Antibodies to the hemagglutinin (HA) and neuraminidase (NA) glycoproteins are the major mediators of protection against influenza virus infection. Here, we report that current influenza vaccines poorly display key NA epitopes and rarely induce NA-reactive B cells. Conversely, influenza virus infection induces NA-reactive B cells at a frequency that approaches (H1N1) or exceeds (H3N2) that of HA-reactive B cells. NA-reactive antibodies display broad binding activity spanning the entire history of influenza A virus circulation in humans, including the original pandemic strains of both H1N1 and H3N2 subtypes. The antibodies robustly inhibit the enzymatic activity of NA, including oseltamivir-resistant variants, and provide robust prophylactic protection, including against avian H5N1 viruses, in vivo. When used therapeutically, NA-reactive antibodies protected mice from lethal influenza virus challenge even 48 hr post infection. These findings strongly suggest that influenza vaccines should be optimized to improve targeting of NA for durable and broad protection against divergent influenza strains.

Here, we report that unlike vaccination, natural influenza virus infection readily induces a high proportion of NA-reactive B cells. Thus, from infected patients, we were able to isolate and characterize protective antibodies binding NA epitopes, informing on the design of an NA-based component for influenza vaccination. The NA-reactive antibodies can be induced in humans or mice by infection or immunization with whole virions but bind epitopes not efficiently detected in the Fluarix or Fluzone influenza vaccines. Importantly, these NA-reactive mAbs bind a broad spectrum of influenza virus strains, often spanning the entire circulation history in humans for that NA group. Moreover, these antibodies have robust NA inhibition (NI) activity and provide prophylactic as well as therapeutic protection in vivo. Our data suggest that the next-generation of influenza vaccines should be optimized to improve the NA humoral immune response to induce broadly cross-reactive and protective NA-reactive antibody responses.

There is a long history of literature suggesting that immunity to NA could protect from influenza infection. The polyclonal antibody response to NA was broadly reactive and conferred protection against heterologous viruses in mice (). This cross-reactivity is evident even when there is substantial change within strain specific NA epitopes, resulting in a phenomenon of one-way drift (). NA-reactive monoclonal antibodies (mAbs) isolated from mice and rabbits protected against both homologous and heterologous influenza infection in vivo (). Several conserved amino acids were identified in these studies as the basis for the broad reactivity of NA-reactive mAbs (). Previous studies in humans have also shown that preexisting NA-reactive antibodies can reduce the number of cases of infection and decrease disease severity (). However, on the whole, little is known about human antibody responses to NA, and most influenza vaccine development efforts both past and present are focused on targeting HA.

There are several mechanisms of NA-reactive antibody inhibition of influenza virus infection (). NA-reactive antibodies can prevent virus budding and egress from infected cells. These antibodies similarly inhibit viral escape from the natural defense proteins that trap the virus via HA-sialic acid interactions on mucosal surfaces. Moreover, NA-reactive antibodies bound to NA at the surface of infected cells might aid in the clearance of the virus through antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) ().

Historically, NA has served as an important target for antivirals or therapeutics due to its critical role in the influenza virus replication cycle (). Inhibition of NA activity is the basis of commonly used influenza therapeutics, including oseltamivir (Tamiflu), zanamivir (Relenza), laninamivir (Inavir), and peramivir (Rapivab). Oseltamivir can reduce the median duration of influenza illness by 1.3 days and markedly reduces symptoms compared to placebo if given within 48 hr of symptom onset. In a prophylactic study, oseltamivir decreased rates of influenza infection 5-fold from 5% (25/519) for the placebo group to 1% (6/520) for the oseltamivir-treated group (). Thus, inhibition of NA activity has become a standard of care for the treatment of influenza virus infections. The limitations of neuraminidase inhibitors such as oseltamivir are that resistant strains of influenza virus have readily emerged (), and the window for efficacy is limited to the first 48 hr of symptom onset.

In the shadow of hemagglutinin: a growing interest in influenza viral neuraminidase and its role as a vaccine antigen.

Currently, the seasonal influenza virus vaccine is the most widely available method to reduce the annual impact of influenza infection (). HA-reactive antibodies are typically considered the de facto mediators of protection from influenza infection; indeed, inhibition of HA activity has been the primary measure of influenza vaccine efficacy for decades. Therefore, most of the current approaches for vaccine design focus on inducing an antibody response to influenza virus HA. Influenza vaccine effectiveness can vary widely from season to season such that protection is always limited. For example, vaccine effectiveness ranged from only 19%–48% during the past three influenza seasons (). Studies have shown that HA antigenic drift (viral genome point mutations) is the primary reason for the limited effectiveness of the seasonal influenza vaccine (). Due to viral mutations, preexisting antibodies often show limited neutralization against currently circulating viruses (). Although point mutations also occur in the NA protein, the rate of antigenic drift around the active site of NA in the head domain is slower than that for HA among influenza A viruses ().

