Oral Delivery of a Novel Attenuated Salmonella Vaccine Expressing Influenza A Virus Proteins Protects Mice against H5N1 and H1N1 Viral Infection

Attenuated strains of invasive enteric bacteria, such as Salmonella, represent promising gene delivery agents for nucleic acid-based vaccines as they can be administrated orally. In this study, we constructed a novel attenuated strain of Salmonella for the delivery and expression of the hemagglutinin (HA) and neuraminidase (NA) of a highly pathogenic H5N1 influenza virus. We showed that the constructed Salmonella strain exhibited efficient gene transfer activity for HA and NA expression and little cytotoxicity and pathogenicity in mice. Using BALB/c mice as the model, we evaluated the immune responses and protection induced by the constructed Salmonella-based vaccine. Our study showed that the Salmonella-based vaccine induced significant production of anti-HA serum IgG and mucosal IgA, and of anti-HA interferon-γ producing T cells in orally vaccinated mice. Furthermore, mice orally vaccinated with the Salmonella vaccine expressing viral HA and NA proteins were completely protected from lethal challenge of highly pathogenic H5N1 as well as H1N1 influenza viruses while none of the animals treated with the Salmonella vaccine carrying the empty expression vector with no viral antigen expression was protected. These results suggest that the Salmonella-based vaccine elicits strong antigen-specific humoral and cellular immune responses and provides effective immune protection against multiple strains of influenza viruses. Furthermore, our study demonstrates the feasibility of developing novel attenuated Salmonella strains as new oral vaccine vectors against influenza viruses.     

Evaluation of live attenuated influenza a virus h6 vaccines in mice and ferrets

Avian influenza A virus A/teal/HK/W312/97 (H6N1) possesses seven gene segments that are highly homologous to those of highly pathogenic human influenza H5N1 viruses, suggesting that a W312-like H6N1 virus might have been involved in the generation of the A/HK/97 H5N1 viruses. The continuous circulation and reassortment of influenza H6 subtype viruses in birds highlight the need to develop an H6 vaccine to prevent potential influenza pandemics caused by the H6 viruses. Based on the serum antibody cross-reactivity data obtained from 14 different H6 viruses from Eurasian and North American lineages, A/duck/HK/182/77, A/teal/HK/W312/97, and A/mallard/Alberta/89/85 were selected to produce live attenuated H6 candidate vaccines. Each of the H6 vaccine strains is a 6:2 reassortant ca virus containing HA and NA gene segments from an H6 virus and the six internal gene segments from cold-adapted A/Ann Arbor/6/60 (AA ca), the master donor virus that is used to make live attenuated influenza virus FluMist (intranasal) vaccine. All three H6 vaccine candidates exhibited phenotypic properties of temperature sensitivity (ts), ca, and attenuation (att) conferred by the internal gene segments from AA ca. Intranasal administration of a single dose of the three H6 ca vaccine viruses induced neutralizing antibodies in mice and ferrets and fully protected mice and ferrets from homologous wild-type (wt) virus challenge. Among the three H6 vaccine candidates, the A/teal/HK/W312/97 ca virus provided the broadest cross-protection against challenge with three antigenically distinct H6 wt viruses. These data support the rationale for further evaluating the A/teal/HK/W312/97 ca vaccine in humans.     

Temperature-sensitive mutants of the exosome subunit Rrp43p show a deficiency in mRNA degradation and no longer interact with the exosome

Rrp43p is a Saccharomyces cerevisiae exosome subunit involved in pre-rRNA processing which is found both in the nucleus and in the cytoplasm. So far, no function has been assigned to the cytoplasmic fraction of Rrp43p. We have addressed Rrp43p function by analyzing mRNA stability in three rrp43 temperature-sensitive (ts) strains, which carry different ts alleles (rrp43-1, rrp43-2 and rrp43-3), and by analyzing Rrp43p interactions with the remaining exosome subunits. In the ts strains, endogenous mRNAs (ACT1 and PAB1), as well as a heterologous reporter mRNA (CATpG) showed longer half-lives, relative to a control strain carrying wild-type RRP43. The mutants also accumulated a degradation intermediate of the reporter mRNA that is typical of defective mRNA decay. These results allow us to propose that Rrp43p is required for mRNA degradation. Rrp43p interacts with the exosome complex via Rrp46p, as determined by two-hybrid analyses. Interestingly, the rrp43 ts mutant proteins do not interact with Rrp46p, indicating that the ts phenotype may be caused by disruption of the Rrp43p– Rrp46p interaction. The ts strains also showed a pre-rRNA processing defect, which is consistent with previous studies on Rrp43p function.     

