Design of H1N1 CBC NA genes

The pairwise identity matrix for a non-redundant set of 1796, full-length, human N1 NA protein sequences were used as the input to partition sequence space by classical multidimensional scaling (MDS). Five clusters of similar protein sequences were defined representing historical seasonal-like sequences (1933–1950; 1948–1997; 1998–2009), swine-like sequences (1976–2008) and pandemic-like sequences (2009–2011) (Fig. 1a). Cluster-based consensus (CBC) NA sequences were generated by the combination of (i) swine-like and pandemic-like sequences yielding NA5200, (ii) historical seasonal-like sequence clusters to yield NA7900 and (iii) all five sequence clusters to yield NA9100, as depicted in Fig. 1b.

Fig. 1 CBC design and production of recombinant tetrameric proteins. a Visualization of the first two dimensions from a multidimensional scaling analysis of the pairwise identity matrix of influenza A subtype H1N1 neuraminidase sequences. Host species represented by colour; Swine (green), Avian (orange), Human (purple). Consensus designs are highlighted in red. b Conceptual overview of CBC NA designs. c, d Purification profile and specific enzymatic activity of recombinant NAs. Soluble NA5200, NA7900 and NA9100 NAs were produced recombinantly in HEK 293 T cells. Supernatant was first affinity purified followed by size exclusion chromatography. c UV absorption profile after size exclusion chromatography for tetrameric soluble NAs. Vertical lines on top of the graph represents standard of known protein sizes. d Recombinant proteins, along with Bel/09 wild-type NA, were tested for enzymatic activity by MUNANA assay. Specific activity was calculated from a standard curve of 4-methylumbelliferone Full size image

Characterization of CBC recombinant soluble tetrameric NAs

As the NA proteins were designed according to criteria different from natural evolutionary processes, it was important to confirm correct folding and maintenance of epitope integrity. We tested NA enzymatic activity as a proxy for structural integrity, since it has been shown that while NA inhibiting antibodies can be induced by immunization with NA antigen that is enzymatically inhibited by the addition of zanamivir,14 the NA is still required to be in its native tetrameric form.15 We therefore produced and purified the computationally designed NA5200, NA7900 and NA9100 NA constructs as soluble tetrameric proteins (rNAs) in a mammalian expression system. Soluble tetrameric NA derived from H1N1pdm09 was produced as well and served as a naturally occurring control.16 Size exclusion chromatography analysis revealed a dominant peak for recombinant NA5200, NA7900 and NA9100 with a retention time that corresponded to the predicted molecular weight of a soluble tetrameric NA (Fig. 1c). For the NA5200 and NA9100 recombinant proteins, minor peaks were observed, which eluted faster from the size-exclusion column than the dominant peaks. These fractions likely corresponded to aggregate forms of NA, and were discarded. All three recombinant tetrameric NAs were enzymatically active as determined by the release of 4-methylumbelliferone from a small molecule sialic acid conjugate precursor. The specific activity of the three CBC recombinant tetrameric NAs was similar to or slightly higher than that of 2009 H1N1pdm-derived soluble tetrameric NA (Fig. 1d). Next, the potential N-glycosylation sites in the head domain of the CBC NAs were compared with those in relevant N1s. NA5200, NA7900 and NA9100 only carried three potential N-glycosylation sites in the head domain, which are found 100% conserved in nearly all N1 NAs.17 These are located at amino acid positions 88, 146 and 235 (Supplementary Table 1).

CBC NAs elicit broad NA-inhibiting antibodies

It is known from previous studies that protection by vaccination with NA largely depends on the induction of antibodies that can mediate neuraminidase inhibition (NI).13 Therefore, as an initial step to examine the potential breadth of the antibody response directed against the CBC NA designs, heat-inactivated sera raised against these molecules in mice were compared with sera from mice that had been immunized with three wild-type (WT) NAs for their capacity to mediate NI against a panel of human H1N1 viruses. Additionally, the H5N1 strain NIBRG-14 (a 6:2 reverse genetics reassortment containing HA, with the polybasic cleavage site deleted, and NA from A/Vietnam/1194/2004) and a swine H1N1 influenza isolate were included in the panel, as these strains represent potential pandemic-causing viruses. An IC 50 titre (1:x dilution resulting in 50% NA inhibition) above a log 2 value of 4.3 (i.e., 20-fold dilution; the lowest dilution of sera tested) was considered as a positive response. As observed with previous studies,13 immune sera raised against natural NAs possessed some cross-reactivity (Fig. 2). PR8/34 NA anti-sera mediated NI against PR8/34, and to a lesser extent against USSR/77 and Sw/Bel/98 (on average just above the cut-off point). Likewise, anti-sera induced by vaccination with purified recombinant tetrameric NA derived from NC/99 reacted with itself and Bris/07. Bel/09 NA anti-sera mediated the strongest NI titres against itself and Sw/Bel/98, followed by NIBRG-14. For the CBC NAs, the design strategy of the NA, for the most, dictated the panel of reactivity. Anti-NA5200 (H1N1 pdm09 and swine-like) anti-sera strongly inhibited the NA activity of Bel/09, Sw/Bel/98 and NIBRG-14. Anti-NA7900 (historical seasonal H1N1-like) immune serum mediated NI against all four seasonal H1N1 viruses tested (PR8/34, USSR/77, NC/99 and Bris/07) and NIBRG-14, displaying a broader NI span than anti-serum raised against NC/99 rNA (Fig. 2a,b). Finally, anti-NA9100 (H1N1 viruses 1933–2009) NA serum showed substantial NI against all N1 viruses tested.

