The generation of the DNA vaccine constructs containing the targeting unit, the dimerization unit consisting of h1 + h4 + C_H3 domains derived from human IgG3, and antigen has been previously described (9, 10). The constructs either expressed amino acids 18–541, the extracellular domain, and part of the transmembrane domain, of influenza A/California/07/2009 (H1N1) hemagglutinin or the conserved IYSTVASSL epitope of H1 (533–541) as the antigen payload, and anti-I-E^d MHC class II single chain variable fragment (scFv) from the 14-4-4S monoclonal antibody that binds the conserved E alpha chain, or murine XCL1 as the targeting unit. All sequences were synthesized by Eurofins MWG (Germany) or GenScript (USA). The synthesized inserts were subcloned into the expression vector pUMVC4a on NotI and BglII, all including either an Ig VH signal peptide or the murine XCL-1 signal peptide to ensure secretion. The αMHCII:HA (Cal/07) construct has been described previously (8).

Six- to eight-week-old female CB6F1, BALB/c, or C57BL/6 mice were obtained from Harlan UK Ltd. (Bradford, UK) and kept in specific pathogen-free conditions in accordance with the UK’s Home Office guidelines, and all work was approved by the Animal Welfare and Ethical Review Board (AWERB) at Imperial College London. Studies followed the ARRIVE guidelines. Animals were immunized in a prime (d0)-boost (d21)-challenge (d42) regime and culled on day 7 of challenge (d49 relative to prime). For protein immunization, mice were immunized intramuscularly (i.m.) with 0.1 μg purified surface antigens from influenza strain H1N1 A/California/7/2009 (GSK Vaccines, Siena, Italy) in 50 μl. For DNA vaccination, mice were injected i.m. into the anterior tibialis with 5 μg plasmid in 50 μl of sterile PBS followed by electroporation (EP). Two lots of 5 pulses of 150 V with switched polarity between pulses were delivered using a CUY21 EDIT system (BEX, Japan). For infections, mice were anesthetized using isoflurane and infected intranasally (i.n.) with 5 × 10⁴ PFU of influenza A H1N1 (strain A/England/195/2009). Where used, CD8⁺ T cells were depleted using two intraperitoneal injections of 0.25 mg anti-murine CD8 antibody clone YTS156, and CD4⁺ T cells were depleted with 0.125 mg each of YTA3 and YTS191 (a kind gift of S. Cobbold, Oxford University) on day −1 and +1 of infection (11).

H1N1 influenza (strain A/England/195/2009), isolated by Public Health England in the UK, April 2009 (12), was grown in Madin–Darby Canine Kidney (MDCK) cells, in serum-free DMEM supplemented with 1 μg/ml trypsin. The virus was harvested 3 days after inoculation and stored at −80°C. Viral titer was determined by plaque assay as previously described (13).

Antibodies specific to influenza H1N1 were measured using a standardized ELISA (14). IgG responses were measured in sera and IgA responses in bronchoalveolar lavage. MaxiSorp 96-well plates (Nunc) were coated with 1 μg/ml H1N1 surface proteins or a combination of anti-murine lambda and kappa light chain-specific antibodies (AbDSerotec, Oxford, UK) and incubated overnight at 4°C. Plates were blocked with 1% BSA in PBS. Bound IgG was detected using HRP-conjugated goat anti-mouse IgG (AbD Serotec). Bound IgA was detected using a biotinylated anti-IgA and a streptavidin-HRP. A dilution series of recombinant murine IgG or IgA was used as a standard to quantify specific antibodies. TMB with H₂SO₄ as stop solution was used to detect the response and optical densities read at 450 nm.

Mice were culled using 100 μl intraperitoneal pentobarbitone (20 mg dose, Pentoject, Animalcare Ltd., UK) and tissues collected as previously described (15). Blood was collected from carotid vessels and sera isolated after clotting by centrifugation. Lungs were removed and homogenized by passage through 100 μm cell strainers, then centrifuged at 200 × g for 5 min. Supernatants were removed, and the cell pellet treated with red blood cell lysis buffer (ACK; 0.15M ammonium chloride, 1M potassium hydrogen carbonate, and 0.01 mM EDTA, pH 7.2) before centrifugation at 200 × g for 5 min. The remaining cells were resuspended in RPMI 1640 medium with 10% fetal calf serum and viable cell numbers determined by trypan blue exclusion.