In the shadow of hemagglutinin: a growing interest in influenza viral neuraminidase and its role as a vaccine antigen.

Influenza is an acute respiratory illness that causes up to 5 million cases of influenza virus infection and 250,000–640,000 deaths annually around the world (). The influenza virus has two main surface glycoproteins, hemagglutinin (HA) and neuraminidase (NA). HA, the more abundant protein, mediates binding to sialic acid receptors and subsequent fusion between the virus and host cell membranes. The less-abundant tetrameric NA protein is essential for cleaving terminal sialic acid residues present on host cell surfaces, allowing the release of the newly formed viral particles ().

The therapeutic efficacy of the NA-reactive mAbs that were protective as prophylactics was also analyzed. Mice that were lethally infected with 10 LDof influenza virus were treated with 10 mg/kg of NA-reactive mAbs 48 hr post infection. All four of the N1-reactive mAbs fully rescued infected mice from severe weight loss and mortality after 2009 pandemic H1N1 influenza virus challenge ( Figure 7 D). Similarly, 88% (7/8) of the N2-reactive mAbs proffered full recovery to the mice challenged with an H3N2 virus ( Figure 7 E). In sharp contrast, all mice in the control mAb group had to be euthanized around day nine post infection because of severe weight loss. These results show that the NA-reactive mAbs can be used therapeutically, even after 48 hr of influenza virus infection, suggesting they could be capable alternatives to NA inhibitors, such as oseltamivir. With improved vaccine formulations to induce NA antibodies, the same benefits as NA-inhibiting drugs could be prophylactically elicited without the need for early administration. Further, unlike NA-inhibiting medications, which lose effectiveness due to the emergence of resistant strains, administration of booster vaccines would control viral resistance.

Variants of the 2009 pandemic H1N1 influenza strains and viruses expressing H5 and N1 from highly pathogenic avian strains are infectious to mice. First, mice treated with N1-reactive mAbs were challenged with a 2009 pandemic H1N1 isolate (A/Netherlands/602/2009). Five out of eight of the mAbs from the 2009–2010 cohort completely protected mice against weight loss and mortality after challenge, whereas mice treated with control mAb lost weight rapidly and were euthanized by day eight post infection ( Figure 7 B). Four out of five of the mAbs that prophylactically protected against H1N1 infection (4/8 in total) also provided 100% protection from a reverse genetic PR8 variant virus expressing the H5 and N1 of a highly divergent avian influenza virus strain (A/Vietnam/1203/2004, H5N1) ( Figure 7 C). Thus, half of all mAbs induced against N1 in individuals infected with the 2009 pandemic H1N1 strain provided broad protection against an H5N1 strain. This frequency far exceeded the 10% of HA-reactive mAbs that arose against this H1N1 strain that even bound to H5 (). Together, these results suggest that when induced against common infectious influenza virus strains, NA-reactive mAbs are outstanding mediators of broadly protective immunity, even to a virus expressing the H5 and N1 glycoproteins from a divergent avian influenza virus strain with pandemic potential.

The broad cross-reactivity, as well as widespread in vitro NI activity of NA-reactive mAbs, suggest that they will be broadly protective in vivo. To test this, we measured the prophylactic protection against challenge with divergent strains in vivo. Half-maximal lethal dosages (LD) of the influenza virus were determined. Mice received 5 mg/kg of NA-reactive mAb or the same dose of a non-binding control mAb by intraperitoneal (i.p.) injection. 2 hr later, the mice were lethally challenged with 10 LDof influenza virus by intranasal inoculation. Recent H3N2 isolates do not replicate well in the mouse model, but historical strains like A/Philippines/2/1982 (H3N2, X-79) infect mice readily. This virus is phylogenetically distant from those that caused the human infections from which the mAbs are derived, providing insight into the breadth of protection as well. A selection of N2-reactive mAbs representing all overlapping epitopes were tested. Surprisingly, 84% (11/13) of the N2-reactive mAbs showed partial or full protection in the prophylactic challenge experiment against this H3N2 influenza strain that was isolated 35 years ago ( Figure 7 A). The protection conferred was consistent with the breadth of binding and NI activity of these mAbs. Moreover, non-neutralizing NA-reactive mAbs also provided in vivo prophylactic protection. These data show that neutralizing and non-neutralizing N2-reactive mAbs provide broad prophylactic protection against H3N2 influenza strains in vivo.

6-week-old female BALB/c mice (five per experimental condition) were injected intraperitoneally (i.p.) with 5 mg/kg of each NA-reactive mAb individually or with an irrelevant negative control human mAb either 2 hr prior to challenge (A–C) or 48 hr after challenge (D and E) with a lethal dose (10 LD 50 ) of virus. The percentage of initial body weight and survival were plotted for each antibody and compared to untreated mice. Data are represented as mean ± SD. Influenza-non-reactive human mAb 003-15D3 (anti-anthrax protective antigen) was used as a negative control in all experiments.