Fig. 2 Anti-sera raised to CBC-designed NAs mediates NI against a broader range of influenza A viruses (IAV) than WT NA sera. Mice were vaccinated twice with 1 µg of PR8/34 rNA, NC/99 rNA, Bel/09 rNA, NA5200, NA7900, NA9100 or PBS alone, 3-weeks apart via the subcutaneous route in the presence of SAS. Three weeks following the boost anti-sera was collected and tested for their ability to inhibit the NA activity of a panel of N1 viruses. Increasing twofold serial dilutions of heat-inactivated sera raised to rNAs were mixed with a panel of N1 viruses and NA activity was determined at 18 h on fetuin coated plates by ELLA as described in Materials and Methods. Data shows IC 50 values as determined by non-linear regression analysis and plotted as log 2 values of the 1:x dilution resulting in 50% NA inhibition. a Data are reported as the mean of the experiment performed in triplicate and is representative of 2 independent experiments. The dotted line on the y axis represents the highest concentration of the sera tested, 4.3 (i.e., log 2 of 1:20) and the cut-off of the assay. b Data represented as a heat map of IC 50 values next to a phylogeny tree of NAs clustered by amino acid sequence of the NA head region Full size image

Next, the percent sequence identity shared between the CBC NAs or wild-type (WT) NAs and the N1 viruses used in the ELLA was determined, considering only amino acids from 75 onwards, as the NA designs lack the native NA stalk sequence. NA9100 shared the highest percent identity with the N1 viruses tested, followed by NA7900, then NA5200. In comparison, the identity of WT NAs to other N1 NA varied greatly. In general, a higher degree of sequence identity between the WT viruses and the CBC-designed NAs, correlated with greater breadth of coverage in the NI (as shown in bold, Supplementary Table 2). Interestingly, although NC/99 NA shared 92% identity with USSR/77 there was no NI detected, even though the majority of the CBC NAs displayed NI at a ≥88% identity. This approach of percent sequence identity, however, does not take into account that some antigenic or conserved epitopes are discontinuous and bridge multiple regions of the monomer and even the tetrameric structure.11,18 Taken together, while the WT NAs do induce a degree of cross-reactivity, by using the consensus-based approach the breadth of cross-reactivity can be significantly enhanced, likely by targeting conserved epitopes within the N1 NA or combining epitope regions from multiple NA sequences into one molecule.

Vaccination with CBC NAs provide protection against influenza A viruses in mice

Next, the protective capability of the CBC NAs was examined in the mouse model. Mice were primed and boosted with NA5200, NA7900 or NA9100 rNA with adjuvant. Mice that had been mock-vaccinated with buffer only plus adjuvant were included as controls. Three weeks following the boost, mice were challenged with 5 LD 50 of either PR8/34, USSR/77, NC/99, Bel/09, Sw/Bel/98 or NIBRG-14 and assessed over 14 days for body weight change and survival (Fig. 3). All challenged mice that received adjuvant alone succumbed to the infection by day 9 (Fig. 3). Vaccination with NA5200 significantly protected mice from weight loss and death, due to infection, with all N1 viruses tested, except USSR/77, compared with the controls. NA5200-vaccinated mice challenged with USSR/77 displayed transient weight loss over time and were only partially protected against mortality (Fig. 3b). NA7900 vaccination significantly protected mice from challenge with PR8/34, USSR/77, NC/99 or NIBRG-14, compared with mock-vaccinated animals, but only partially against Bel/09 and Sw/Bel/98. NA9100 showed a broad range of protection, significantly protecting mice from weight loss and death when infected with PR8/34, NC/99, Bel/09, Sw/Bel/98 and NIBRG-14. Mice infected with USSR/77 displayed no significant difference compared with mock-vaccinated mice, when considering main columns effects of a two-way ANOVA (P-value = 0.08). However, on days 6 through 9, there was a significant difference in weight loss compared with mock-vaccinated mice (p < 0.01, two-way ANOVA) and 100% of the mice survived the infection (Fig. 3b). Taking these results together, we can conclude that NA5200 and NA9100 rNAs provide broad protection potentially spanning a long time frame (approximately 80 years) and corresponding antigenic space.