Viral load in vivo was assessed by Trizol extraction of RNA from frozen lung tissue disrupted in a TissueLyzer (Qiagen, Manchester, UK). RNA was converted into cDNA, and quantitative RT-PCR was carried out using bulk viral RNA, for the influenza M gene and mRNA using 0.1 μM forward primer (5′-AAGACAAGACCAATYCTGTCACCTCT-3′), 0.1 μM reverse primer (5′-TCTACGYTGCAGTCCYCGCT-3′), and 0.2 μM probe (5′-FAM-TYACGCTCACCGTGCCCAGTG-TAMRA-3′) on a Stratagene Mx3005p (Agilent technologies, Santa Clara, CA, USA). M-specific RNA copy number was determined using an influenza M gene standard plasmid.

Live cells were suspended in Fc block (Anti-CD16/32, BD) in PBS-1% BSA and stained with surface antibodies: influenza A H1 HA_533–541 IYSTVASSL Pentamer R-PE (Proimmune, Oxford, UK), CD3-FITC (BD, Oxford UK), CD4-APC (BD), and CD8-APC Alexa75 (Invitrogen, Paisley, UK). Analysis was performed on an LSRFortessa flow cytometer (BD). FMO controls were used for surface stains.

Calculations as described in figure legends were performed using Prism 6 (GraphPad Software Inc., La Jolla, CA, USA).

Vaccine-induced, antibody-mediated protection against influenza is well characterized, but CD8⁺ T cells are also important. DNA vaccines allow the induction of strong cellular responses (16), and the use of different targeting modules allows us to compare the relative contributions of different effectors (17). We compared the response to immunization using a DNA vaccine encoding dimeric APC-targeted antigen alone or in combination with protein antigens. The DNA vaccine construct for these studies encoded the HA gene from influenza Eng/195 (H1N1) dimerized to an anti-I-E^d MHC class II scFv with a dimerization unit consisting of h1 + h4 + C_H3 domains from human IgG3. CB6F1 mice were used for these studies, and they are the F1 cross of BALB/c (I-E^d) and C57BL/6 (I-E^b) strains. Mice were immunized once with 5 μg DNA encoding the dimeric vaccine construct (αMHCII:HA) i.m. with EP, with or without a boost (on day 21), using a sub-protective dose of H1N1 proteins (0.1 μg) from CAL/09. Three weeks after the boost immunization (on day 42), mice were challenged i.n. with H1N1 influenza (strain A/England/195/2009) and culled 7 days later (day 49).

Blood was collected prior to infection to determine anti-influenza antibodies. αMHCII:HA primed-protein boosted animals had significantly more antibody than protein alone or αMHCII:HA alone (p < 0.05, Figure 1A). All immunizations gave some reduction of weight loss following influenza infection. The αMHCII:HA alone group recovered faster on days 6 and 7 after infection than PBS control mice, and a similar phenotype was seen after immunization with protein alone. However, prime immunization with DNA and then protein boost led to significantly improved recovery from d4 after infection (p < 0.05 compared to DNA or protein alone on d5 and d6, Figure 1B). After infection, antibody responses in the αMHCII:HA-Protein group were the same as the PBS-protein group, and levels were 10-fold higher than before infection (Figure 1C). There was some detectable antibody after immunizing with αMHCII:HA alone, which was slightly boosted by infection. However, αMHCII:HA alone immunized animals had a significant influenza-specific CD8⁺ T cell response in the lungs, as measured by pentamer-positive cells, greater than the protein alone or naive animals (p < 0.05, Figure 1D). These cells were also induced in the prime-boost group. These data suggest that while antibody is protective against influenza infection, antigen-specific CD8⁺ T cells contribute to recovery in the absence or near absence of antibodies.