In a previous study, 26 single-amino-acid escape mutations were identified for common binding sites on N1 (). To map the epitopes recognized by the N1-reactive mAbs identified herein, these 26 single-amino-acid mutant A/California/7/2009 N1 proteins were expressed in HEK293. Cell-based ELISAs were carried out to test the binding of all of the NI-active mAbs to the mutant proteins, identifying the binding sites of four antibodies. A G249K mutation significantly affected the binding of 1000-3B06 (70% decrease compared to the wild-type N1). The N273D mutation reduced the binding of 1000-1D05 compared to the wild-type N1 protein. Furthermore, the N309S mutation affected both 294-A-1C02 and 294-A-1D05 binding ( Figure 6 A). Amino acids N273 and N309 are 99.7% (6835/6855 H1 influenza strains) conserved in H1N1 viruses isolated from 1918 until now in the United States. The G249 site is also conserved in H1N1 viruses (90.3%, 6196/6855 H1 influenza strains). These residues are all located on the NA head ( Figure 6 B). To map the epitope(s) targeted by the N2-reactive mAbs, ELISA was used to test the binding avidity of all of the N2-reactive NI-active mAbs against 12 single-amino-acid mutants of N2 expressed on an A/Minnesota/11/2010 (H6N2-PR8 backbone) purified virus. The 12 amino acids mutated were chosen based on a solved N2-mAb crystal structure and are important for the N2 enzymatic site (). Three amino acids (N221, G248, and G429) on the NA enzymatic conserved domain are critical for the binding of three of the NI-positive N2-reactive mAbs ( Figures 6 C and 6D). Consistently, all three of these mAbs were also positive in the NA-STAR assay ( Figure 4 A). Notably, as indicated by the red asterisks in Figures 6 B and 6D, these key epitopes on N1 and N2 are disrupted in the inactivated vaccine compositions because the mAbs that bind these sites have substantially reduced or no reactivity to the vaccine.

The mutated sites within epitopes that are also disrupted in the inactivated vaccines ( Figure 1 ), disrupting mAb binding, are indicated with red asterisks (mAbs 1000-1D05, 294-A-1C02, and 294-A-1D05 did not bind to either Fluarix or Fluzone, and 1000-3B06 and 235-1C02 bound poorly).

(D) Modeling of N2 protein was done using PyMOL to show the three critical amino acids involved in the binding of the N2-reactive mAbs (PDB: 4K1J ) ().

(C) Binding of three N2-reactive mAbs (229-1D05, 235-1C02, and 235-1E06) to 12 A/Minnesota/11/2010 (H6N2-PR8 backbone) NA mutant viruses. Data are represented as mean ± SD. Data are representative of two independent experiments performed in duplicate.

(B) Modeling of N1 was done using PyMOL to show the four critical amino acids involved in the binding of the N1-reactive mAbs (PDB: 3TI6 ) ().

(A) Binding of four N1-reactive mAbs (1000-3B06, 1000-1D05, 294-A-1C02, and 294-A-1D05) to A/California/7/2009 (H1N1) NA mutant proteins transiently expressed on the surface of 293T cells. Hyper-immune mouse serum against A/California/7/2009 (H1N1)-X179A virus was used to verify the expression of NA. Binding to A/California/7/2009 wild-type NA is shown in the last bar labeled “WT.” Data are represented as mean ± SD. Data are representative of two independent experiments performed in duplicate.

Identification of NA Residues that Are Crucial for mAb Binding and that Are Disrupted in the Inactivated Vaccine Compositions

Microneutralization (MN) measures the inhibition of influenza virus replication in vitro, providing another measure of protective activity. Unlike for HA mAbs, this assay underestimates the potency of anti-NA mAbs because the mechanism of action of these antibodies inhibits viral egress, not the initial infection of individual cells. However, MN assays do allow a measure of the frequency of NA antibodies that have protective activity. In total, 45% of the NA-reactive mAbs, including 43% (6/14) of the N2-reactive mAbs and 47% (7/15) of the N1-reactive mAbs, were able to neutralize viruses related to the infecting strain ( Figure 5 A). The five N2-reactive antibodies that bound the enzymatic site of NA and had NI-activity against oseltamivir-resistant strains were also able to neutralize the resistant A/Texas/12/2007 E119V (H3N2) influenza strain in vitro ( Figure 5 B). Conversely, oseltamivir had virtually no neutralization activity against this strain. To ensure that the anti-NA antibody response was contributing to long-term serum immunity, we isolated NA-reactive polyclonal antibodies by affinity purification from day 21 and day 63 post-infection serum samples and tested them by using MN assays (). The isolated NA-reactive polyclonal antibodies also readily protected Madin-Darby canine kidney( MDCK) cells from infection in vitro ( Figure 5 C). These data show that nearly half of the NA-reactive antibodies characterized herein exhibit neutralization activity, inhibiting virus replication, and suggest that they contribute to long-term serum immunity.

