Influenza Viruses Expressing Chimeric Hemagglutinins: Globular Head and Stalk Domains Derived from Different Subtypes

293T and MDCK cells were obtained from the American Type Culture Collection (ATCC) and were maintained either in Dulbecco's minimal essential medium (DMEM) or in MEM (Gibco, Invitrogen) supplemented with 10% fetal calf serum (HyClone; Thermo Scientific) and penicillin-streptomycin (Gibco, Invitrogen). Wild-type A/Puerto Rico/8/1934 (PR8) and A/Perth/16/2009 (Perth/09) (kindly provided by Alexander Klimov, CDC) and the recombinant viruses were grown in 10-day-old specific-pathogen-free embryonated hen's eggs (Charles River) at 37°C for 2 days.

Plasmids encoding the different chimeric hemagglutinins were constructed using a strategy similar to those described previously (7, 8, 13). Briefly, the different segments of chimeric HA were amplified by PCR with primers containing SapI sites, digested with SapI, and cloned via multisegmental ligation into the SapI sites of the ambisense expression vector pDZ, in which viral RNA (vRNA) transcription is controlled by the human RNA polymerase I promoter and the mouse RNA polymerase I terminator, while mRNA/cRNA transcription is controlled by the chicken beta-actin polymerase II promoter (17). We thank Daniel Perez (University of Maryland) for the kind gift of the H7 HA plasmid (GenBank accession number DQ017504). The plasmids encoding PR8 genes were used as described previously (8).

To assess levels of hemagglutinin protein expression at the cell surface, 293T cells were transfected with 1 μg of the appropriate plasmid by using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions, or MDCK cells were infected with cHA-expressing recombinant viruses. At 48 h posttransfection, 293T cells were trypsinized and resuspended in phosphate-buffered saline (PBS) containing 2% fetal bovine serum (FBS) prior to staining with monoclonal antibody (MAb) 6F12 (5 μg/ml), a MAb generated in our laboratory that is broadly reactive to the stalk domain of group 1 HAs (data not shown), or with MAb 12D1 (5 μg/ml) against H3 HAs (25). At 12 h postinfection, MDCK cells were resuspended by trypsinization and were stained with MAb 12D1. Stained cells were analyzed on a Beckman Coulter Cytomics FC 500 flow cytometer, and the results were analyzed using FlowJo software.

The procedure for pseudoparticle production was adapted from previous studies (6, 23). Briefly, we cotransfected 293T cells with four plasmids encoding (i) a provirus containing the desired reporter (V1-Gaussia luciferase) (6), (ii) HIV Gag-Pol (6), (iii) chimeric hemagglutinin protein, and (iv) B/Yamagata/16/88 virus neuraminidase (NA). Supernatants were collected 72 h posttransfection and were subsequently filtered (pore size, 0.45 μm). The presence of pseudotype virus-like particles (VLPs) was evaluated through hemagglutination assays. Different VLP preparations were adjusted to the same 4 hemagglutination units prior to inoculation of MDCK cells. All of the assays using pseudoparticles described below were performed in the presence of 1 μg/ml Polybrene (Sigma) to increase the efficiency of transduction (23).

The entry assay was performed by transducing MDCK cells with pseudoparticles that expressed different chimeric hemagglutinins and contained the Gaussia luciferase reporter. Twenty-four hours posttransduction, cells were washed three times with fresh medium to remove any residual Gaussia luciferase protein present in the inoculum. Forty-eight hours posttransduction, luciferase assays were performed (6).

Influenza A viruses were rescued from plasmid DNA as described previously (7, 8, 13). To generate the recombinant wild-type (rWT) PR8 virus, 293T cells were cotransfected with 1 μg of each of the eight pDZ PR8 rescue plasmids using Lipofectamine 2000 (Invitrogen). The wild-type HA plasmid was replaced with a plasmid encoding the desired chimeric HA in order to generate cHA-expressing recombinant viruses. At 6 h posttransfection, the medium was replaced with DMEM containing 0.3% bovine serum albumin (BSA), 10 mM HEPES, and 1.5 μg/ml TPCK (l-1-tosylamide-2-phenylethyl chloromethyl ketone)-treated trypsin (Sigma). After 24 h posttransfection, 8-day-old embryonated chicken eggs were inoculated with the virus-containing supernatant. Allantoic fluid was harvested after 2 days of incubation at 37°C and was assayed for the presence of virus by hemagglutination of chicken red blood cells. The titers of virus stocks were determined by plaque assays on MDCK cells as described previously (7, 8).