Having observed that heterologous prime-boost immunization led to faster recovery, and the DNA vaccines induced both an influenza-specific CD8 and antibody response, we wished to determine the role of the CD8 cells. Mice were immunized with αMHCII:HA with a protein boost or protein alone, and responses compared between animals treated with CD8 depleting antibody and control during infection. As seen before, αMHCII:HA-Protein immunization induced more antibody than protein alone 21 days after the boost immunization (Figure 2A). αMHCII:HA-Protein-immunized, CD8⁺-depleted mice lost significantly more weight than the immunized animals with intact CD8⁺ responses (p < 0.05 on day 6 and 7, Figure 2B). CD8 depletion had no effect on protein alone immunization. At day 7 after infection, αMHCII:HA-Protein-immunized mice had no detectable viral load, and CD8 depletion had no effect on this (Figure 2C). CD8 depletion also had no effect on the antibody response (Figure 2D) or CD4⁺ T cell number in the lungs (Figure 2E) but led to a significant reduction in both total (Figure 2F) and influenza-specific CD8⁺ T cells (Figure 2G). From this, we conclude that the improved recovery seen after αMHCII:HA priming before protein vaccination is partially mediated by CD8⁺ cells.

Since we observed that CD8 cells contribute to the accelerated resolution of disease in the prime-boost immunization, we wished to determine whether vaccines inducing influenza-specific CD8 alone could also improve disease resolution. A pilot study was performed to determine the immune response vaccine constructs using different targeting unit/antigen combinations, in order to select the ones that gave the greatest CD8⁺ T cell responses. Mice were immunized with constructs encoding either anti-I-E^d scFv or the XCL1-targeting module with either the full HA surface domain (of Cal/07) or the K^d immunodominant epitope alone in H1 hemagglutinin (HA_533–541 IYSTVASSL). The groups immunized with constructs encoding the epitope alone were not protected against influenza infection (Figures 3A,B). The more complete HA constructs offered modest protection with αMHCII:HA-immunized animals recovering slightly faster than the naive animals, and the XCL1:HA-immunized animals gaining weight on d7 post infection. There were striking differences in the antibody responses: only animals immunized with a construct expressing the whole HA had detectable antibody responses, and the response to the MHCII-targeting construct was greater than the XCL1 (Figure 3C). While the antibody responses were poor to these constructs, there was substantial recruitment of influenza-specific CD8⁺ T cells. All immunized groups had influenza-specific T cells in the lungs, but there were greater responses in the epitope-immunized animals (Figure 3D). From this pilot study, we conclude that the epitope-only vaccines induce a stronger CD8 response.

To assess the relative contributions of CD8⁺ cells versus antibody, we took advantage of the differential responses to the αMHCII:HA or αMHCII:Epitope constructs, with either a DNA or protein boost, prior to infection with influenza. Prime-boost regimes with protein or αMHCII:HA (Eng/195) led to significant protection against infection, with little difference between the homologous or heterologous prime-boost regimes in weight loss (Figure 4A). Protein-containing regimes (Protein–Protein or αMHCII:HA-Protein) had slightly less detectable viral RNA in the lungs than the αMHCII:HA homologous regime (Figure 4B). The groups receiving a protein vaccination had more antibody than the other groups (Figure 4C), though it was surprising that there was no boost in antibody response after the second protein immunization. The regimes using the αMHCII:Epitope induced the greatest level of CD8⁺ cells in the lungs after infection (p < 0.05, Figure 4D), but the αMHCII:Epitope-immunized animals were not protected against infection, losing a similar amount of weight as naive animals and having an equivalent viral load. Priming with αMHCII:Epitope followed by protein did lead to significantly more CD8⁺ T cells than Protein–Protein but had little effect on protection. As seen before, the protein-only immunization regime did not induce any influenza-specific CD8⁺ T cells. These data suggest influenza-specific CD8⁺T cells targeting the IYSTVASSL epitope of H1 are not sufficient to protect against infection.