(B) The N2-reactive mAbs were tested for neutralization by MN assay using A/Washington/01/2007 (oseltamivir-sensitive strain) and A/Texas/12/2007 E119V (oseltamivir-resistant strain) H3N2 viruses. Data are represented as IC 50 (nM).

(A) NA-reactive mAbs were tested for neutralization by microneutralization (MN) assay using A/Switzerland/9715293/2013 (H3N2) and A/California/7/2009 (H1N1) viruses. Data are represented as IC 50 (μg/mL). Positive control mAbs 229-1C01 (anti-H3N2) and EM-4C04 (anti-H1N1) bind HA and neutralize these influenza virus strains.

NA-reactive mAbs may be improved alternatives to therapeutic NA-inhibitor drugs such as oseltamivir or even more efficacious when efficiently elicited by vaccination. Using biolayer interferometry, we devised an assay to competitively measure the binding of oseltamivir versus NA-reactive mAbs to the NA protein. Binding of three of the enzymatic domain-targeting mAbs (NA-STAR assay positive, 229-1D05, 229-1F06, and 229-1G03) can be inhibited by prior saturation of NA of an oseltamivir-sensitive strain with oseltamivir ( Figures 4 C and S4 ), demonstrating overlap binding of the mAb binding footprints with the pocket occupied by oseltamivir. Because oseltamivir acts by blocking the enzymatic domain, its activity against influenza strains can be assessed by the NA-STAR assay. While oseltamivir had virtually no NI activity, all five of the enzymatic domain-binding mAbs on hand—which is 36% of the N2-reactive mAbs isolated—inhibited the NA activity of typical oseltamivir-resistant strains (A/Bethesda/956/2006 R292K and A/Texas/12/2007 E119V). For 229-1G03 and 235-1E06, the ICis nearly identical against the sensitive and resistant A/Texas/12/2007 E119V strains ( Figure 4 D). These studies demonstrate that the majority of human antibodies against NA analyzed herein can inhibit the enzymatic activity of this protein on highly divergent influenza strains.

The enzymatic function of NA is to cleave the terminal sialic acid residues, allowing viral egress from infected cells. To better access the protective capacity of the NA-reactive mAbs, inhibition of sialic acid cleavage was evaluated using ELLA and NA-STAR assays. For ELLA, the sialic acid substrate is found on bulky glycans on the fetuin glycoprotein. If an antibody binds to NA, even outside of the enzymatic site, it sterically blocks access of the enzymatic site to sialic acid, making this assay more sensitive to multiple mechanisms of NI activity. Conversely, the NA-STAR assay uses a small, soluble chemiluminescent substrate that easily accesses the enzymatic site unless an antibody binds in close proximity to the enzymatic site. Using ELLA, 79% (11/14) of the N2-reactive mAbs—of which about half (5 /14) were also positive in the NA-STAR assay, likely demonstrating activity through blockage or allosteric mechanisms of inhibition—inhibited NA activity against A/Switzerland/9715293/2013 (H3N2). Importantly, by either assay, all of these mAbs inhibited the first pandemic H3N2 strain A/Hong Kong/1/1968 as well as A/Philippines/2/1982 (X-79) ( Figure 4 A). The antibody 229-1D05 also had broad NI activity against swine influenza virus strain A/swine/Missouri/4296424/2006 (H6N3, H6 is from A/mallard/Sweden/81/2002, PR8 backbone) ( Figure S3 ). Therefore, these mAbs have broad NI activity spanning five decades of H3N2 virus evolution. For mAbs reactive to the 2009 pandemic H1N1 strain, 53% (8/15) had NI activity by any means as detected by ELLA, and 20% blocked the enzymatic domain, showing inhibition via the NA-STAR assay. As with the N2-reactive mAbs, N1-reactive mAbs had broad activity against the 1918 pandemic strain A/Brevig Mission/1/1918, against A/New Caledonia/20/99, and against the avian A/Vietnam/1203/2004 (H5N1) and A/rhea/NC/39482/93 (H7N1) strains ( Figure 4 B).

(C) Cross N1-reactive mAbs were tested for inhibiting NA enzymatic activity via ELLA assay against A/Northern shoveler/Alaska/7MP1708/07 (H3N8). Bar graphs represent IC 50 (nM) values. Oseltamivir was used as a positive control and influenza-non-reactive mAb 003-15D3 was used as a negative control in the assays. Data are representative of two independent experiments.