A Novel M2e Based Flu Vaccine Formulation for Dogs

Background

The USA 2004 influenza virus outbreak H3N8 in dogs heralded the emergence of a new disease in this species. A new inactivated H3N8 vaccine was developed to control the spread of the disease but, as in humans and swine, it is anticipated that the virus will mutate shift and drift in the dog population. Therefore, there is a need for a vaccine that can trigger a broad protection to prevent the spread of the virus and the emergence of new strains.

Methodology and Principal Findings

The universal M2e peptide is identical in almost all the H3N8 influenza strains sequenced to date and known to infect dogs. This epitope is therefore a good choice for development of a vaccine to provide broad protection. Malva mosaic virus (MaMV) nanoparticles were chosen as a vaccine platform to improve the stability of the M2e peptide and increase its immunogenicity in animals. The addition of an adjuvant (OmpC) purified from Salmonella typhi membrane in the vaccine formulation increased the immune response directed to the M2e peptide significantly and enlarged the protection to include the heterosubtypic strain of influenza in a mouse model. An optimal vaccine formulation was also shown to be immunogenic in dogs.

Conclusions and Significance

The MaMV vaccine platform triggered an improved immune response directed towards the universal M2e peptide. The adjuvant OmpC increased the immune response to the M2e peptide and protection to a heterosubtypic influenza strain that harbors a different M2e peptide in a mouse model. Antibodies generated by the vaccine formulation showed cross-reactivity with M2e peptides derived from influenza strains H9N2, H5N1 and H1N1. The vaccine formulation shows a potential for commercialization of a new M2e based vaccine in dogs.     

Molecular Basis of Live-Attenuated Influenza Virus

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

Alterations in glycoprotein gB specified by mutants and their partial revertants in herpes simplex virus type 1 and relationship to other mutant phenotypes

The tsB5 mutant of herpes simplex virus type 1 (HSV-1) strain HFEM was shown previously to be temperature sensitive for accumulation of the mature form of glycoprotein gB, for production or activity of a factor required in virus-induced cell fusion, and for production of virions with normal levels of infectivity. In addition, a previous study showed that virions produced by tsB5 at permissive temperature were more thermolabile than HFEM virions and contained altered gB that did not assume the dimeric conformation characteristic of HFEM. Results presented here demonstrate that, at permissive temperature, tsB5 differs from HFEM in another respect: plaques formed by tsB5 are syncytial on Vero cells (but not on HEp-2 cells), whereas plaques formed by HFEM are nonsyncytial on both cell types. In addition, our results indicate that tsB5 produces an oligomeric form of gB, but that it differs in electrophoretic mobility and stability from the gB dimers of HFEM. The major purpose of this study was to investigate the dependence of the various tsB5 mutant phenotypes on the temperature sensitivity of gB accumulation and on the alterations in oligomeric conformation of gB produced at permissive temperature. For this work the following HSV-1 strains related to tsB5 or HFEM were analyzed: (i) phenotypic revertants selected from tsB5 stocks for nonsyncytial plaque morphology on Vero cells or for ability to form plaques at restrictive temperature (38.5°C); (ii) a plaque morphology variant of HFEM selected for its syncytial phenotype on Vero cells; (iii) temperature-sensitive recombinants previously isolated from a cross between tsB5 and the non-temperature-sensitive syncytial strain HSV-1(MP); and (iv) a phenotypic revertant selected from one of the recombinant stocks for its ability to form plaques at 39°C. These strains were all compared with tsB5 and HFEM at three different temperatures in two different cell lines with respect to plaque formation, yield of infectious progeny, virus-induced cell fusion, and accumulation of gB. The results of our analyses on all the strains tested revealed the following correlations between mutant phenotypes and the accumulation and oligomeric conformation of gB. (i) There was a direct and quantitative relationship between the accumulation in infected cells of infectious progeny and of the mature form of gB, providing strong support for the hypothesis that this form of gB is necessary to the production of infectious virions. The oligomeric conformation of gB characteristic of HFEM is apparently not required for virion infectivity; nor was virion thermostability necessarily related to the presence of the HFEM-like oligomeric form of gB. (ii) The previously reported correlation between temperature sensitivity of gB accumulation and virus-induced cell fusion was confirmed for tsB5 and extended to other virus strains, and coordinate reversion of these traits was also demonstrated, providing support for the hypothesis that gB has a role in virus-induced cell fusion. At 37°C, intermediate between permissive and restrictive temperatures, some of the mutants and partial revertants induced cell fusion despite reduced accumulations of the mature form of gB, suggesting that the amount of mature gB present did not determine the extent of fusion and that other forms of gB as well as other factors should be investigated with regard to the process of cell fusion. (iii) Some of the mutants and partial revertants could form plaques at 38.5°C despite reduced ccumulations of gB and infectious progeny, indicating that the cell-to-cell transmission of viral infection may be at least in part independent of these factors.     

