3.1. Generation of experimental H1N1pdm live attenuated influenza vaccines (LAIVs)
We created the recombinant wild type H1N1pdm virus (A/New York/1682/2009 (rNY1682)) directly from an original clinical specimen using M-RTPCR genomic amplification and recombination-based cloning techniques [36
]. LAIV candidate rNY1682-TS1 was designed to incorporate five mutations in the PB1, PB2, and NP genes that were identified as the major determinants responsible for the temperature sensitive phenotype of ca
]. These amino acid mutations include three mutations in PB1 (PB1-Mut3: K391E, D581G, A661T) and one mutation in PB2 (PB2-Mut1: N265S) (). No mutation was introduced into the NP vRNA because the NY1682 wild-type virus already contained NP-D34G, which was shown to play a role in the ts
phenotype of ca
Since the temperature sensitive (ts)
phenotype of the ca
A/AA/6/60 master donor strain is a polygenic trait and is related to the gene constellation, the four mutations introduced to create rNY1682-TS1 might not be sufficient to achieve an ideal ts
phenotype in the novel H1N1pdm virus gene constellation. Thus, to enhance attenuation and safety of the TS-LAIV prototype (NY1682-TS1) we also introduced three additional PB2 mutations (P112S, N556D, Y658H), which were previously identified and confirmed as ts
markers in ts
A/Ud/307/72 and ts
]. The rNY1682-TS2 LAIV candidate was designed to incorporate these additional PB2 mutations (PB2-Mut4: P112S, N265S, N556D, Y658H) into the rNY1682 genome (). This panel of engineered viruses consists of rNY1682-WT, rNY1682-TS1 and rNY1682-TS2, and they are all encoded entirely by vRNAs derived from NY1682. The presence of desired mutations in the TS-LAIVs rescued by reverse genetics was confirmed by sequencing (data not shown).
3.2. Influence of the various mutations on the viral RNA polymerase activity at various temperatures
A mini-genome replication assay system was used to determine if the mutations introduced into the PB1 and PB2 genes of NY1682 H1N1pdm confer temperature sensitivity to the viral RNA dependent RNA polymerase (RDRP). A pPolI-NS-EGFP plasmid expressing vRNA-like EGFP RNA under the control of a human PolI promoter was co-transfected with four plasmids expressing PB1 (PB1-WT or PB1-Mut3), PB2 (PB2-WT, PB2-Mut1, or PB2-Mut4), PA and NP proteins into HEK-293T cells and they were incubated at various temperatures. At 48 h post-transfection, EGFP expression in the cells was examined using fluorescence microscopy. All combinations of the RNA polymerase complex resulted in similar EGFP expression levels at 30°C and 33°C (). Reduction of EGFP expression was not observed at any temperature when PB2-Mut1 was used in the polymerase complex (). In contrast, at 37°C, EGFP expression in PB2-Mut4 complex was weaker than that expressed by the PB2-WT or PB2-Mut1 (). Additionally, the PB1-Mut3/PB2-Mut4 combination showed very little EGFP expression at 37°C (). At 39°C, PB1-Mut3 alone and the PB1-Mut3/PB2-Mut1 combination were weaker than the PB2-WT, but still visible; whereas, PB2-Mut4 and the PB1-Mut3/PB2-Mut4 combination had no visible EGFP expression (). To better quantitate the influence of the specific ts mutations and the synergistic effects observed in the EGFP expression experiments we analyzed the same combination of wild type and mutant polymerase subunits using a luciferase-mediated mini-genome replication assay (). This quantitative data is consistent with EGFP expression data and shows that PB2-Mut4 alone causes a 1000-fold reduction in activity at 37°C and 10,000-fold reduction at 39°C (). Furthermore, the PB1-Mut3/PB2-Mut4 combination acts synergistically resulting in 10,000-fold reduction at 37°C (). Collectively the mini-genome data demonstrate that the PB1-Mut3 and PB2-Mut4 each demonstrated ts phenotypes in the context of the other WT RDRP subunits (PB2-Mut4 > PB1-Mut3) and combining the PB1-Mut3 and PB2-Mut4 resulted in a synergistic effect at 37°C, and 39°C ().