Influenza-non-reactive human mAb 003-15D3 is specific for anthrax protective antigen and was used as a negative control in (A), (B), and Figure 5

(D) The N2-reactive mAbs were tested for inhibiting NA enzymatic activity by NA-STAR assay against oseltamivir-sensitive strains A/Brisbane/10/2007 (H1N1) and A/Washington/01/2007 (H3N2) and oseltamivir-resistant strains A/Bethesda/956/2006 R292K (H1N1) and A/Texas/12/2007 E119V (H3N2).

(C) Binding competition between the N2-reactive mAb 229-1D05 and oseltamivir to A/Texas/50/2012 rNA was measured by biolayer interferometry.

(B) N1-reactive mAbs were tested for inhibiting NA enzymatic activity in ELLA assays and NA-STAR assays against A/California/7/2009 (H1N1) virus, A/Brevig Mission/1/1918 (H1N1) rNA protein, A/New Caledonia/20/99 (H1N1) virus, avian A/Vietnam/1203/2004 (H5N1) virus, and A/rhea/NC/39482/93 (H7N1) virus. Data are represented as IC 50 (nM). Oseltamivir was used as a positive control.

(A) N2-reactive mAbs were tested for inhibiting NA enzymatic activity via ELLA assays and NA-STAR assays against A/Switzerland/9715293/2013 (H3N2), A/Hong Kong/1/1968 (H3N2), and A/Philippines/2/1982 (H3N2-X-79) viruses. Data are represented as half-maximum inhibitory concentration IC 50 (nM). Oseltamivir was used as a positive control.

To determine the breadth of binding of the NA-reactive mAbs induced by infection, ELISA was used to test binding against a diverse panel of rNA proteins ( Figure 3 A). All of the N2-reactive mAbs cross-reacted to all contemporary H3N2 influenza strains, and also, a surprising 86% (12/14) reacted to the first pandemic H3N2 virus strain known to infect humans (A/Hong Kong/1/1968). Considering that the 1968 H3N2 pandemic virus expressed the NA protein from a reassortant strain with the 1957 H2N2 strain (), it is not surprising that 71% (10/14) of the antibodies reacted to the H2N2 influenza strain that circulated since 1957—11 years prior. By comparison, only 40% of infection-induced and half of the vaccine-induced H3-reactive mAbs were cross-reactive to this 1968 H3N2 strain ( Figure 3 B). Moreover, 64% (9/14) of the N2-reactive mAbs induced by infection, including two mAbs with cross-reactivity to heterosubtypic subtypes (N3 and N9), were able to bind to avian N2 proteins ( Figure 3 A). Of the N1-reactive mAbs, 67% of cross-reacted to the 1918 pandemic H1N1 strain, 33% reacted to various human H1N1 strains spanning the entire century, and 20% bound to heterosubtypic strains ( Figure 3 A). Additionally, demonstrating durability of the NA-epitopes, incubating H3N2 (A/Switzerland/9715293/2013) virus with mAb concentrations up to 250 μg did not induce viral escape mutants even after 8 (229-1A02, 229-1D05, and 229-1G03) or 13 (228-2D04, 229-2B04, and 229-2C06) passages—an approach that does induce escape to arise from highly conserved HA-stalk mAbs (). On the whole, the NA-reactive mAbs induced during influenza virus infections are significantly more broadly reactive than antibodies against HA.

(B) Binding of 32 HA-reactive mAbs isolated from infected or vaccinated subjects to historical past H3N2 strain (A/Hong Kong/1/1968) rH3 were measured by ELISA. Pie charts represent the comparative frequency of HA-reactive mAbs against A/Hong Kong/1/1968 rH3 protein between the infected and vaccinated individuals.

(A) Binding of NA-reactive mAbs to rNA proteins was measured by ELISA. Representative minimum positive concentrations (μg/mL) from three independent experiments are plotted as a heatmap. The different NAs were clustered by amino acid sequence phylogeny. The top panel shows N2-reactive mAbs binding to a panel of NA proteins, except for strain A/Switzerland/9715293/2013 (H3N2), which was whole virus. The bottom panel shows N1-reactive mAbs binding to a panel of NA proteins. Pie charts represent the frequency of NA-reactive mAbs binding to historic strains (A/Hong Kong/1/1968 rN2 and A/Brevig Mission/1/1918 rN1).