To analyze the replication characteristics of recombinant viruses, 10-day-old embryonated chicken eggs were inoculated with 100 PFU of wild-type or cHA-expressing recombinant viruses. Allantoic fluid was harvested and was subsequently assayed for viral growth at 0, 9, 24, 48, and 72 h postinfection (hpi). The titers of virus present in allantoic fluid were determined by plaque assays on MDCK cells as referenced above.

Plaques were visualized by immunostaining with MAb HT103 against the influenza A virus nucleoprotein (NP) by use of a previously described protocol (1, 19).

Confluent MDCK cells either were infected (multiplicity of infection [MOI], 2) with recombinant influenza viruses or were mock infected with PBS for 1 h at 37°C. At 12 hpi, cells were lysed in 1× SDS loading buffer as described previously (8, 29). The reduced cell lysates were analyzed by Western blot analysis using MAbs against influenza A virus NP (HT103) (14), the PR8 HA head domain (PY102), the Cal/09 HA head domain (29E3) (11), and the VN/04 HA head domain (MAb 8) (19), as well as 12D1, a pan-H3 antibody reactive against the HA stalk (25). In order to detect H7 head domains, polyclonal goat serum NR-3152 (raised against the A/FPV/Dutch/27 [H7] virus; BEI Resources) was used. An anti-glyceraldehyde-3-phosphate dehydrogenase (anti-GAPDH) antibody (Abcam) was used as the loading control. Proteins were visualized using an enhanced chemiluminescence protein detection system (Perkin-Elmer Life Sciences).

For immunofluorescence analysis, confluent monolayers of MDCK cells on 15-mm-diameter coverslips were infected with recombinant viruses at an MOI of 2. At 15 hpi, cells were fixed and permeabilized with methanol-acetone (ratio, 1:1) at −20°C for 20 min. After being blocked with 1% bovine serum albumin in PBS containing 0.1% Tween 20, cells were incubated for 1 h with the antibodies described above, as well as with MAb 6F12. After three washes with PBS containing 0.1% Tween 20, cells were incubated for 1 h with Alexa Fluor 594-conjugated anti-mouse IgG (Invitrogen) or Alexa Fluor 594-conjugated anti-goat IgG (Invitrogen). Following the final three washes, infected cells were analyzed by fluorescence microscopy with an Olympus IX70 microscope.

The plaque reduction assay was performed as described previously (25). Approximately 60 to 80 PFU of recombinant viruses expressing cHA made up of a Cal/09 or VN/04 globular head domain atop a PR8 stalk was incubated with or without different concentrations (100, 20, 4, 0.8, 0.16, and 0.032 μg/ml) of MAb KB2, a broadly neutralizing anti-HA stalk antibody generated in our laboratory (data not shown), for 60 min in a total volume of 240 μl at room temperature. A confluent layer of MDCK cells in 6-well plates was washed twice with PBS and was then incubated with the antibody-virus mixture for 40 min at 37°C. A TPCK-trypsin agar overlay either with no antibody or supplemented with the antibody at the concentrations described above was then added to each well after the inoculum had been aspirated off. Plates were incubated for 2 days at 37°C. Plaques were then visualized by immunostaining (1, 19) with anti-influenza A virus NP antibody HT103.

The procedure for pseudotype particle production was the same as that described above and used the cHA construct comprising either a VN/04 (H5) or a Cal/09 (H1) head and a PR8 (H1) stalk with the influenza B/Yamagata/16/88 virus NA. Particles were then incubated with different concentrations of MAb KB2 at 5-fold dilutions from 100 to 0.032 μg/ml. Then these mixtures were added to MDCK cells. Transductions proceeded for 6 h before cells were washed and fresh medium was placed over cells. All transductions using pseudotype particles were performed in the presence of 1 μg/ml Polybrene (Sigma, St. Louis, MO) (23). Forty-eight hours posttransduction, luciferase assays were performed in order to assay the degree in which entry was blocked by MAb KB2.