Fig. 3 Vaccination with CBC NAs protects mice against a lethal infection with influenza A viruses. Mice were primed and boosted at a 3-week interval via the subcutaneous route with 1 µg of NA5200 (black circles), NA7900 (white squares) or NA9100 (white circles) rNA in SAS or mock vaccinated (white triangles). Following vaccination, mice were infected with 5 LD 50 of either a PR8/34, b USSR/77, c NC/99, d Bel/09, e Sw/Bel/98 or f the H5N1 strain NIBRG-14 intranasally and monitored for weight loss (left panels) and mortality (right panels). When a mouse had lost ≥25% of its original body mass the animal was culled. Weight loss data are shown as the mean percentage (±SEM) of original body weight over time (n = 8–10), survival data are shown as the percentage of survival over time (n = 8–10). The data shown are pooled from two independent experiments. Weight loss was examined by two-way ANOVA, main column effects, and survival proportions were assessed using a two-tailed, log-rank (Mantel Cox) test. *P < 0.05, **P < 0.01 in comparison to mock-vaccinated mice Full size image

Previous studies have shown that viral loads within the lung are decreased when anti-NA immunity is induced.19 Therefore, in separate experiments, we assessed if vaccination with NA5200, NA7900 or NA9100 could also reduce viral lung loads. Accordingly, mice were primed and boosted with either NA5200, NA7900 or NA9100 CBC NA and infected with 5 LD 50 of PR8/34, NC/99 or Bel/09, 3 weeks following the boost (Fig. 4). On day 3 and day 7 post-infection, lungs were collected and viral loads examined. On day 3, viral loads did not show any significant reduction in the NA-vaccinated groups compared with mock-treated animals. By day 7 after challenge, however, all mice that had been vaccinated with consensus rNAs had significantly lower titres than the mock-vaccinated mice. Since vascular leakage and pulmonary oedema are indicative of a severe influenza infection, we also investigated the possible benefit of NA vaccination for these parameters based on the total protein content within cell-free Broncoalveolar lavage (BAL) fluids.20,21 Mice vaccinated with NA5200, NA7900 or NA9100 rNA and infected with either PR8/34, NC/99 and Bel/09 displayed no significant difference in the protein levels in BAL fluids isolated on day 3 after challenge, but had significantly less total protein on day 7 post infection compared with control mice (Fig. 4a–c, right panels).

Fig. 4 Protection mediated by CBC NAs results in a decrease of lung viral titres and inflammation within the lung milieu. Mice were primed and boosted with a 3-week interval via the subcutaneous route with 1 µg of NA5200, NA7900 or NA9100 rNA in SAS or mock-vaccinated with PBS in SAS. Following vaccination, mice were infected with 5 LD 50 of either a PR8/34, b NC/99 or c Bel/09 intranasally. On day 3 or day 7 post-infection BAL fluids and lungs were taken and lung homogenates were assessed for viral titres by TCID 50 (left panels). Cell-free BAL fluids were investigated for total levels of protein (right hand panels). The dashed line, in viral titre graphs, indicates the cut-off limit of the TCID 50 assay (0.7). For protein concentrations; bars represent the mean. Data are pooled from two independent experiments. Significance was assessed using one-way ANOVA. *P < 0.05, **P < 0.01 in comparison to mock-vaccinated mice Full size image

Passive transfer of CBC NA anti-sera protects against lethal challenge with influenza A virus