The greater induction of NA-reactive plasmablasts during natural infection may be because the live intact virus displays epitopes not present in the inactivated vaccines. Memory to conserved epitopes appears to play a role in the observed bias, as serological studies have shown an induction of NA-reactive antibodies to past strains (). Both HA and NA antibodies were encoded from highly mutated variable genes, supporting a memory cell recall origin ( Figure S1 ). Furthermore, first exposure to the 2009 pandemic influenza virus strain induced NA-reactive plasmablasts at detectable frequencies in only two of the five infected patients that we characterized (black versus red lines in the top row of Figure 1 D). Conversely, exposure to that strain seven years later in 2016 or to H3N2 strains that have circulated since 1968 readily induced NA-reactive plasmablasts ( Figures 1 A and 1D). To determine if infection or exposure to whole virus particles could account for the increased NA targeting, we infected mice with live H1N1 or killed H3N2 whole virions as opposed to split/subunit vaccine. For this, mice were infected intranasally with a sublethal dose of live 2009 pandemic H1N1 virus or immunized intranasally with intact virions of inactivated H3N2 influenza virus followed by an intranasal boost with the respective virus strains 30 days later. Similar to what we observed in infected humans, serum responses in mice to NA were dramatically increased after immunization with whole influenza virions versus the 2015–2016 Fluarix quadrivalent influenza vaccine ( Figures 2 A and 2B ). To verify the preferred targeting of NA by whole virions at the cellular level, ELISPOT assays on whole splenocytes 8 days after secondary infection or immunization were used to measure the proportions of HA- and NA-reactive immunoglobulin G (IgG)-secreting cells that were activated. The frequency of NA-reactive cells was high after exposure to whole virions for both the H1N1 and H3N2 strains ( Figures 2 C and 2D). This observation was not dependent on viral replication, as the H3N2 influenza strain was inactivated. As with human infections with an H3N2 virus, more plasmablasts were specific to N2 than to H3 ( Figure 2 D). Although mice are not an ideal model of human immunity to influenza, it was surprising how accurately the response to whole virions reflected that of humans to both N1 and N2. Together, these experiments suggest that NA epitopes present on whole virions are not efficiently targeted by current influenza vaccines. To address this possibility directly, we tested the NA- and HA-reactive mAbs generated from infection-induced plasmablasts for binding to inactivated influenza virus vaccines. While HA-reactive mAbs bound the rHA protein with equal avidity to the vaccine, the NA-reactive mAbs had only negligible binding to the Fluarix ( Figures 2 E and 2G) or Fluzone ( Figures 2 F and 2H) vaccines. There was no notable difference in binding avidity by subtype (H1N1 versus H3N2) or by vaccine batch. Five mAbs that bound poorly to the vaccines maintained full avidity against inactivated the H3N2 virus used for the mouse immunization experiments ( Figures 2 A–2D), demonstrating that gentle inactivation protocols can maintain key NA epitopes ( Figure S2 ). We conclude from these studies that current influenza virus vaccines have insufficient NA content or NA protein structural integrity to induce NA-reactive antibody responses efficiently.

(B) NA-reactive mAbs (spots colored in red are the NA-STAR positive mAbs binding around the enzymatic site in Figure 6 C). Data are representative of two independent experiments, data are represented as median.

Several representative HA-reactive or NA-reactive mAbs were tested for binding by ELISA to A/Switzerland/9715293/2013 virus or inactivated A/Switzerland/9715293/2013 virus.

(E–H) HA- and NA-reactive mAbs were tested for binding by ELISA to HA, NA, and two influenza virus vaccine preparations. Binding avidities (K D ) were estimated by Scatchard plot analyses of ELISA data for 35 anti-H1, 15 anti-N1, 10 anti-H3, and 14 anti-N2 mAbs. (E) H1-mAb binding was compared between A/California/7/2009 (H1N1) rHA and Fluarix vaccine (2015–2016) and for H3-mAbs to A/Texas/50/2012 (H3N2) rHA and Fluarix vaccine (2014–2015). (F) Binding of H1-mAbs to A/California/7/2009 (H1N1) rHA compared to the Fluzone vaccine (2016–2017). (G) Binding N1-mAbs to A/California/7/2009 (H1N1) rNA compared to Fluarix vaccine (2015–2016) and N2-mAbs binding to A/Texas/50/2012 (H3N2) rNA compared to Fluarix vaccine (2014–2015). (H) Binding of N1-mAbs to A/California/7/2009 (H1N1) rNA compared to Fluzone vaccine (2016–2017). The red points indicate H3- and N2-reactive mAbs. Data are representative of three independent experiments. Statistical significance was determined using the paired nonparametric Wilcoxon test. The line represents the median. n.s., not significant. ∗ p < 0.05, ∗∗ p < 0.001, ∗∗∗ p < 0.0001.

(C and D) The proportion of HA and NA-reactive IgG secreting cells (ASCs) in mice after infection (C, H1N1) or immunization (D, H3N2). Pie charts show the average frequency of HA versus NA-reactive B cells. Bars indicate mean values.

(A and B) Serum responses in immunized mice were determined by ELISA. (A) HA1 and N1 serum endpoint titers (n = 5) were tested by A/California/07/2009 rHA and rNA, respectively. (B) HA3 and N2 serum endpoint titers (n = 5) were tested by A/Switzerland/9715293/2013 rHA and A/Texas/50/2012 rNA, respectively. Data are represented as mean ± SD.

