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. 2012 May 21;30(24):3691-702.
doi: 10.1016/j.vaccine.2012.03.025. Epub 2012 Mar 24.

Engineering temperature sensitive live attenuated influenza vaccines from emerging viruses

Bin Zhou  1 , Yan Li, Scott D Speer, Anju Subba, Xudong Lin, David E Wentworth
Affiliations

Engineering temperature sensitive live attenuated influenza vaccines from emerging viruses

Bin Zhou et al. Vaccine. .

Abstract

The licensed live attenuated influenza A vaccine (LAIV) in the United States is created by making a reassortant containing six internal genes from a cold-adapted master donor strain (ca A/AA/6/60) and two surface glycoprotein genes from a circulating/emerging strain (e.g., A/CA/7/09 for the 2009/2010 H1N1 pandemic). Technologies to rapidly create recombinant viruses directly from patient specimens were used to engineer alternative LAIV candidates that have genomes composed entirely of vRNAs from pandemic or seasonal strains. Multiple mutations involved in the temperature-sensitive (ts) phenotype of the ca A/AA/6/60 master donor strain were introduced into a 2009 H1N1 pandemic strain rA/New York/1682/2009 (rNY1682-WT) to create rNY1682-TS1, and additional mutations identified in other ts viruses were added to rNY1682-TS1 to create rNY1682-TS2. Both rNY1682-TS1 and rNY1682-TS2 replicated efficiently at 30°C and 33°C. However, rNY1682-TS1 was partially restricted, and rNY1682-TS2 was completely restricted at 39°C. Additionally, engineering the TS1 or TS2 mutations into a distantly related human seasonal H1N1 influenza A virus also resulted pronounced restriction of replication in vitro. Clinical symptoms and virus replication in the lungs of mice showed that although rNY1682-TS2 and the licensed FluMist(®)-H1N1pdm LAIV that was used to combat the 2009/2010 pandemic were similarly attenuated, the rNY1682-TS2 was more protective upon challenge with a virulent mutant of pandemic H1N1 virus or a heterologous H1N1 (A/PR/8/1934) virus. This study demonstrates that engineering key temperature sensitive mutations (PB1-K391E, D581G, A661T; PB2-P112S, N265S, N556D, Y658H) into the genomes of influenza A viruses attenuates divergent human virus lineages and provides an alternative strategy for the generation of LAIVs.