3.3. The replication of H1N1pdm rNY1682 TS-LAIVs was restricted in vitro
To test if the temperature sensitive polymerases can result in impaired growth of the viruses at restrictive temperatures, we rescued recombinant viruses to analyze the ts effects of these mutations on a complete viral infection. The replication kinetics of rNY1682-TS1 and rNY1682-TS2 were compared to the kinetics of rNY1682-WT using Madin-Darby canine kidney (MDCK) cell cultures at various temperatures (). At lower temperatures (30°C and 33°C), all of the viruses replicated with similar kinetics and reached uniformly high titers (~108 TCID50/ml), which is important for the development of vaccine candidates (). At 37°C, rNY1682-WT and rNY1682-TS1 replicated with similar kinetics while rNY1682-TS2 displayed slower replication kinetics. Nevertheless, all of the viruses eventually reached fairly high titers (~107.5 TCID50/ml) (). Although rNY1682-WT replicated efficiently at 39°C (108 TCID50/ml), rNY1682-TS1 was restricted and required 4 days to reach a peak titer of 105 TCID50/ml (). Furthermore, rNY1682-TS2 was not detectable at any of the time points collected at 39°C (), indicating that the additional mutations (P112S, N556D, Y658H) engineered into the PB2 gene of rNY1682-TS2 conferred an enhanced ts phenotype and restricted the virus completely.
Replication of H1N1pdm rNY1682-WT, rNY1682-TS1, rNY1682-TS2 viruses, and the FluMist®-H1N1pdm vaccine at various temperatures
To compare the replication kinetics of the rNY1682 LAIVs to the licensed live attenuated monovalent H1N1pdm vaccine (FluMist®-H1N1pdm, Medimmune, CA), we infected MDCK cells with high MOI of each virus and the viral titers were determined at various time points. The replication of both the rNY1682-TS2 and FluMist®-H1N1pdm were partially restricted at 37°C () and completely restricted at 39°C (). No statistically significant difference between these two viruses was observed at any time points compared.
3.4. The TS1 and TS2 constellation of mutations also restricted a human seasonal H1N1 virus
To demonstrate that combining critical substitutions responsible for the ts phenotypes of ca A/AA/6/60, A/Ud/307/72, A/GL/0389/65 would also restrict other human virus lineages we used the same strategy to generate LAIVs from a seasonal H1N1 influenza A virus that co-circulated with the H1N1pdm virus. An infectious clone set was created directly from clinical specimen using M-RTPCR and recombination-based cloning technologies, and the TS1 mutations (PB1-K391E, D581G, A661T, and PB2-N265S) or TS2 mutations (PB1-K391E, D581G, A661T, and PB2-P112S, N265S, N556D, Y658H) were introduced into this seasonal H1N1 influenza A strain (A/New York/1692/2009 [NY1692]), creating LAIV candidates rNY1692-TS1 and rNY1692-TS2. Both rNY1692-TS1 and rNY1692-TS2 showed replication kinetics similar to the wild-type rNY1692 virus at lower temperatures (30°C and 33°C) (). However, in contrast to the H1N1pdm TS-LAIVs, the rNY1692-TS2 was severely restricted at 37°C and both rNY1692-TS1 and rNY1692-TS2 were completely restricted at 39°C (). These results illustrate that to some extent, the temperature sensitivity rendered by the mutations depends on the different virus strains, but that the TS2 mutations are sufficient to thoroughly restrict both the pandemic and seasonal strains at higher temperatures (e.g., 39°C).
Replication of seasonal H1N1 rNY1692-WT, rNY1692-TS1, and rNY1692-TS2 viruses at various temperatures
3.5. H1N1pdm rNY1682-TS2 LAIV candidate was attenuated in mice
Because the seasonal influenza virus is less pathogenic than the H1N1pdm virus in mice [44
] and the seasonal H1N1 TS-LAIVs are more restricted than the corresponding H1N1pdm TS-LAIVs in vitro
, only the H1N1pdm TS-LAIVs were examined in vivo
. A licensed live attenuated monovalent H1N1pdm vaccine (FluMist®-H1N1pdm, Medimmune, CA) was also included in these experiments for comparison. For consistency, we propagated the original FluMist®-H1N1pdm vaccine stock in MDCK cells using the same conditions used for the generation of the rNY1682-TS1 and rNY1682-TS2 working stocks. Six-week-old female BALB/cJ mice were inoculated intranasally with 104
of the rNY1682-WT, rNY1682-TS1, rNY1682-TS2, or FluMist®-H1N1pdm viruses, or were mock-inoculated. Clinical signs of disease, such as ruffled fur, were observed in mice inoculated with rNY1682-WT virus as early as 2 days post-inoculation (dpi), but were not observed in the mice inoculated with rNY1682-TS1, rNY1682-TS2, or FluMist®-H1N1pdm (data not shown). Weight loss became evident in rNY1682-WT inoculated mice at 3 dpi and they did not recover until 10-11 dpi (). In contrast, mice inoculated with rNY1682-TS2 and FluMist® LAIVs continued to gain weight at the same rate as the mock-inoculated mice (). Statistical analysis showed that the weight loss caused by rNY1682-WT virus was statistically different from rNY1682-TS2 from 4-10 dpi (p < 0.001, ANOVA analysis). Interestingly, mice inoculated with the rNY1682-TS1 virus experienced slight and transient weight loss (2~3%) at 7-8 dpi, which was significantly different from rNY1682-TS2 inoculated mice (p < 0.001, ANOVA analysis). However, statistical analysis showed that the weight loss caused by rNY1682-TS1 was significantly less than that caused by rNY1682-WT from 4-9 dpi (p < 0.001, ANOVA analysis).