(A–D) Mice were infected with live H1N1 virus or immunized with inactivated H3N2 virus (as detailed in the methods).

Analysis of Anti-Influenza Virus Neuraminidase Antibodies in Children, Adults, and the Elderly by ELISA and Enzyme Inhibition: Evidence for Original Antigenic Sin.

While characterizing the specificity of plasmablasts induced by influenza virus infection, we noted a surprisingly high proportion of NA-reactive cells. The specificity of plasmablasts was evaluated by ELISPOT or mAb characterization from a total of 16 confirmed influenza-infected patients. Notably, as previously reported (), plasmablasts are highly representative of the memory compartment, accounting for almost half of the memory cells activated in future responses to similar influenza strains. These patients included 11 subjects infected with the H1N1 pandemic strain (five from 2009 and six from 2016) and five infected with H3N2 virus strains (three from 2014 and two from 2017) ( Table S1 ). First, we analyzed large numbers of activated plasmablasts in six influenza-virus-infected patients (four infected with H1N1 in 2016 and two infected with H3N2 in 2017). Scoring of thousands of activated plasmablasts by ELISPOT assay detected an average of 24% that were reactive to NA and 38% to HA ( Figure 1 A). Surprisingly, plasmablasts from H3N2-infected patients predominantly targeted NA. To more rigorously assess the frequency of NA-reactive B cells activated during infection, we characterized mAbs obtained from patients. For this, the isolated variable region genes from single plasmablasts activated by infection were used to express 128 influenza-binding mAb proteins from 12 of the patients using methods previously described (). The NA-reactive mAbs were more often encoded by VH3 family genes but used variable genes that were otherwise similar to HA antibodies ( Figure S1 ). Consistent with the ELISPOT assays, 22.6% (29/128)—and on average, 24% by year and strain—of plasmablast mAbs activated by influenza virus infection were reactive to recombinant NA (rNA) ( Figures 1 B–1D). These mAbs included 15/88 that were N1 reactive and 14/40 that were N2 reactive (mAbs by subject indicated in Table S1 ). Notably, as with the ELISPOT analysis, H3N2 virus infections consistently induced a higher proportion of NA-reactive B cells compared to HA-reactive B cells ( Figures 1 A and 1D [blue dots]). By comparison, activation of NA-reactive B cells was quite rare 7 days after vaccination, accounting for only 1.2% (2/258) of vaccine-induced plasmablasts relative to 87% that targeted HA and 12% binding other influenza antigens ( Figure 1 E). This observation was consistent for several influenza virus vaccine compositions, including 1.5% (2/133) of NA-reactive cells after immunization with a subunit vaccine (from 2006–2008 and in 2010), 1.1% (1/89) after the 2009 H1N1 monovalent vaccine, and none (0/36) induced by split vaccines (2008–2010) ( Figure 1 E). We conclude from this analysis that a quarter of plasmablasts induced by natural influenza virus infection target NA—a percentage that nearly equals that of HA-specific plasmablasts—compared to only 1%–2% from influenza vaccination.

(D) Total heavy chain mutation number of NA and HA-reactive mAbs based on analysis using the NCBI IgBlast tool ( https://www.ncbi.nlm.nih.gov/igblast/ ), data are represented as mean ± SD.

PAPie charts show the percentages of mAbs that bind a given antigen (HA, NA, or other) of the total, indicated in the center circle. Graphed on the right are the percentages of HA- and NA-reactive antibodies per individual. Each dot represents one individual (n = 11). Red indicates patients with no NA B cells detected on first exposure to the pandemic H1N1 strain in 2009 (E). The frequency of NA-reactive mAbs induced by vaccination (the vaccinated cohorts are detailed in the STAR Methods ) is shown. As in (D), pie charts show the percentages of mAbs that bind a given antigen (HA, NA, or other) in individuals vaccinated with influenza virus subunit vaccine (seasons 2006–2008 and 2010–2011), influenza virus split vaccine (2008–2010), or monovalent pandemic H1N1 vaccine (2009–2010). For the panels (A) and (D), the blue dots indicate patients infected with an H3N2 virus.

(B and C) Binding of NA-reactive mAbs to rNA proteins by ELISA. Represented are ELISA binding curves. The assays were performed in duplicate at least three times for each antibody. Binding to A/California/7/2009 (H1N1) rN1 protein (B) or A/Texas/50/2012 (H3N2) rN2 protein (C) is shown. Numbers of antibodies per subject are indicated in Table S1

(A) The proportions of HA- and NA-reactive antibody-secreting cells (ASCs) out of the total virus-reactive cells were determined by ELISPOT assay. Individuals infected with an H1N1 influenza virus (in black) were compared to individuals infected with an H3N2 influenza virus (in blue). Each dot represents a subject (n = 6). Bars indicate mean values.