Three Amino Acid Substitutions in the L Protein of the Human Parainfluenza Virus Type 3 cp45 Live Attenuated Vaccine Candidate Contribute to Its Temperature-Sensitive and Attenuation Phenotypes

Studies were initiated to define the genetic basis of the temperature-sensitive (ts), cold adaptation (ca), and attenuation (att) phenotypes of the human parainfluenza virus type 3 (PIV3) cp45 live attenuated vaccine candidate. Genetic data had previously suggested that the L polymerase protein of cp45, which contains three amino acid substitutions at positions 942, 992, and 1558, contributed to its temperature sensitivity (R. Ray, M. S. Galinski, B. R. Heminway, K. Meyer, F. K. Newman, and R. B. Belshe, J. Virol. 70:580–584, 1996; A. Stokes, E. L. Tierney, C. M. Sarris, B. R. Murphy, and S. L. Hall, Virus Res. 30:43–52, 1993). To study the individual and aggregate contributions that these amino acid substitutions make to the ts, att, and ca phenotypes of cp45, seven PIV3 recombinant viruses (three single, three double, and one triple mutant) representing all possible combinations of the three amino acid substitutions were recovered from full-length antigenomic cDNA and analyzed for their ts, att, and ca phenotypes. None of the seven mutant recombinant PIVs was cold adapted. The substitutions at L protein amino acid positions 992 and 1558 each specified a 10⁵-fold reduction in plaque formation in cell culture at 40°C, whereas the substitution at position 942 specified a 300-fold reduction. Thus, each of the three mutations contributes individually to the ts phenotype. The triple recombinant which possesses an L protein with all three mutations was almost as temperature sensitive as cp45, indicating that these mutations are the major contributors to the ts phenotype of cp45. The three individual mutations in the L protein each contributed to restricted replication in the upper or lower respiratory tract of hamsters, and this likely contributes to the observed stability of the ts and att phenotypes of cp45 during replication in vivo. Importantly, the recombinant virus possessing L protein with all three mutations was as restricted in replication as was the cp45 mutant in both the upper and lower respiratory tracts of hamsters, indicating that the L gene of the cp45 virus is a major attenuating component of this candidate vaccine.Human parainfluenza virus type 3 (PIV3), a member of the genus Paramyxovirus of the family Paramyxoviridae, has a single-stranded, negative-sense RNA genome that is 15,462 nucleotides (nt) in length. PIV3 is a major cause of serious lower respiratory illness requiring hospitalization of infants and young children (4). A vaccine is needed to prevent the severe disease caused by this virus, and two live attenuated candidate PIV3 vaccines are currently being evaluated in humans (21, 22). One of these is a bovine strain of PIV3 that is discussed elsewhere (21). The other was produced by passaging the human PIV3 wild type (wt), JS strain, at low temperature for 45 passages to yield the PIV3 cold-passaged 45 (cp45) candidate vaccine virus (1). The cp45 vaccine virus possesses temperature-sensitive (ts), cold adaptation (ca), and attenuation (att) phenotypes (1, 6). The att phenotype is manifested by attenuation of replication in the upper and lower respiratory tracts of rodents and nonhuman primates (6, 15, 16). In addition, the virus appears to be satisfactorily attenuated, phenotypically stable, and immunogenic in seronegative infants and children (22) and therefore is a promising vaccine candidate. Comparison of the complete nucleotide sequences of the cp45 and wt (JS strain) viruses indicated that cp45 possesses multiple point mutations in coding and noncoding regions of the genome, including three point mutations in the L polymerase gene that each encode an amino acid substitution (34).Previously, we recovered recombinant PIV3 from a full-length antigenomic cDNA clone of wt PIV3 (JS strain), the parent of cp45, and demonstrated that the recovered virus was not temperature sensitive and replicated to a level in the respiratory tract of rodents comparable to that of the biologically derived wt (JS strain) virus (9). This meant that it was now possible to systematically examine the genetic basis of the att phenotype of PIV3 candidate vaccines such as cp45. Since the polymerase genes are the sites of many att and ts mutations for influenza virus and respiratory syncytial virus (RSV) (8, 11, 20, 24, 32) and since preliminary data suggested that the L gene of cp45 possesses a ts mutation (30), we initiated our studies to examine the genetic basis of attenuation of cp45 by introducing the mutations yielding the seven possible combinations of the three amino acid substitutions present in the L gene of cp45 into the cDNA clone of its wt (JS strain) parent. Seven recombinant viruses (three single, three double, and one triple mutant) were isolated and analyzed for their ts and att phenotypes. Analysis of these mutants indicated that each of the three mutations in the L protein is a major separate contributor to the ts and att phenotypes of this promising vaccine candidate. Furthermore, this study illustrates the usefulness of the newly developed reverse-genetics systems for characterizing and manipulating a nonsegmented negative-strand virus.     