All constructed cHA genes used in this study have been deposited in the Influenza Research Database and can be accessed under GenBank accession numbers CY110921 to -24. The chimeric cH1/1, cH5/1, cH7/3, and cH5/3 viruses are listed as A/Puerto Rico/8-RGcH1-1/34, A/Puerto Rico/8-RGcH5-1/34, A/Perth/16-RGcH7-3/09, and A/Perth/16-RGcH5-3/09, respectively.

We generated an alignment of influenza A virus HA sequences of the H1, H3, H5, and H7 subtypes used in this study in order to see if the cysteine residues forming the Cys52-Cys277 disulfide bond were conserved. Because these cysteine residues are highly conserved across HA subtypes, for both group 1 and group 2 HAs, we hypothesized that we could use this bond as a delineating point between the head and stalk domains. By defining the sequence between Cys52 and Cys277 as the head region and the remainder of the molecule as the stalk, we rationalized that we could engineer constructs that encode novel head and stalk combinations from a variety of HA subtypes (Fig. 1A and B).

The degree of amino acid identity that exists between the stalk regions of hemagglutinin subtypes further encouraged us to think that the swapping of head domains might be possible. Higher percentages of amino acid identity were seen in the stalk domains across all subtypes than in the head domains (Fig. 1C).

All 16 subtypes of influenza virus HA are classified into two phylogenetic groups (15). Because we saw higher percentages of amino acid identity within the stalk regions of a particular group (Fig. 1C), and because we had success in generating one cHA virus that contained head and stalk domains from group 1 viruses (16), we attempted to make intragroup cHAs. For group 1, we generated two chimeric hemagglutinin constructs that encode either the pandemic H1 Cal/09 or VN/04 globular head domain with the stalk region from PR8 HA (H1) (cH1/1 and cH5/1, respectively) (Fig. 1B). We applied a similar strategy to generate a chimeric HA that expressed head and stalk domains from different group 2 influenza strains: the head from Alb/01 HA (H7, group 2) and the stalk region from Perth/09 HA (H3, group 2) (cH7/3) (Fig. 1B). Finally, we evaluated whether we could swap the head and stalk domains to make an intergroup chimeric HA containing the head domain of VN/05 HA (H5, group 1) atop a Perth/09 HA (H3, group 2) stalk (cH5/3) (Fig. 1B).

Following the construction of these plasmids, we tested whether the different chimeric HA constructs could be expressed and transported to the cell surface like wild-type HAs. Fluorescence-activated cell sorter (FACS) analysis of transiently transfected 293T cells was performed following surface staining with H1 and H3 stalk domain-specific antibodies. Using this method, we were able to detect the expression of all four chimeric constructs on the cell surface (Fig. 2A). However, compared to that for the wild-type PR8 HA, less surface protein expression was detected for the cH1/1 construct, which could be attributed to the inherent character associated with the head domain of the Cal/09 HA or to a lower transfection efficiency for this chimeric DNA construct. In addition, it is noteworthy that the cell surface expression patterns of the cH7/3 and cH5/3 constructs were different. This “double-peak” expression pattern was observed only under transfection conditions and was reproducible. It was not detected upon infection with either cH7/3- or cH5/3-expressing recombinant viruses (Fig. 2A). Therefore, these data indicate that the cHAs can be transported through the Golgi complex to the cell surface.

Next, we examined the entry characteristics of the different cHAs through transduction of MDCK cells with retroviral pseudotype particles that contained a luciferase reporter construct and expressed the cHA and wild-type B/Yamagata/16/88 virus NA on the particle surface. The entry efficiency mediated by the cHA proteins was detected by the luciferase readout. Comparable levels of pseudotype particle-mediated luciferase expression were observed for cH5/1, cH7/3, and cH5/3 chimeric HAs and the corresponding wild-type proteins (Fig. 2B). Particles encoding the cH1/1 HA expressed lower luciferase levels than did the other HA constructs, which could be due either to the lower expression of cH1/1 in the producer cell line (leading to fewer HA trimers per particle) or to the less efficient entry properties of the cH1/1 HA. It is also possible that when one is normalizing the pseudotype particles to 4 hemagglutinin units, the actual number of pseudotype particles may differ due to differences in binding to red blood cells.