In previous studies using similar DNA vaccine constructs in BALB/c mice, complete protection against Cal/07 infection was observed after a single DNA vaccination (8). Possible sources of differences include the amount of DNA delivered (25 μg in published, 5 μg in current), the route of delivery (i.d. in published, i.m. in current), viruses used for challenge (Cal/07 in published, Eng/195 in current: the HA genes from Cal/07 and Eng/195 are 99% identical, with 4 amino acid changes), the mouse strains used (BALB/c in published, CB6F1 in current), or the antigens inserted into the MHCII-targeted construct. To ensure that there was no difference between constructs used in the current study and the published constructs, we compared immunization with the construct used in the previous study (8) and a construct expressing the HA from Eng/195. CB6F1 mice were immunized with 5 μg of each construct with EP, and 28 days later, they were infected i.n. with 5 × 10⁴ PFU of ENG195. Weight was measured daily after infection, and there was no difference between mice immunized with the two vaccine constructs; immunized mice recovered faster than naive mice on day 7 after infection (Figure 5A). Significantly, more viral RNA was detected in the lungs of previously naive animals than in immunized animals, and there was no difference in viral load between mice immunized with either construct (Figure 5B). Both constructs induced an immune response, as there was detectable specific IgG in the sera at d7 (Figure 5C) and flu-specific CD8⁺ T cells in the lung (Figure 5D) in immunized but not naive animals. From this, we conclude that the incomplete protection observed in the initial studies was not due to the construct, the antigen targeted, or the challenge virus, suggesting that mouse strain may be important, though the dose and route may also contribute to differences seen.

The targeting unit of the MHC vaccine construct is based on an scFv, derived from the 14-4-4S monoclonal antibody that binds the conserved E alpha chain of the I-E^d MHCII molecule, which is expressed in mouse strains that are H-2^d. We have previously observed that mouse strain is critical in the recall immune response to respiratory viral infection (18). Previously published studies with similar MHC-targeting vaccine constructs used BALB/c mice (H-2^d), and the current studies used CB6F1 mice, which are mixed H-2^d and H-2^b. To test whether mouse strain has an effect on the immune response to the vaccine, we immunized BALB/c (H-2^d), C57BL/6 (H-2^b), and CB6F1 (mixed H-2^d and H-2^b) with the αMHCII:HA construct. These animals were then challenged with influenza. Naive animals started losing weight on day 2 after infection, and this weight loss continued to day 7, at which point the animals were culled (Figure 6A). There was no significant difference in the magnitude or the profile of the weight loss between the naive animals regardless of strain, indicating that baseline susceptibility to influenza was similar. However, there was a striking difference in protection based on MHC genotype. BALB/c were more protected than F1 mice, which were more protected than the C57BL/6 mice, directly reflecting the amount of I-E^d MHC (Figures 6B–D) and reflecting the previously published study (8). Likewise, there was only a reduction in viral load in the BALB/c and F1-immunized mice (Figure 6E). There was detectable influenza-specific antibody (Figure 6F) and CD8⁺ T cells (Figure 6G) in both the BALB/c and CB6F1 mice, and there was no difference between the two strains, suggesting that there are other components that contribute to protection against infection. The BALB/c mice had a higher proportion of CD4⁺ T cells in the lungs, which may have contributed to protection (Figure 6H), but in a separate study when treated with CD4-depleting antibody during challenge, there was no effect on resolution of disease after depletion (Figure 6I).

In the CB6F1 mice, the regimes that induced CD8⁺ T cells alone did not protect against infection. Since we observed a difference between BALB/c and CB6F1 mice in protection following immunization with the αMHCII:HA construct, we wished to determine whether there was a difference in the protective capacity of the CD8⁺ T cells induced in H-2^b mice. BALB/c were immunized twice with the MHCII-epitope construct prior to infection with influenza. Mice were not protected against infection (Figure 6J) despite inducing an extremely high influenza-specific CD8 response (Figure 6K). As with the CB6F1 mice, no antibody response was seen after immunization with this construct (data not shown). These studies clearly demonstrate the effect of the targeting module on the response.