Matrix-M™ adjuvation broadens protection induced by seasonal trivalent virosomal influenza vaccine

Background

Influenza virus infections are responsible for significant morbidity worldwide and therefore it remains a high priority to develop more broadly protective vaccines. Adjuvation of current seasonal influenza vaccines has the potential to achieve this goal.

Methods

To assess the immune potentiating properties of Matrix-M?, mice were immunized with virosomal trivalent seasonal vaccine adjuvated with Matrix-M?. Serum samples were isolated to determine the hemagglutination inhibiting (HAI) antibody titers against vaccine homologous and heterologous strains. Furthermore, we assess whether adjuvation with Matrix-M? broadens the protective efficacy of the virosomal trivalent seasonal vaccine against vaccine homologous and heterologous influenza viruses.

Results

Matrix-M? adjuvation enhanced HAI antibody titers and protection against vaccine homologous strains. Interestingly, Matrix-M? adjuvation also resulted in HAI antibody titers against heterologous influenza B strains, but not against the tested influenza A strains. Even though the protection against heterologous influenza A was induced by the adjuvated vaccine, in the absence of HAI titers the protection was accompanied by severe clinical scores and body weight loss. In contrast, in the presence of heterologous HAI titers full protection against the heterologous influenza B strain without any disease symptoms was obtained.

Conclusion

The results of this study emphasize the promising potential of a Matrix-M?-adjuvated seasonal trivalent virosomal influenza vaccine. Adjuvation of trivalent virosomal vaccine does not only enhance homologous protection, but in addition induces protection against heterologous strains and thus provides overall more potent and broad protective immunity.

     

Identification of a gene for alpha-tubulin in Aspergillus nidulans.