Because we had determined that our cHA constructs were efficiently expressed and transported to the cell surface, we wanted to ascertain whether a recombinant influenza virus that encodes a cHA could be rescued. Using previously published protocols (7, 8), we succeeded in generating viruses containing the different cHAs. The resulting viruses were plaque purified and were amplified in 10-day-old embryonated eggs, and the chimeric segments were analyzed by reverse transcription-PCR (RT-PCR) and sequenced. In all cases, the virus was found to have the expected chimeric HA segment and no other HA segment (data not shown).

We further confirmed the presence of the cHAs in rescued viruses by Western blotting and indirect immunofluorescence of infected cells (Fig. 3A and B). MDCK cells were infected with the rWT PR8, wild-type Perth/09, cH1/1, cH5/1, cH7/3, and cH5/3 viruses (Fig. 3A and B). cH1/1 and cH5/1 chimeric HA proteins were detected in the corresponding samples using antibodies reactive against the head domain of Cal/09 (H1) HA (29E3) (11) or VN/04 (H5) HA (MAb 8) (19), respectively (Fig. 3A). Using 12D1, a pan-H3 anti-stalk MAb (25), we could observe comparable expression levels among the cH7/3, cH5/3, and wild-type Perth HAs. The wild-type Perth HA showed slower migration on the gel, likely due to a higher number of glycosylation sites in the globular head domain. We confirmed that the correct HA head domain was expressed atop an H3 stalk using an anti-H7 polyclonal (NR-3152) or anti-H5 monoclonal (MAb 8) antibody on cH7/3 or cH5/3 infection samples, respectively. Positive bands were detected in both cases.

For the immunofluorescence study, the infection conditions were similar to those used for Western blot analysis. Infected cells were stained with corresponding antibodies as used in Fig. 3A. All infected cells showed the expected expression of chimeric and wild-type HAs, as well as of influenza A virus NP (Fig. 3B).

The growth properties of wild-type and recombinant viruses were assessed in 10-day-old embryonated chicken eggs at 37°C (Fig. 4A). The rWT PR8 virus was included for comparison of the growth kinetics of the recombinant viruses expressing chimeric HAs. The cH5/1 and cH5/3 viruses displayed replication kinetics comparable to that of the rWT PR8 virus. The cH7/3 virus grew to peak titers similar to those of rWT PR8 at 48 hpi (1 × 10⁹ PFU/ml), although the cH7/3 viral titer was 2 log units lower than that of the rWT PR8 virus at 9 hpi. The cH1/1 virus was attenuated compared to the rWT PR8 virus, as shown by reduced viral titers at all time points. Nonetheless, the cH1/1 virus reached a respectable peak titer of approximately 10⁸ PFU/ml. The Perth/09 wild-type virus grows to comparable peak titers in embryonated eggs (data not shown).

The plaque phenotype of each of the chimeric viruses was also evaluated in MDCK cells. All viruses formed comparably sized plaques, as shown in Fig. 4B. These data, taken together, confirm that the chimeric HA constructs fold correctly and are biologically functional.

Finally, we wanted to test whether stalk-specific antibodies were able to neutralize our newly generated recombinant viruses expressing cHAs. We performed plaque reduction assays either without antibodies or in the presence of MAb KB2, an HA stalk-specific antibody with broad group 1 reactivity generated in our laboratory. We were able to show that MAb KB2 neutralizes all cHA-expressing viruses with similar efficiencies and in a dose-dependent manner. At 100 μg/ml, MAb KB2 was able to neutralize the cH1/1 and cH5/1 viruses with 100% efficiency, and it retained some neutralizing activity at concentrations as low as 4 μg/ml (Fig. 5A).

We confirmed these results by using a pseudotype particle inhibition assay with MAb KB2. Pseudotype particles expressing cH1/1 or cH5/1 and influenza B virus NA were added to MDCK cells either in the presence of MAb KB2 or without an antibody. Forty-eight hours posttransduction, the supernatant was collected, and luciferase activity was analyzed. As expected, MAb KB2 blocked the entry of cH1/1 and cH5/1 pseudotype particles in a dose-dependent manner at concentrations above 4 μg/ml. While lower concentrations of MAb KB2 than those effective in the plaque reduction assay were sufficient to inhibit the entry of pseudotype particles, this result was expected due to the assumed lower incorporation of HA trimers on the surfaces of pseudotype particles (3). This phenomenon of different neutralizing potencies of MAbs in assays that involve whole virus versus pseudotype particles has been recognized in other studies (3, 21).