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Figures

Fig. 1
Fig. 1. Engineering TS-LAIV candidates and effects of temperature on the activity of viral RNA polymerase complex with various PB1 and PB2 gene segments
(A) Schematic diagram of PB1 and PB2 proteins depicting the putative attenuating temperature sensitive mutations designed to create TS-LAIV candidates. TS-1 candidates contain a PB1 with three mutations (PB1-Mut3) and a PB2 with one mutation (PB2-Mut1); TS2 candidates contain a PB1 with three mutations (PB1-Mut3) and a PB2 with four mutations (PB2-Mut4). (B) Viral polymerase activity was analyzed at various temperatures. HEK-293T cells were transfected with pPolI-NS-EGFP plasmid that expresses vRNA-like EGFP RNAs, which can be transcribed by the viral RNA polymerase. The HEK-293T cells were also co-transfected with plasmids expressing the NY1682 PB1 (WT or PB1-Mut3), PB2 (WT, PB2-Mut1, or PB2-Mut4), PA and NP, to generate different viral RNA polymerase complexes. Transfected cells were incubated at 30°C, 33°C, 37°C, and 39°C. EGFP expression was examined using fluorescence microscopy at 48 h post-transfection. The name of the mutant subunit (shown on the top of the panel) in the polymerase complex is used to represent the entire polymerase complex. Representative pictures of at least three independent experiments are shown. (C) Similar to (B) but pPolI-NS-EGFP plasmid was replaced by the pPolI-NS-Luc plasmid that expresses negative-sense virus-like RNA encoding a destabilized firefly luciferase enzyme that can be transcribed by the viral RDRP. Cells were also cotransfected with a Renilla luciferase expression plasmid to control for transfection efficiency. Forty-eight hours posttransfection, both firefly and Renilla luciferase production levels were measured, and Renilla expression was used to normalize the data. At each temperature, the firefly/Renilla relative activity of WT polymerase was set as 100%, and the relative activity of mutant polymerases was expressed of percentage of that. The averages of triplicate experiments are shown, with error bars that represent SD.
Fig. 2
Fig. 2. Replication of H1N1pdm rNY1682-WT, rNY1682-TS1, rNY1682-TS2 viruses, and the FluMist®-H1N1pdm vaccine at various temperatures
MDCK cells were inoculated with an MOI of 0.01 TCID /cell and incubated at 30° 50 C (A), 33°C (B), 37°C (C), and 39°C (D), and culture supernatants were collected at 2, 24, 48, 72, and 96 hpi. To compare the rNY1682-TS2 to the replication of FluMist®-H1N1pdm LAIV, MDCK cells were inoculated with an MOI of 10 TCID /cell and incubated at 37° 50 C (E), and 39°C (F). Culture supernatants were collected at 1, 3, 6, 9, 12, and 24 hpi. Note, the 1 hpi supernatants were collected prior to removal and washing the inocula from the cell monolayers. Viral titers were determined by TCID50 assay using MDCK cells and the limit of detection (LOD) of these assays is indicated by the dotted line. Error bars represent the standard deviation. In (A), (B), (C), and (D), “*” indicates viral titer of rNY1682-TS2 is lower than that of rNY1682-WT (P < 0.001, Bonferroni post-test of ANOVA analysis). In (E) and (F), viral titer of rNY1682-TS2 was not statistically different from that of FluMist®-H1N1pdm vaccine.
Fig. 3
Fig. 3. Replication of seasonal H1N1 rNY1692-WT, rNY1692-TS1, and rNY1692-TS2 viruses at various temperatures
MDCK cells were inoculated with an MOI of 0.01 TCID /cell and incubated at 30° 50 C (A), 33°C (B), 37°C (C), and 39°C (D). Culture supernatants were collected at 2, 24, 48, 72, and 96 hpi and viral titers were determined by TCID50 assay using MDCK cells. *, viral titer of rNY1682-TS2 is lower than that of rNY1682-WT (P < 0.001, Bonferroni post-test of ANOVA analysis). The limit of detection (LOD) of these assays is indicated by the dotted line.
Fig. 4
Fig. 4. Replication of rNY1682-WT, rNY1682-TS1, rNY1682-TS2 and FluMist®-H1N1pdm in the respiratory tracts of mice
Six-week-old female BALB/cJ mice were inoculated intranasally with 104 TCID50 of rNY1682-WT (WT), rNY1682-TS1 (TS1), rNY1682-TS2 (TS2), FluMist®-H1N1pdm (FluMist®), or were mock inoculated. (A) Body weight of inoculated mice (n = 11/group) was recorded daily and is represented as the percent of each animals weight on the day of inoculation (day 0). *, WT caused significant weight loss compared to mock infected mice (P < 0.01, Bonferroni post-test of ANOVA analysis). (B) Viral titers in the lung homogenates of inoculated mice (n = 3/group) were determined for each mouse by TCID50 assay at 2 and 4 dpi. (C) Viral titers from nasal airway of inoculated mice (n = 3/group). The average of each group is shown with error bars determined by standard deviation (A, B, C). Data were analyzed by ANOVA analysis and selected P values are shown for specific comparisons. The limit of detection for virus titration is indicated by the dotted line (B, C).
Fig. 5
Fig. 5. TS-LAIV candidates protect mice from lethal homologous infection
Groups of mice (n = 11/group) were immunized by intranasal inoculation with rNY1682-WT (WT), rNY1682-TS1 (TS1), rNY1682-TS2 (TS2), FluMist®-H1N1pdm (FluMist®), or inoculation media alone (Mock). (A) H1N1pdm viral HA-specific antibody levels in sera collected at 21dpi were determined by Luminex assay. (B) Mice were challenged intranasally, at 30 dpi, with 5 × 104 TCID50 (~100 MLD50) of a lethal mouse-adapted variant of NY1682, and their weight was monitored for 14 days post-challenge, error bars represent ± SD (days 1-4, n=8; days 5-14, n=5). All mice in the mock-immunized group were euthanized for humane reasons by day 5. *, Challenge in FluMist® immunized mice caused significant weight loss compared to that of the TS1, TS2, and WT immunized mice. (P < 0.01, Bonferroni post-test of ANOVA analysis). (C) Viral titers in the lungs were determined at 2 and 4 days post challenge. Data were analyzed by ANOVA analysis and selected P value is shown to compare the TS2 and FluMist® immunized mice at 2 dpc. Each dot represents the antibody titer (A) or virus titer (C) of an individual mouse, and horizontal lines indicate the average of the group. Mock indicates animals that were mock immunized and subsequently challenged. The limit of detection of the assays is indicated by the dotted line.
Fig. 6
Fig. 6. TS2-LAIV candidate protects mice from heterologous challenge
Groups of mice were immunized by intranasal inoculation with rNY1682-TS2 (TS2), FluMist®-H1N1pdm (FluMist®), or diluent alone (Mock). (A) Mice were challenged intranasally, at 30 dpi, with 5 × 104 TCID50 of a lethal reassortant virus that comprises the internal protein expressing gene segments from NY1682 and the HA and NA glycoprotein gene segments from A/PR/8/1934. The weight of the mice was monitored for 14 days post-challenge and it is displayed as the percentage of the animals starting weight. Error bars represent ± the standard deviation. *, Challenge of FluMist® immunized mice caused significant weight loss compared to that TS2 immunized mice. (P < 0.01, Bonferroni post-test of ANOVA analysis). (B) Viral titers in the lungs of the mice were determined at 3 and 6 days post challenge. No significant differences were observed between the TS2 and FluMist® groups (ANOVA analysis). Each dot represents the virus titer of an individual mouse, and horizontal lines indicate the average of the group. Mock indicates animals that were mock immunized and subsequently challenged. The limit of detection is indicated by the dotted horizontal line.
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