Discussion

Angeletti and Yewdell, 2017 Angeletti D.

Yewdell J.W. Is It Possible to Develop a “Universal” Influenza Virus Vaccine? Outflanking Antibody Immunodominance on the Road to Universal Influenza Vaccination. Sandbulte et al., 2011 Sandbulte M.R.

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Cox M.M. Influenza viral neuraminidase: the forgotten antigen. The results presented herein demonstrate that with the right immunogen, NA is capable of inducing a potent, broadly cross-reactive, and protective humoral immune response. In fact, the NA-reactive mAbs were more broadly reactive; the potency of protection and neutralization rivaled that of HA-reactive mAbs. For H3N2 infections, there were more NA-reactive than HA-reactive B cells activated. This response is consistent with a recent report that by molar composition, NA is the most immunogenic influenza protein (). The relative conservation of NA epitopes () also drives a back-boost effect against NAs of historical isolates (). In contrast, after vaccination, we find that there is only a 1:87 ratio of NA to HA plasmablasts activated. The NA-reactive mAbs induced by infection reported here have substantially reduced binding to the inactivated vaccines tested, suggesting that the vaccines do not efficiently present important conserved and protective NA epitopes. This observation can be explained by several factors. First, the inactivated influenza vaccines are optimized only for the HA antigen, as the FDA requires that licensed influenza virus vaccines contain at least 15 μg of each HA subtype (). Second, antigenic competition between HA and NA may affect the NA humoral immune response (). However, this mechanism did not appear to preclude the response to NA during infection or to whole virions in mice as reported herein. Third, although influenza vaccine compositions contain varying amounts of NA (), it is unclear if the NA antigen retains its natural tetramer structure, which is important to maintain immunogenicity (). Conversely, during an influenza virus infection, NA replicates along with the virus so that B cells can respond to intact NA on whole virions and infected cells.

Sandbulte et al., 2011 Sandbulte M.R.

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Eichelberger M.C. Discordant antigenic drift of neuraminidase and hemagglutinin in H1N1 and H3N2 influenza viruses. The rate of NA antigenic drift is slower than that of HA, which helps to explain the high frequency of broadly cross-reactive antibodies (). The NA-reactive mAbs isolated herein typically cross-bind to heterologous NA proteins from most human influenza A virus strains, and a subset also bound to avian H5N1 and H7N9 and had some reactivity to H7N3, H4N4, and H3N8 strains. This breadth was evident for the antibodies that were used to map the epitopes. On N1, two of the primary amino acids targeted (N309 and N273) are 99.7% conserved (present in 6835/6855 strains) in H1N1 virus from 1918 to 2017 H1N1 strain in the United States ( https://www.fludb.org/brc/home.spg?decorator=influenza ). Also, N1-reactive mAbs that selected changes at two conserved epitopes (G249 and N273) shared between the human and avian strains were able to mediate prophylactic protection against H5N1 challenge in vivo in mice. Five of the N2-reactive mAbs bind to the conserved enzymatic active site on the head of the NA. The broad reactivity and conservation of the targeted epitopes suggest that NA may be an essential component of universal influenza virus vaccine compositions.

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et al. Both Neutralizing and Non-Neutralizing Human H7N9 Influenza Vaccine-Induced Monoclonal Antibodies Confer Protection. Wohlbold et al., 2017 Wohlbold T.J.

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et al. Broadly protective murine monoclonal antibodies against influenza B virus target highly conserved neuraminidase epitopes. Both NA-inhibiting and non-inhibiting mAbs to N2 protected from influenza virus challenge in vivo. While inhibition of viral release is the typical mechanism of protection, anti-NA antibodies may also mediate protection by other means. For example, several antibodies exhibited high NA-inhibition activity but had low or no microneutralization activity in vitro. However, most that protected in vivo had some degree of NA-inhibiting activity. Thus, the NA-reactive mAbs may alter the functional balance of opposing actions between HA and NA to disrupt efficient viral replication (). For non-NI mAbs, there are several mechanisms that likely account for protection. Fc-FcR interactions have been shown to be required for full protection by some NA-reactive mAbs ().

In conclusion, NA-reactive antibodies can be readily or even dominantly induced, protecting at levels comparable to HA-reactive antibodies. While particular antibodies to HA have been isolated with immense breadth of activity, those to NA appear to have greater breadth than antibodies to HA on a per antibody basis and based on the sampling herein. The data presented suggest that inclusion of an improved NA component to future influenza vaccine compositions would reduce the severity of infections. With a robust response to NA, the degree of protection conferred might protect from any influenza infection occurring at all and possibly provide broad-ranging protection against potential pandemic strains that express N1 or N2 NAs.