This paper demonstrates that revertants of temperature-sensitive benA (β-tubulin) mutations in Aspergillus nidulans can be used to identify proteins which interact with β-tubulin. Three benomyl-resistant benA (β-tubulin) mutants of Aspergillus nidulans, BEN 9, BEN 15 and BEN 19, were found to be temperature-sensitive (ts^?) for growth. Temperature sensitivity co-segregated with benomyl resistance among the progeny of outcrosses of BEN 9, 15 and 19 to a wild-type strain, FGSC#99, indicating that temperature sensitivity was caused by mutations in the benA gene in these strains. Eighteen revertants to ts⁺ were isolated by selection at the restrictive temperature. Four had back-mutations in the benA gene and fourteen carried extragenic suppressor mutations. Two of the back-mutated strains had β-tubulins which differed from the β-tubulins of their parental strains by one (1^?) or two (2^?) negative charges on two-dimensional gel electrophoresis. Although the β-tubulins of the extragenic suppressor strains were all electrophoretically identical to those of the parental strains, one of the suppressor strains, BEN 9R7, had an electrophoretic abnormality in α1-tubulin (1⁺). A heterozygous diploid between this strain and a strain with wild-type α1-tubulin was found to have both wild-type and mutant (1⁺) α1-tubulins. This experiment rules out post-translational modification as a possible cause of the α1-tubulin abnormality. Thus the suppressor mutation in BEN 9R7 must be in a structural gene for α1-tubulin. We propose that this gene be designated tubA to denote that it is a gene for α1-tubulin in A. nidulans.     

A physiological connection between tmRNA and peptidyl-tRNA hydrolase functions in Escherichia coli

The bacterial ssrA gene codes for a dual function RNA, tmRNA, which possesses tRNA-like and mRNA-like regions. The tmRNA appends an oligopeptide tag to the polypeptide on the P-site tRNA by a trans-translation process that rescues ribosomes stalled on the mRNAs and targets the aberrant protein for degradation. In cells, processing of the stalled ribosomes is also pioneered by drop-off of peptidyl-tRNAs. The ester bond linking the peptide to tRNA is hydrolyzed by peptidyl-tRNA hydrolase (Pth), an essential enzyme, which releases the tRNA and the aberrant peptide. As the trans-translation mechanism utilizes the peptidyl-transferase activity of the stalled ribosomes to free the tRNA (as opposed to peptidyl-tRNA drop-off), the need for Pth to recycle such tRNAs is bypassed. Thus, we hypothesized that tmRNA may rescue a defect in Pth. Here, we show that overexpression of tmRNA rescues the temperature-sensitive phenotype of Escherichia coli (pth^ts). Conversely, a null mutation in ssrA enhances the temperature-sensitive phenotype of the pth^ts strain. Consistent with our hypothesis, overexpression of tmRNA results in decreased accumulation of peptidyl-tRNA in E.coli. Furthermore, overproduction of tmRNA in E.coli strains deficient in ribosome recycling factor and/or lacking the release factor 3 enhances the rescue of pth^ts strains. We discuss the physiological relevance of these observations to highlight a major role of tmRNA in decreasing cellular peptidyl-tRNA load.     

Isolation of Temperature-Sensitive Conditional Lethal Mutants of “Fixed” Rabies Virus

In an attempt to induce temperature-sensitive (ts) conditional lethal mutants of rabies virus, stocks of a plaque-purified substrain of strain CVS fixed rabies virus were subjected to mutagenesis by HNO₂, 5-fluorouracil, or 5-azacytidine. It was necessary to prepare virus stocks from clones of mutagenized virus selected at random and to test subsequently each stock for possible ts characteristics by measuring its relative capacity for growth at permissive (33 C) and nonpermissive (40.5 C) temperatures. Five ts mutants were detected in tests of 161 clones of mutagenized virus. Each of the mutants exhibited a remarkably low incidence of reversion and little demonstrable “leakiness.” One of the five ts mutants (ts2), which formed formed very small plaques, and another (ts1), which formed plaques of only slightly reduced size, were further characterized. Virus ts1 was more thermostable at 40.5 C than the parental virus, but the ts2 mutant was unchanged in this respect. The ts1 virus exhibited normal pathogenicity for mice, but ts2 virus caused a very irregular death pattern. Both deaths and survivors immune to rabies virus challenge were noted in all groups of mice inoculated with ts2 virus, regardless of the virus dose.     

