered to horses, and recipient horses when challenged with wild-type influenza have reduced physiological and virological correlates of disease. METHODS: Vaccination and challenge studies were done in horses, with measurement of pyrexia, clinical signs, virus shedding and systemic proinflammatory cytokines. RESULTS: Aerosol or intranasal inoculation of horses with the viruses produced no adverse effects. Seronegative horses inoculated with the NS1-73 and NS1-126 viruses, but not the NS1-99 virus, shed detectable virus and generated significant levels of antibodies. Following challenge with wild-type influenza, horses vaccinated with NS1-126 virus did not develop fever (>38.5 degrees C), had significantly fewer clinical signs of illness and significantly reduced quantities of virus excreted for a shorter duration post challenge compared to unvaccinated controls. Mean levels of proinflammatory cytokines IL-1beta and IL-6 were significantly higher in control animals, and were positively correlated with peak viral shedding and pyrexia on Day +2 post challenge. CONCLUSION AND CLINICAL RELEVANCE: These data suggest that the recombinant NS1 viruses are safe and effective as modified live virus vaccines against equine influenza. This type of reverse genetics-based vaccine can be easily updated by exchanging viral surface antigens to combat the problem of antigenic drift in influenza viruses.

Genetic analysis of influenza virus.

The newly developed ribonucleoprotein reconstitution and transfection systems have facilitated the characterization of cis and trans functions required for transcription and replication of the influenza virus genome. For the first time, the genome of a negative-strand RNA virus can be manipulated using recombinant DNA techniques.

Antitumor properties of influenza virus vectors.

We are investigating the potential use of influenza virus vectors expressing selected tumor-associated antigens (TAAs) as therapeutic agents in anticancer strategies. Previously, we have shown that recombinant influenza viruses expressing a model TAA mediated the regression of established pulmonary metastases in mice through the induction of cytotoxic T-cell responses (N. P. Restifo et al., Virology, 249: 89-97, 1998). We have now expanded these observations in the mouse model using survival as the end point of the assay. Animals with a high tumor burden showed extended survival times when treated with a recombinant influenza virus expressing a TAA, but they finally succumbed to death. Death was associated with the presence of a small number of large tumors in lungs. Interestingly, these tumors were found to express undetectable levels of the TAAs because of a down-regulation in the TAA-specific mRNA levels. On the other hand, mice with five times lower tumor burden showed complete tumor regression and survival for >6 six months when treated with the recombinant virus. These animals showed protection against a tumor challenge 6 months after treatment. Our results suggest that recombinant influenza viruses may be useful as therapeutic agents for the prevention and treatment of cancers with known TAAs.

A genetically engineered influenza A virus with ras-dependent oncolytic properties.

The NS1 protein of influenza virus is a virulence factor that counteracts the PKR-mediated antiviral response by the host. As a consequence, influenza NS1 gene knockout virus delNS1 (an influenza A virus lacking the NS1 open reading frame) fails to replicate in normal cells but produces infectious particles in PKR-deficient cells. Because it is known that oncogenic ras induces an inhibitor of PKR, we addressed the question of whether the delNS1 virus selectively replicates in cells expressing oncogenic ras. We show that upon transfection and expression of oncogenic N-ras, cells become permissive for productive delNS1 virus replication, suggesting that the delNS1 virus has specific oncolytic properties. Viral growth in the oncogenic ras-transfected cells is associated with a reduction of PKR activation during infection. Moreover, treatment of s.c. established N-ras-expressing melanomas in severe combined immunodeficiency mice with the delNS1 virus revealed that this virus has tumor-ablative potentials. The delNS1 virus does not replicate in nonmalignant cell lines such as melanocytes, keratinocytes, or endothelial cells. The apathogenic nature of the delNS1 virus combined with the selective replication properties of this virus in oncogenic ras-expressing cells renders this virus an attractive candidate for the therapy of tumors with an activated ras-signaling pathway.

An avian tumor virus promoter directs expression of plasmid genes in Escherichia coli.