Replication of rNY1682-WT, rNY1682-TS1, rNY1682-TS2 and FluMist®-H1N1pdm in the respiratory tracts of mice
Average viral titers in the lungs of rNY1682-TS2 and FluMist®-H1N1pdm infected mice were approximately 5-6 log10 lower than those of rNY1682-WT infected mice at 2 and 4 dpi (). Whereas, the titers of rNY1682-TS1 in the lungs of infected mice were similar to that of rNY1682-WT infected mice, indicating that rNY1682-TS1 was only modestly attenuated in vivo. Although the viral titers in the upper respiratory tracts of mice showed similar LAIV-dependent trends, the effects of the ts mutations were less dramatic. The average titer of rNY1682-TS1 in nasal washes was approximately 1 log10 lower than rNY1682-WT, whereas the rNY1682-TS2 and FluMist®-H1N1pdm showed 2-3 log10 reductions in titer compared to rNY1682-WT (). Nevertheless, both the rNY1682-TS2 and FluMist®-H1N1pdm replicated more efficiently in the upper respiratory tracts than in the lower respiratory tracts, which was an objective of this approach because replication at the cooler temperatures within the respiratory tract will promote a strong adaptive immune response.
To assess the stability of the ts phenotype of the rNY1682-TS2, viruses recovered from mouse lungs and nasal washes at 4 dpi (mice in ) were examined and found to maintain a ts phenotype in vitro (data not shown). Furthermore, sequence analysis of the PB1, PB2 and PA genes of these viruses showed that the ts mutations were present, and that there were no potential compensatory mutations at a second site within these vRNAs.
3.6. The H1N1pdm rNY1682-TS2 LAIV candidate protected mice from lethal homologous challenge
Mice were immunized by intranasal inoculation with 104 TCID50 of the rNY1682-WT, rNY1682-TS1, rNY1682-TS2, FluMist®-H1N1pdm, or were mock immunized. Luminex assays demonstrated that the rNY1682-WT virus infection elicited the highest level of antibodies to HA (6400 Units), followed by rNY1682-TS1 (4200 Units), rNY1682-TS2 (2000 Units) and FluMist®-H1N1pdm (800 Units) at 21 dpi (). The reason that rNY1682-TS2 elicited higher antibody titer than FluMist®-H1N1pdm did may be that the former replicated to higher titers in the cooler part of the respiratory tract than the latter did ().