Generation of Influenza Virus from Avian Cells Infected by Salmonella Carrying the Viral Genome

Domestic poultry serve as intermediates for transmission of influenza A virus from the wild aquatic bird reservoir to humans, resulting in influenza outbreaks in poultry and potential epidemics/pandemics among human beings. To combat emerging avian influenza virus, an inexpensive, heat-stable, and orally administered influenza vaccine would be useful to vaccinate large commercial poultry flocks and even migratory birds. Our hypothesized vaccine is a recombinant attenuated bacterial strain able to mediate production of attenuated influenza virus in vivo to induce protective immunity against influenza. Here we report the feasibility and technical limitations toward such an ideal vaccine based on our exploratory study. Five 8-unit plasmids carrying a chloramphenicol resistance gene or free of an antibiotic resistance marker were constructed. Influenza virus was successfully generated in avian cells transfected by each of the plasmids. The Salmonella carrier was engineered to allow stable maintenance and conditional release of the 8-unit plasmid into the avian cells for recovery of influenza virus. Influenza A virus up to 10⁷ 50% tissue culture infective doses (TCID₅₀)/ml were recovered from 11 out of 26 co-cultures of chicken embryonic fibroblasts (CEF) and Madin-Darby canine kidney (MDCK) cells upon infection by the recombinant Salmonella carrying the 8-unit plasmid. Our data prove that a bacterial carrier can mediate generation of influenza virus by delivering its DNA cargoes into permissive host cells. Although we have made progress in developing this Salmonella influenza virus vaccine delivery system, further improvements are necessary to achieve efficient virus production, especially in vivo.     

Genetic Analyses of ENDOTHIA PARASITICA: Linkage Data for Four Single Genes and Three Vegetative Compatibility Types

The loci cre, met and ts segregate independently in Endothia parasitica. The phenotype brown (br) seems to be determined by an allele at or very near the cre locus. The vegetative compatibility types (v-c) 5 and 39 are determined by different alleles at a locus that is not linked to cre, met or ts. Analysis of two crosses of v-c 5 strains by v-c 10 strains provides evidence that these two v-c groups are different at 5 or more v-c loci.     

Bacterially Produced Recombinant Influenza Vaccines Based on Virus-Like Particles

Although current influenza vaccines are effective in general, there is an urgent need for the development of new technologies to improve vaccine production timelines, capacities and immunogenicity. Herein, we describe the development of an influenza vaccine technology which enables recombinant production of highly efficient influenza vaccines in bacterial expression systems. The globular head domain of influenza hemagglutinin, comprising most of the protein''s neutralizing epitopes, was expressed in E. coli and covalently conjugated to bacteriophage-derived virus-like particles produced independently in E.coli. Conjugate influenza vaccines produced this way were used to immunize mice and found to elicit immune sera with high antibody titers specific for the native influenza hemagglutinin protein and high hemagglutination-inhibition titers. Moreover vaccination with these vaccines induced full protection against lethal challenges with homologous and highly drifted influenza strains.     

Strain identification of commercial influenza vaccines by mass spectrometry

Current influenza vaccine manufacturing and testing timelines require that the constituent hemagglutinin (HA) and neuraminidase (NA) strains be selected each year approximately 10 months before the vaccine becomes available. The threat of a pandemic influenza outbreak requires that more rapid testing methods be found. We have developed a specialized on-filter sample preparation method that uses both trypsin and chymotrypsin to enzymatically digest peptide-N-glycosidase F (PNGase F)-deglycosylated proteins in vaccines. In tandem with replicate liquid chromatography-mass spectrometry (LC-MS) analyses, this approach yields sufficient protein sequencing data (>85% sequence coverage on average) for strain identification of HA and NA components. This has allowed the confirmation, and in some cases the correction, of the identity of the influenza strains in recent commercial vaccines as well as the correction of some ambiguous HA sequence annotations in available databases. This method also allows the identification of low-level contaminant egg proteins produced during the manufacturing process.     