A sequence in the long terminal repeat (LTR) of avian tumor virus (ATV) DNA was shown to contain a promoter acute in Escherichia coli. For this analysis the bacterial promoters for the tetracycline (Tc) and neomycin (Nm) resistance genes were deleted from different plasmids and replaced with various fragments derived from the ATV DNA. Expression of the drug-resistant phenotype in the recombinant plasmids at levels comparable to or greater than those found with parental bacterial promotes was shown to be dependent on the presence of an intact sequence ranging from nucleotide +19 to -23 (relative to the cap site) in the ATV DNA. Comparison of the consensus bacterial promoter with the nucleotide sequence in this region revealed strong similarities.

The 3' and 5'-terminal sequences of influenza A, B and C virus RNA segments are highly conserved and show partial inverted complementarity.

The 3'- and 5'-terminal nucleotides of the genome segments of an influenza A, B, and C virus were identified by directly sequencing viral RNA using two different sequencing techniques. A high degree of conservation at the 3' ends as well as at the 5' ends was observed among the genome segments of each virus and among the segments of the three different virus types. A uridine-rich region was observed from positions 17 through 22 at the 5' end of each segment. Moreover, the conserved 3' and 5'-terminal sequences showed partial and inverted complementarity. This feature results in very similar sequences at the 3' ends of the plus and minus strand RNAs and may also enable single-strand RNAs of influenza virus to form "panhandle" structures. Inverted complementary repeats may play an important role in initiation of viral RNA replication.

Binding of Plasmodium falciparum 175-kilodalton erythrocyte binding antigen and invasion of murine erythrocytes requires N-acetylneuraminic acid but not its O-acetylated form.

Sialic acid on human erythrocytes is involved in invasion by the human malaria parasite, Plasmodium falciparum. Mouse erythrocytes were used as a reagent to explore the question of whether erythrocyte sialic acid functions as a nonspecific negative charge or whether the sialic acid is a necessary structural part of the receptor for merozoites. Human erythrocytes contain N-acetylneuraminic acid (Neu5Ac), whereas mouse erythrocytes, which are also invaded by P. falciparum merozoites, contain 9-O-acetyl-N-acetylneuraminic acid (Neu5,9Ac2) and N-glycoloylneuraminic acid (Neu5Gc), in addition to Neu5Ac. We compared the effects of sialidase and influenza C virus esterase treatments of mouse erythrocytes on invasion and the binding of a 175-kDa P. falciparum protein (EBA-175), a sialic acid-dependent malaria ligand implicated in the invasion process. Sialidase-treated mouse erythrocytes were refractory to invasion by P. falciparum merozoites and failed to bind EBA-175. Influenza C virus esterase, which converts Neu5,9Ac2 to Neu5Ac, increased both invasion efficiency and EBA-175 binding to mouse erythrocytes. Thus, the parasite and EBA-175 discriminate between Neu5Ac and Neu5,9Ac2, that is, the C-9 acetyl group interferes with EBA-175 binding and invasion by P. falciparum merozoites. This indicates that sialic acid is part of a receptor for invasion.

Alterations of the stalk of the influenza virus neuraminidase: deletions and insertions.

The neuraminidase (NA) of influenza viruses cleaves sialic acids from receptors, prevents self-aggregation and facilitates release of virus during budding from host cells. Although the structure and function of the globular head of the influenza virus NA has been well studied, much less is known about the stalk of the NA, the region between the viral membrane and the globular head. Applying a reverse genetics system, we altered the stalk of the influenza A/WSN/33 virus NA by making deletions, insertions and mutations in this region of the gene. Our data show that the length of the NA stalk can be variable. Deletions of up to 28 amino acids and insertions of up to 41 amino acids in the stalk region did not abolish formation of infectious progeny virus. The data also indicate that the cysteine at position 76 is essential for formation of infectious virus, and that deletions beyond the cysteine did not result in infectious virus. Interestingly, shortening of the length of the stalk region by 28 amino acids resulted in a virus with a markedly reduced growth rate in MDCK cells as compared to that in MDBK cells. An insertion of 41 extra amino acids into the stalk did not significantly interfere with viral growth in MDCK or MDBK cells, which suggests that the stalk region would tolerate the introduction of long foreign sequences.