TS-LAIV candidates protect mice from lethal homologous infection
The immunized mice were challenged with 100 MLD50
of a mouse-adapted variant of NY1682 (NY1682-MAP7) [42
] at 30 dpi. The challenge virus NY1682-MAP7 was derived from rNY1682-WT and the HA used in the FluMist®-H1N1pdm vaccine was derived from A/CA/07/09. To identify amino acid differences between the two viruses and determine if they influence antigenicity we sequenced the virus stocks and preformed hemagglutinin inhibition assays. The nucleotide sequence of the virus stocks used in these experiments showed that the proteins were 99% identical (560/566). There are six amino acid differences (S100P, K136E, A203D, D239G, D293N, and V338I, amino acid positions are based on the use of the start codon and include the signal peptide) between the HA’s of rNY1682-viruses and the MDCK propagated FluMist®-H1N1pdm LAIV. Hemagglutinin inhibition assays performed with H1N1pdm (rNY1682) antisera showed viruses possessing either rNY1682 HA or FluMist HA had the same hemagglutinin inhibition titer (data not shown). This illustrates that these two viruses show equivalent cross neutralization and is consistent with previous reports indicating that these substitutions don’t influence antigenicity of H1N1pdm viruses [45
Immunization with rNY1682-TS1 and rNY1682-TS2 or prior infection by the rNY1682-WT virus protected the mice from the lethal challenge. No disease symptoms were observed in the mice immunized with the rNY1682-TS1 or rNY1682-TS2 LAIV candidates or in control mice infected with the rNY1682-WT. In contrast, disease symptoms, including ruffled fur, hunched posture, and weight loss, were observed in the mock-immunized mice as early as 2 days post challenge (dpc); the symptoms progressed to severe disease, and the animals showed statistically significant weight loss (p<0.001, ANOVA analysis) compared to the mice immunized by the TS-LAIVs or by infection with rNY1682-WT (). The mock-immunized mice became moribund, and succumbed to infection by 5-6 dpc. Interestingly, the FluMist®-H1N1pdm immunized mice experienced intermediate weight loss after lethal challenge, which was significant compared to the mice immunized with rNY1682-TS1, rNY1682-TS2, or by rNY1682-WT infection from 2-6 dpc (p < 0.01, ANOVA analysis). The differences in the protection generated by the various vaccine candidates were more obvious when viral titers in the mouse lungs post challenge were determined. Compared to the high titers of virus found in the lungs of mock-immunized mice at 2 dpc (1 × 108 TCID50/ml) and at 4 dpc (3 × 107 TCID50/ml), average viral titers in the FluMist®-immunized mice were reduced by approximately 2 log10 at 2 dpc (3 × 106 TCID50/ml) and at 4 dpc (1 × 105 TCID50/ml) (). The rNY1682-TS2-immunized mice showed better protection from lung replication after lethal challenge than those immunized by the FluMist®-H1N1pdm vaccine. At 2 dpc, the viral titer was 2 × 105 TCID50/ml in one mouse and close to the limit of detection (3 × 101 TCID50/ml) in the other mice immunized with rNY1682-TS2. Furthermore, the challenge virus was not detectable in any of the rNY1682-TS2 immunized mice at 4 dpc (). Finally, the rNY1682-TS1-immunized mice showed protection that was nearly identical to WT infection (). These results demonstrate that both the rNY1682-TS1 and rNY1682-TS2 LAIV candidates induced strong immunity to H1N1pdm viruses, and that they provided better protection than the currently licensed live attenuated H1N1pdm vaccine under these experimental conditions.
3.7. The H1N1pdm rNY1682-TS2 LAIV candidate provided stronger heterologous protection than FluMist®-H1N1pdm
We used a heterologous challenge study to further compare the highly attenuated rNY1682-TS2 LAIV with the licensed FluMist®-H1N1pdm vaccine. We used reverse genetics to create a lethal reassortant challenge virus (rNY1682:PR8-HA/NA) that contained 6 gene segments (PB2, PB1, PA, NP, M, NS) from rNY1682-MAP7 and the HA and NA from an antigenically distinct H1N1 virus (A/Puerto Rico/8/1934) isolated in 1934. Briefly, mice were immunized with 104 TCID50 of rNY1682-TS2, FluMist®-H1N1pdm, or were mock immunized. At 30 dpi, the immunized mice were challenged with 5 × 104 TCID50 of rNY1682:PR8-HA/NA. Mice were monitored for clinical disease for 14 days and lungs were collected at 3 dpc and 6 dpc. Although the rNY1682-TS2 immunized mice showed weight loss (maximum 16% at 5 dpi), they survived the normally lethal challenge. In contrast the FluMist®-H1N1pdm and mock immunized mice showed more dramatic weight loss and had to be euthanized for humane reasons 5-7 dpc (). However, unlike the homologous challenge experiments, the viral titers in the lungs of mice immunized with FluMist®-H1N1pdm were similar to that of the rNY1682-TS2 or mock immunized groups at 3 dpc (P>0.05, ANOVA analysis) (). This indicates that antibodies to the HA and NA induced from immunization didn’t significantly inhibit the initial replication of the antigenically unrelated challenge virus and suggests that the difference in disease symptoms between rNY1682-TS2 and FluMist®-H1N1pdm immunized mice () results from enhanced T cell mediated immunity.
TS2-LAIV candidate protects mice from heterologous challenge