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«DISSERTATION INFLUENZA B NS1 TRUNCATION MUTANTS: A LIVE ATTENUATED VACCINE APPROACH Doktor/in der Naturwissenschaften (Dr. rer.nat.) Verfasserin / ...»

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HA and NA genes of B/Thüringen/02/06 virus and the remaining 6 genes of the B/Vienna/33/06 master strain including non-coding regions were sequenced and each cloned into a phW2006 vector, a synthetically produced vector similar to pHW2000 (Hoffmann et al., 2002). It contains a PolI and PolII promoter and terminator for the bicistronic expression of influenza genes. The NS gene was modified by site-directed mutagenesis (Stratagene) to express an NS1 protein of 14, 38, 57 or 80 aa, respectively. Translation was terminated by two consecutive in-frame stop codons. The cDNA downstream of the stop codon cassette up to the splicing signal of NS2/NEP (position 634nt) was deleted by inverse PCR. A mutation at nt position 280 (A-G) in the M gene resulting in a change at amino acid position 86 (M-V) of the M1 protein was introduced by similar way. Reassortants having HA and NA genes from B/Thüringen/02/06 and all other genes from B/Vienna/33/06 (6:2 composition) were rescued by transfection of Vero cells as described previously (Kittel et al., 2005). This 6:2 constellation was chosen because wt isolates containing all 8 genes from B/Vienna/33/06 lack replication in mice. Viruses were named according to the size of the NS1 protein, i.e. NS1-14, NS1-38, NS1-57, NS1-80 and NS1-wt, respectively. The expected sequences of HA, NA and NS genes of rescued viruses were confirmed by sequencing and for NS additionally by analysis of amplified RT-PCR products. All sequences are available upon request.

6.3.3. Isolation, generation and infection of immature monocytederived macrophages Peripheral blood monocytes (PBMCs) obtained from leukocyte-rich buffy coats of healthy donors were purified by standard gradient centrifugation with Ficol-Paque (GE HealthCare). The CD14-positive cells were separated by immunomagnetic sorting using VARIOMACS technique (Miltenyi BiotecGmbH) according to the manufacturer’s procedure. Isolated CD14cells were cultured in polystyrene 6-well plates with hydrophobic surface (Greiner bio-one). 2 x 106 cells per well were cultivated in 2ml RPMI 1640 medium (Invitrogen) containing 10% FCS (Hy Clone) at 37°C in a humidified 5% CO2 atmosphere in the presence of 250 U/ml recombinant human granulocyte-macrophage colony stimulating factor (Berlex) for 7 days. Every second day, cells were fed with 1ml RPMI-1640 medium containing 10% FCS. At day 7, 1 x 106 macrophages were collected and transferred to polystyrene tubes (Falcon), washed with serum-free medium and infected with the viruses described above at an MOI of 2.

After incubation for 30 min, cells were spun down and resuspended in 1ml RPMI-1640 medium (Gibco) containing 10% FCS and incubated at 37°C in 5% CO2. Supernatants were harvested at 24h p.i. and analyzed for the presence of IFN-α, TNF-α, IL-1β and IL-6.

6.3.4. Cytokine measurement in cell-culture supernatants

For cytokine measurement (TNF-α, IL-1β and IL-6) a Luminex 100 system was used (Beadlyte Human Multi-Cytokine Detection System 2) according to the manufacturer’s instructions. The amounts of IFN-α/β were determined by quantitative cytokine-specific ELISA kits (PBL Biomedical Laboratories), following manufacturer’s instructions and shown as one representative of three independent experiments.

6.3.5. Immunization and challenge of mice

Seven animals per group of 6 to 8-week-old female BALB/c mice were i.n.

infected under ether anesthesia with the indicated viruses at 5 x 105 TCID50/mouse or with serum-free Optipro media (control group). Three days post immunization, 3 animals per group were sacrificed to determine the viral load in lungs and nasal turbinates. For this purpose, a 10% tissue extract in SPGN buffer was prepared by grinding the tissue sample with a rotor homogenizer. The suspension was then centrifuged at 2000 x g for 10 min and supernatants were analyzed for viral yield by TCID50/ml. Blood was collected from the murine retro-orbital venous plexus 29 days following priming, the sera were prepared and stored at -20°C. The remaining animals were challenged with influenza NS1 wt strain (5 x 105 TCID50/mouse) 32 days post immunization and were sacrificed 3 days post challenge to analyze the viral load in their lungs as described above.