The major correlate of protection induced by vaccination with rNA is the ability to induce NA inhibiting antibodies.12 To examine if antibodies were the major mediators of protection induced by vaccination with CBC NAs in this mouse model, heat-inactivated anti-sera raised separately against NA5200, NA7900, NA9100 or buffer alone (PBS) were passively transferred intranasally to mice 1 day prior to infection with 2 LD 50 of either PR8/34, NC/99 or Bel/09 (Fig. 5). Anti-sera raised to individual WT rNAs were included as homologous-positive controls. Anti-sera were the same sera used previously in Fig. 2 to assess the broadening of NAI responses. In all viral challenges, the positive control anti-sera fully protected mice from weight loss and survival following a potentially lethal infection with the homologous virus. Although active vaccination with NA5200 rNA protected against PR8/34, NC/99 and Bel/09 infection, passive transfer of NA5200 anti-serum only provided significant protection against weight loss and mortality in mice infected with Bel/09 or PR8/34, and not with NC/99 (p < 0.01 two-way ANOVA or log-rank test) (Fig. 5). NA7900 rNA immune serum recipient mice were significantly protected against morbidity and mortality when infected with PR8/34 and NC/99, in comparison with the negative control group (p < 0.01 two-way ANOVA or log-rank test). Similar to the poor protection by NA7900 rNA active vaccination against Bel/09 challenge, protection against this challenge virus by passive transfer of NA7900 anti-serum was not significantly different from the negative control group. Finally, the passive transfer of NA9100 anti-serum provided significant protection against all three challenge viruses for both weight loss and survival (p < 0.01 two-way ANOVA or log-rank test). In all passive transfer experiments (Fig. 5), full protection against mortality correlated with the ability of the anti-sera to mediate NI (Fig. 2). As such, it can be concluded that antibodies play a major role in the protection induced by the CBC NAs.

Fig. 5 Antibodies play a major role in the protection provided by vaccination with CBC NAs. Anti-sera raised to CBC rNAs, WT PR8/34 rNA, WT NC/99 rNA, WT Bel/09 rNA (same as used in Fig. 2) were assessed for their ability to control influenza virus infection via passive transfer. Mice were treated intranasally with 20 µl of heat-inactivated anti-sera raised against (i) NA5200, (ii) NA7900, (iii) NA9100, (iv) PBS (negative; -ve control) or (v) respective homologous rNA positive (+ve) control. Twenty-four hrs later they were challenged with 2 LD 50 of a PR8/34, b NC/99 or c Bel/09 via the intranasal route and monitored over 14 days for weight loss (left panels) and survival (right panels). If mice had lost ≥25% of their original body weight they were euthanized. Weight loss data displays the mean percentage (±SEM) of original body weight over time (n = 8) and survival data is shown as the percentage of survival over time (n = 8). The data are pooled from two independent experiments. A two-way ANOVA was used to analyse weight loss over time, assessing main column effects, whilst a two-tailed, log-rank (Mantel Cox) test was used to assess survival. *P < 0.05 in comparison with negative control mice Full size image

CBC NAs provide broader protection than WT NAs and increase the breadth of a split inactivated vaccine

To test if the CBC NAs could (i) offer broader protection in vivo compared with WT rNAs and (ii) increase upon the protection provided by a conventional split inactivated vaccine, mice were vaccinated with NA5200 and NA9100 or WT soluble recombinant NAs derived from Bel/09 and NC/99 alone or in combination with a monovalent H1N1 pdm09 vaccine. Subsequently, the mice were challenged with 5 LD 50 of Bel/09 or NC/99. Mice vaccinated with NC/99 rNA were not protected from weight loss and mortality following Bel/09 challenge. Also following Bel/09 challenge, both NA5200 and NA9100-vaccinated mice displayed slightly more, yet significant, weight loss in comparison with homologous-vaccinated mice. However, 100% of these mice survived the infection (Fig. 6a, left and middle panels). Furthermore, although viral loads in the lungs on day 7 of NC/99-vaccinated mice were on average similar to mock-vaccinated mice, viral titres on day 7 of NA5200 and NA9100-vaccinated mice were significantly reduced compared with mock-vaccinated mice (p < 0.01, one-way ANOVA), and both homologous Bel/09 rNA and NA5200 out-performed NA9100 NA (Fig. 6a, right panels). A similar, but reversed pattern was observed for mice challenged with seasonal NC/99 when receiving the more ‘seasonal’-based NA9100 (Fig. 6c). In this case, Bel/09 rNA-vaccinated mice lost significantly more weight, displayed increased mortality and higher viral loads at day 6 post-infection compared with NC/99-vaccinated mice (Fig. 6c left and middle panels). Compared with the data presented in Fig. 3c, NA5200 did not protect NC/99 challenged mice to the same degree, but NA9100 was not significantly different in its ability to protect mice compared with homologous-vaccinated mice. In addition, Bel/09 rNA could also not protect mice from morbidity and mortality when infected with PR8/34 virus (data not shown), while all CBC tetNA induced protection (Fig. 3). Taken together, these data show that NA9100, in particular, shows broader protection than the WT NAs tested.