The cytoplasmic tail of the neuraminidase protein of influenza A virus does not play an important role in the packaging of this protein into viral envelopes.

We have rescued a transfectant influenza virus, NA/TAIL(-), whose neuraminidase (NA) protein lacks the predicted cytoplasmic tail. The virus was attenuated (one log10 reduction) both in tissue culture and in mouse lungs. Attenuation correlated with a 50% reduction of the level of NA in infected cells and levels of incorporation of the tail-less NA protein into viral particles paralleled that in infected cells. This result indicates that the signal for packaging of the NA protein into the viral envelope is not located in its cytoplasmic domain.

The influenza C virus NS gene: evidence for a spliced mRNA and a second NS gene product (NS2 protein).

It has previously been shown that the shortest RNA (RNA 7) of influenza C viruses codes for a nonstructural (NSI) protein (Nakada et al. (1985) J. Virol. 56, 221-226). Experiments reported here indicate that RNA 7 also directs the synthesis of a second nonstructural protein via a spliced mRNA. The amino terminal 62 codons of this NS2 protein appear to be shared with the NS1 protein and the carboxyl terminal 59 amino acids are unique (derived from a +1 open reading frame in the mRNA). Although the size of the C virus NS2 protein is comparable to that of the A and B virus NS2 proteins, the overall arrangement of the C virus NS gene is quite different from that of the A and B virus NS genes. The second (+1) open reading frame of the C virus NS gene is completely overlapped by that of the NS1 protein, whereas the second (+1) open reading frame of the A and B virus NS genes extends to the 3' end of the RNA and only partially (or in some strains not at all) overlaps the NS1 open reading frame.

Mutational analysis of the influenza virus vRNA promoter.

The influenza virus vRNA promoter was characterized: a complete set of single substitution mutants was generated in the fifteen 3' terminal nucleotides of a synthetic model RNA containing the reporter gene chloramphenicol acetyl transferase (CAT). The contribution of each nucleotide to the function of the promoter was tested by an in vitro assay. This system involves reconstitution of template (mutant) RNAs and purified viral polymerase; the system is primer-dependent and yields full-length complementary (c)RNA and not poly A-containing mRNA. The results of this in vitro replication assay suggest that (1) nucleotides 1 to 14 at the 3' terminus comprise the promoter sequence of the vRNA, (2) not all the mutations in the first 14 nucleotides affect vRNA promoter activity equally and (3) changes in positions 2 and 11 have the greatest effect on this promoter activity. In addition, the template (mutant) RNAs were examined in an in vivo assay. This system involves transfection of plasmid DNA-derived template (mutant) RNAs into helper virus-infected cells and measurement of levels of CAT activity. The expression of template RNAs was found to be highly sensitive to mutations in almost any of the first 14 positions. Differences in the results of the in vivo and the in vitro system are possibly due to the presence of overlapping cis-acting signals which are required for replication of vRNA and the expression of mRNA. Deletion and addition of nucleotides at the 3' end of the promoter resulted in a drastic reduction in template activity in both the in vitro and in vivo assays.

Complementation and analysis of an NP mutant of influenza virus.

Cell lines were constructed so as to express the influenza A virus nucleoprotein (NP) at levels approximating 5% of the total NP made throughout virus infection. Two types of cell lines were analyzed. One cell line (NP-5) expresses only the NP while another cell line was constructed which expresses the three viral polymerase proteins in addition to the NP (3PNP-4). Both cell lines were able to complement the growth of an NP mutant, ts56, at the non-permissive temperature. The 3PNP-4 cell line, constructed by transfecting a cell line already expressing the three polymerase proteins, continued to be able to complement viral PB2 mutants. In addition, sequence analysis was performed on the NP gene segment of A/WSN/33 and ts56 viruses. This analysis revealed that the mutant phenotype exhibited by ts56 at non-permissive temperature is due to a single serine to asparagine change (at codon 332) within the protein.