6.3.6. Influenza-specific IgG ELISA

96-well microtiter plates were coated with influenza B/Thüringen/02/06 adjusted to 50 HA units per well in a carbonate buffer (pH 9.6). Coated plates were incubated over night at 4°C, then washed with PBS containing 0.1% Tween20 (PBS/Tween) and blocked with PBS/Tween plus I-block (Applied Biosystems) 5mg/ml (PBS/Tween/I-block). Serial dilutions of sera from immunized and, as a control, from naïve mice in PBS/Tween/I-block were applied to the plates (50µL/well) and incubated for 1.5h at room temperature. After washing, secondary rabbit anti-mouse IgG1 or IgG2a antibodies conjugated with horseradish peroxidase (Invitrogen) were added. After an additional washing step, plates were stained with UltraTMB substrate (Thermo). The reaction was stopped with 4M H2SO4 and absorbance was measured at a wavelength of 450nm. The cut-off value was defined as the mean value of absorption of blank plus three standard deviations and shown as one representative of two independent experiments.

6.4. RESULTS

Rescue of influenza B mutant viruses encoding C-terminally truncated NS1 proteins in Vero cells. Eight plasmids expressing HA and NA from B/Thüringen/02/06 and the remaining six genes from B/Vienna/33/06 master strain were used to generate influenza B virus NS1 truncation mutants by reverse genetics. We found that this 6:2 gene constellation leads to high rescue efficiency in Vero cells (data not shown).





Translation of NS1 was terminated by two consecutive in-frame stop codons at aa positions 14, 38, 57 and 80, respectively. The non-translated part downstream of the stop codons up to the splicing signal of NS2/NEP was deleted to prevent reversion to wt NS1. A schematic representation of the constructs is shown in Fig. 1a. The NS1 truncation mutants were rescued in Vero cells as was the recombinant wt virus. The resulting rescued truncation viruses containing the N-terminal NS1-specific 14, 38, 57 and 80 aa, respectively, were designated NS1-14, NS1-38, NS1-57, NS1-80 and NS1-wt. The different sizes of the NS gene of generated mutant viruses analyzed by RT-PCR confirmed the identity of the specific truncation as shown in Fig. 1b. Despite several attempts, we did not succeed in rescuing a ∆NS1-B virus, in which the ORF of NS1 is completely deleted and NS2/NEP is expressed as monocistronic mRNA.

Figure 1: Generation of recombinant wt influenza B virus and NS1 truncation mutants. (A) Schematic representations of the NS genes and NS specific mRNAs of the wt, NS1-14, NS1-38, NS1-57 and NS1-80 truncation viruses. The asterisks (**) indicate two consecutive in frame stop codons. The part downstream of the stop codons up to the splicing signal of NEP/NS2 was deleted. (B) RT-PCR analysis of viral NS segments. RNA was isolated from the wt influenza B virus and from the NS1 truncated viruses and the NS segments were reverse transcribed and amplified by PCR. The resulting products were separated on a 2% agarose gel and stained with ethidium bromide. Sizes are indicated.

Influenza B NS1 truncation mutants replicate efficiently in IFNdeficient Vero cells but are attenuated in A549 cells and MEFs of PKR ko mice. NS1 truncation mutants and wt virus were evaluated for their potential to grow in IFN-competent A549 and IFN-deficient Vero cells (Fig. 2a+b). All NS1 truncation mutants showed similar growth kinetics in Vero cells reaching titers in the range of 107 to 108 TCID50/ml, which are comparable to those found with the wt virus. Replication of NS1-truncated viruses was severely attenuated in IFN-competent A549 cells as compared to Vero cells. While NS1-wt virus replicated to high titers of 4.4 x 107 TCID50/ml, the growth of NS1-80 virus was significantly impaired with a difference of approximately 4 orders of magnitude. The NS mutants expressing an NS1 protein of less than 80 aa were even more attenuated showing almost complete restriction in growth in A549 cells with titers close to or below the detection limit of 2 x 102 TCID50/ml. Similar results were observed in human macrophages where only wt virus was able to replicate to 6 logs and all NS mutants did not replicate (data not shown).

In the next step we investigated whether knocking out PKR, an antiviral protein known to be counteracted by the N terminal domain of the influenza B NS1 protein, is sufficient to restore viral growth in interferon competent cells. Therefore, we compared the truncated viruses’ ability to grow in mouse embryonic fibroblasts derived from PKR ko mice (Fig. 2c).

This cell line supported the growth of wt virus of more than 7 logs. The NS1-80 virus replicated almost to wt levels whereas NS truncation mutants expressing a NS1 protein of less than 80 aa showed reduced growth properties by approximately 3 logs.

Figure 2: Growth properties of wt influenza B virus and NS1 truncation mutants in different cell lines. Confluent monolayers of (A) A549 cells, (B) Vero cells and (C) PKR knock out MEFs were infected with indicated viruses at an MOI of 0.01 and incubated at 33°C. At different time points, supernatants were harvested and the infectious titer

was determined by TCID50/ml. All values below the detection limit of 1*102 TCID50/ml

were considered to be 100.