Fig. 6 CBC NAs provide broader protection than WT NAs and provide increased breadth when added to monovalent inactivated vaccine. Mice were primed and boosted at a 3-week interval via the subcutaneous route, in the presence of SAS adjuvant, with either 1 µg of WT Bel/09 rNA, WT NC/99 rNA, CBC NAs NA5200, NA9100 or with 0.1 µg of monovalent pdm09 vaccine alone or in combination with 1 µg of WT Bel/09 rNA, WT NC/99 rNA, CBC NAs NA5200, NA9100. A group of mice was also mock vaccinated. Three weeks following the final boost, the mice were challenged with 5 LD 50 of Bel/09 or NC/99 via the intranasal route. Mice were monitored for weight loss (left panels) and survival (middle panels) over 14 days. Mice were sacrificed if they lost ≥25% of their original body mass. On day 7 for Bel/09 infections and day 6 for NC/99 infections lung homogenates were obtained and assessed for viral load. Weight loss data represents the mean percentage (±SEM) of original body weight over time and survival data are shown as the percentage of survival over time (n = 9–10 for a, c pooled from two independent experiments and n = 5 for b, d from one experiment). Weight loss over time was analysed by two-way ANOVA, examining main column effects, and survival proportions were assessed using a two-tailed, log-rank (Mantel Cox) test. Significance was assessed using one-way ANOVA for viral titres. *P < 0.05, **P < 0.01 in comparison to homologous rNA-vaccinated mice for a, c or in comparison to monovalent alone vaccinated mice for b, d Full size image

Mice vaccinated with monovalent H1N1 pdm09 split vaccine (alone or in combination with CBC rNAs) and challenged with Bel/09 showed little signs of weight loss, almost no mortality and no signs of virus in the lungs by day 7 post-infection (Fig. 6b). These results are as expected after immunization with the monovalent vaccine, especially in combination with an adjuvant, and is known to provide protection against homologous viruses, with little room for improvement within this model.22 Protection provided by the monovalent vaccine is most likely attributable to HA content, as the monovalent H1N1 pdm09 vaccine has been shown to contain a low amount of intact NA.23 When mice were challenged with NC/99, there was also a degree of protection provided by the adjuvanted split vaccine alone compared to mock-vaccinated mice (Fig. 6c,d). Both NA5200 and NA9100 showed no additive effect on weight loss and survival in comparison with monovalent only. However, the addition of NC/99 or NA9100 NAs to the monovalent split vaccine significantly reduced viral titres in the lungs compared to monovalent alone (Fig. 6d). As such, while we only saw a modest increase in protection compared to monovalent alone vaccine, these data highlight the ability of rNA to augment protection when combined with the traditional vaccine approach.

NA5200 and NA9100 NA anti-sera inhibits the NA activity of a A(H1N1)pdm09 HA variant

For the 2017 Southern Hemisphere influenza season, the World Health Organization (WHO) recommended to replace the A(H1N1)pdm09-like virus in the seasonal influenza vaccine with a A/Michigan/45/2015-like strain,24 as it was evident this variant possessed a change within the HA that resulted in increased infection rates in middle-aged adults.25,26 It was therefore important to test if the CBC designs could also mediate NI against this variant, as its sequence was not included in the original design strategy. Initially, we investigated the HA antigenic difference using hemagglutination inhibition (HI), between Bel/09 and the A/Michigan/45/2015-like virus A/Singapore/GP1908/2015 (Sing/15). We observed a twofold difference in the ability of anti-sera raised in mice against the monovalent split A(H1N1)pdm09 vaccine to mediate HI against Bel/09 and Sing/15 (1280 HAU vs 640 HAU, data not shown), which is in agreement with previous studies where ferret reference sera did not indicate evidence of significant antigenic drift.25,26 Next, the ability of anti-sera raised against the CBC rNAs, or Bel/09 rNA, to mediate NI against Bel/09 and Sing/15 was tested. Whereas Bel/09 immune serum had a significantly lower NI titre against the drift variant Sing/15 as compared with its homologous titre (p < 0.05, one-way ANOVA), anti-sera raised to NA5200 and NA9100 mediated NI against both Sing/15 and Bel/09 equally well albeit at lower levels than WT Bel/09 anti-NA sera (Supplementary Fig. 1A/B). When considering amino acid identity from sequence position 75 onwards, Sing/15 shared 91%, 87%, 89 and 97% identity to NA5200, NA7900, NA9100 and Bel/09, respectively (Supplementary Table 2). Whether the NA of Bel/09 and Sing/15 are antigenically different is not known and if the significant difference between the ability of anti-Bel/09 sera to mediate NI between Sing/15 and Bel/09 reflects a difference antigenically is also not known. Of importance, though, is that the CBC NAs displayed the same level of inhibition against a strain that was not included in the original design of the proteins but is considered drifted with regards to HA and is now the new vaccine strain.