Optimization and Utilization of Agrobacterium-mediated Transient Protein Production in Nicotiana

Agrobacterium-mediated transient protein production in plants is a promising approach to produce vaccine antigens and therapeutic proteins within a short period of time. However, this technology is only just beginning to be applied to large-scale production as many technological obstacles to scale up are now being overcome. Here, we demonstrate a simple and reproducible method for industrial-scale transient protein production based on vacuum infiltration of Nicotiana plants with Agrobacteria carrying launch vectors. Optimization of Agrobacterium cultivation in AB medium allows direct dilution of the bacterial culture in Milli-Q water, simplifying the infiltration process. Among three tested species of Nicotiana, N. excelsiana (N. benthamiana × N. excelsior) was selected as the most promising host due to the ease of infiltration, high level of reporter protein production, and about two-fold higher biomass production under controlled environmental conditions. Induction of Agrobacterium harboring pBID4-GFP (Tobacco mosaic virus-based) using chemicals such as acetosyringone and monosaccharide had no effect on the protein production level. Infiltrating plant under 50 to 100 mbar for 30 or 60 sec resulted in about 95% infiltration of plant leaf tissues. Infiltration with Agrobacterium laboratory strain GV3101 showed the highest protein production compared to Agrobacteria laboratory strains LBA4404 and C58C1 and wild-type Agrobacteria strains at6, at10, at77 and A4. Co-expression of a viral RNA silencing suppressor, p23 or p19, in N. benthamiana resulted in earlier accumulation and increased production (15-25%) of target protein (influenza virus hemagglutinin).     

Characterization of vaccinia virus DNA replication mutants with lesions in the D5 gene

The vaccinia virus D5 gene encodes a 90 kDa early protein that is essential for viral DNA replication. In this report we map and explore the phenotypes of the temperature sensitive mutants bearing lesions in this gene:ts17,ts24,ts69, (WR strain) andts6389 (IHD strain). Viral DNA synthesis was virtually undetectable during non-permissive infections performed withts17, and incorporation of³H-thymidine ceased rapidly when cultures were shifted to the non-permissive temperature in the midst of replication. The D5 protein may therefore be involved in DNA synthesis at the replication fork. The lesions of the four mutants were localized within the D5orf by marker rescue, and the single nucleotide changes responsible for thets phenotype of the three WR mutants were identified. Unexpectedly, the three alleles with N-terminal mutations were impaired in marker rescue when homologous recombination with small (<2 kb), intragenic DNA fragments at 39.5°C was required. This deficiency was not due to degradation of transfected DNA under non-permissive conditions. Efficient marker rescue could be restored by incubation at the permissive temperature for a brief period after transfection, suggesting a requirement for functional D5 in genome/plasmid recombination. Marker rescue under non-permissive conditions could alternatively be restored by co-transfection of unlinked but contiguous DNA sequences.     

Host genetic background and the innate inflammatory response of lung to influenza virus

Many studies of influenza severity have focused on viral properties that confer virulence, whereas the contributory role of the host genetic background on infection severity remains largely unexplored. In this study, we measure the impact of inoculation with influenza virus in four strains of inbred mice - BALB/cByJ, C57BL/6 J, A/J, and DBA/2 J. To evaluate the extent to which responses are inherent to lung per se, as opposed to effects of the systemic response to lung infection, we also measured cytokines and chemokines in lung slices exposed to the virus in vitro. Finally, we evaluate the in vivo responses of recombinant inbred (RI) and select consomic strains of mice to search for genomic loci that contribute to phenotypic variance in response to influenza infection. We found marked variation among mouse strains after challenge with virus strain A/HKX31(H3N2), consistent with previous reports using more virulent strains. Furthermore, response patterns differ after in vivo versus in vitro exposure of lung to virus, supporting a predominant role of the systemic host inflammatory response in generating the strain differences. These results add to the body of information pointing to host genotype as a crucial factor in mediating the severity of influenza infections.