Influenza B NS1 truncation mutants induce antiviral and proinflammatory cytokines in macrophages and human nasal epithelial cells. It is well known that wt influenza viruses are able to antagonize type 1 IFN response as well as cytokine release from infected cells in various cell types (Dauber et al., 2004, Dauber et al., 2006, Egorov et al., 1998, Garcia-Sastre et al., 1998, Stasakova et al., 2005). In order to demonstrate the influence of influenza B virus NS1 protein on cytokine regulation we evaluated the potential of the NS1 truncation mutants NS1-14, NS1-38, NS1-80 and NS1-wt virus to induce IFN-α or IFN-β and major pro-inflammatory cytokines (TNF-α, IL-6 and IL1β) in 7day-old human macrophages and primary nasal epithelial cells, respectively. The NS1-wt virus was fully competent to inhibit the release of IFN-α/β, TNF-α, IL-1 β and IL-6 whereas all NS1-truncated viruses induced markedly higher levels of indicated cytokines in both, macrophages and nasal epithelial cells (see Fig. 3). Although the NS1-80 mutant virus showed intermediate growth capacity in interferon competent cells, it seems that the first 80 aa of NS1 are not sufficient to block the activation of IFN and other pro inflammatory cytokines. Our data imply that carboxy-terminal deletions of the NS1 protein of influenza B viruses are associated with the loss of functions responsible for inhibiting pro-inflammatory and antiviral cytokine production in human macrophages and nasal epithelial cells.

Figure 3: Cytokine release in human macrophages and HNEC infected with wt influenza B virus or NS1 truncation mutants. Human macrophages (black bars) and HNEC (white bars) were infected at an MOI of 2 with indicated viruses. Supernatants from infected cells were harvested 24h p.i. and assayed for TNF-α, IL-1β, IL-6 and IFN-α for macrophages and IFN-β for HNEC, respectively. One representative of three independent experiments is presented as a mean of two measurements +/- SEM. Mock value is subtracted.

Influenza B NS1 truncation mutants are replication-deficient in mice. Due to the attenuated replication pattern in A549 cells we used a mouse model to examine whether the same attenuating effect was observable in vivo. 6 to 8-week-old female BALB/c mice were infected i.n.

with 5 x 105 TCID50/mouse with either NS1 truncation or wt virus. Viral titers in lungs and nasal turbinates of mice were measured by TCID50/ml of a 10% tissue homogenate in Vero cells 3 days p.i. The geometric mean titers are shown in Table 1. The influenza B NS1-wt virus was replicating to a titer of 3.83 x 104 TCID50/ml of 10% tissue homogenate in lung tissue and 6.29 x 103 TCID50/ml in nasal turbinates. All NS1-truncated viruses failed to be re-isolated from lungs and nasal tissue, which indicates a replication-deficient phenotype.

TABLE 1: Replication of wt influenza B virus or NS1 truncation mutants in mice

–  –  –

Influenza B NS1 truncation mutants induce virus specific IgG response in mice. Subsequently, we investigated whether the replication-deficient NS1 truncation mutants induce a humoral immune response in mice. Animals immunized with any of the NS1 mutant viruses showed substantial virus-specific serum antibody levels even after one single i.n. immunization as determined by serum ELISA 29 days post immunization (Fig. 4). Differently to influenza B wt virus, a tendency to polarize the immune response towards Th1 was detected for the NS1 truncation mutants, reflected by a predominance of IgG2a antibodies over IgG1. Serum of non immunized control mice did not yield in any significant titer and did not show any polarization effect.

Figure 4: Detection of virus specific IgG1/IgG2a in serum of primed mice.

BALB/c Mice were immunized i.n. with indicated viruses (5*105 TCID50/mouse) or medium as a control. Serum samples were obtained 29 days after immunization. Virus specific IgG1 and IgG2a geometric mean titers were determined by ELISA and presented as one representative of two independent experiments.

Mice are protected against wt virus challenge after one single immunization with NS1 truncation mutants. To investigate whether a single, i.n. immunization of the replication-deficient NS1 truncation mutants induces protective immunity, mice were challenged with 5 x 105 TCID50/mouse of homologous influenza NS1-wt 32 days post immunization. The challenge virus was derived from a human isolate and did therefore not induce any symptoms such as body weight loss or lethality in mice (data not shown). Mice were sacrificed 3 days post challenge and viral titers were determined in lungs and reported as geometrical mean titers (GMT) TCID50/ml of a 10% tissue homogenate.

Upon challenge with NS1-wt virus, none of the naïve mice was protected against infection, as indicated by viral loads of 2.47 x 104 TCID50/ml in lung tissue. In contrast all mice immunized either with NS1-wt virus or with any of the NS1-truncated viruses were completely protected as demonstrated by the absence of detectable challenge viruses in their lungs (Table 2).



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