describe monoclonal antibody (MAb) 5J8, which was isolated from a healthy middle-aged woman by hybridoma technology. We found that MAb 5J8 inhibited a ...
JOURNAL OF VIROLOGY, Oct. 2011, p. 10905–10908 0022-538X/11/$12.00 doi:10.1128/JVI.00700-11 Copyright © 2011, American Society for Microbiology. All Rights Reserved.
Vol. 85, No. 20
A Broadly Neutralizing Human Monoclonal Antibody That Recognizes a Conserved, Novel Epitope on the Globular Head of the Influenza H1N1 Virus Hemagglutinin䌤 Jens C. Krause,1† Tshidi Tsibane,3† Terrence M. Tumpey,4 Chelsey J. Huffman,1 Christopher F. Basler,3 and James E. Crowe, Jr.1,2* Departments of Pediatrics1 and of Microbiology and Immunology,2 Vanderbilt University Medical Center, Nashville, Tennessee 37232; Department of Microbiology, Mount Sinai School of Medicine, New York City, New York 100293; and Influenza Division, Centers for Disease Control and Prevention, Atlanta, Georgia 303334 Received 6 April 2011/Accepted 8 August 2011
The conserved influenza virus hemagglutinin (HA) stem domain elicits cross-reactive antibodies, but epitopes in the globular head typically elicit strain-specific responses because of the hypervariability of this region. We isolated human monoclonal antibody 5J8, which neutralized a broad spectrum of 20th century H1N1 viruses and the 2009 pandemic H1N1 virus. Fine mapping of the interaction unexpectedly revealed a novel epitope between the receptor-binding pocket and the Ca2 antigenic site on HA. This antibody exposes a new mechanism underlying broad immunity against H1N1 influenza viruses and identifies a conserved epitope that might be incorporated into engineered H1 virus vaccines. H1N1 influenza viruses circulated in humans from the 1918 pandemic until 1957 and again from 1977 to the present (14). A novel H1N1 virus exhibiting some antigenic similarity to the 1918 virus was reintroduced into humans from animal reservoirs, causing a pandemic in 2009 (1, 7). Elderly people had preexisting antibodies against the 2009 virus (2, 9, 10). We and others have shown that the conservation of 1918-like sequences in the Sa antigenic site in the globular head domain of 2009 virus hemagglutinin (HA) is a likely structural correlate for this cross-reactivity (11, 13, 19, 22). Sequence conservation, including the absence of glycosylation in early-20th-century strains and again in 2009 H1N1 virus HA, make this area on the HA protein surface an important target for human humoral immunity (19, 22). A second class of recently identified antibodies, many of which are encoded by the VH1-69 germ line antibody variable-gene segment, are directed to the conserved H1N1 HA stem region (6, 8, 17, 18, 21). There may be other cross-reactive epitopes on H1N1 viruses (21). Here, we describe monoclonal antibody (MAb) 5J8, which was isolated from a healthy middle-aged woman by hybridoma technology. We found that MAb 5J8 inhibited a broad spectrum of 20thcentury H1N1 strains and the pandemic 2009 H1N1 virus. The epitope of this antibody revealed a novel conserved H1 epitope adjacent to the receptor binding site domain (RBD) on the HA globular head. Hybridoma generation and recombinant antibody expression. Peripheral blood mononuclear cells were isolated from a healthy 47-year-old human subject and Epstein-Barr virus * Corresponding author. Mailing address: Vanderbilt Vaccine Center, Vanderbilt University Medical Center, T-2220 Medical Center North, 1161 21st Avenue South, Nashville, TN 37232-2905. Phone: (615) 343-8064. Fax: (615) 343-4456. E-mail: james.crowe@vanderbilt .edu. † J.C.K. and T.T. contributed equally to the article. 䌤 Published ahead of print on 17 August 2011.
(EBV) transformed in 384-well plates (Nunc) in the presence of 2.5 g/ml of CpG oligodeoxynucleotides (ODNs) 2006 (Invitrogen), 10 M Chk2 inhibitor II (Sigma C3742), and 1 g/ml of cyclosporine A (Sigma) essentially as previously described (24, 25). The supernatant was screened by enzyme-linked immunosorbent assay (ELISA) against a panel of recombinant soluble HA proteins. B cells were fused with HMMA2.5 myeloma cells, cultured in selection medium, and cloned by limiting dilution. The antibody genes were cloned molecularly from mRNA isolated from the cloned hybridoma cell line using previously described primer sets (15) into a pGEM-T Easy vector (Promega) and eventually into pEE12.4/pEE6.4 mammalian expression vectors (Lonza), from which they were expressed (22). They were purified by fast protein liquid chromatography (FPLC) on a protein G column (for IgG1) or via CaptureSelect resin (for Fab; BAC B.V.). Analysis with the international ImMunoGeneTics information system (IMGT) (12) identified MAb 5J8 as an antibody encoded by the IGHV4-b*01, J4*02, D3-3*02, IGLV3-21*02 or *03, J2*01, or J3*01 variable gene segment. The recombinant antibody was used for all of the following studies. VLP expression and HAI assays. Expression plasmids encoding HA protein molecules were coexpressed with neuraminidase to produce virus-like particles (VLPs) in 293T cells (5, 25). Hemagglutination inhibition (HAI) assays were performed as described previously (20) using VLPs (for 1918 influenza) or live virus (for all other strains). MAb 5J8 inhibited all tested H1N1 influenza strains from 1918 to 1977 and the pandemic 2009 virus but not the seasonal H1N1 strains from 1999 or 2007 (Tables 1, 2, and 3). HAI activity was the most potent against 1918 VLPs and the 1930 virus at 40 ng/ml. Microneutralization assay. Different dilutions of antibody were incubated with 5 log10 50% tissue culture infective doses (TCID50) of each virus for 1 h. The mixture was used to infect MDCK cells in triplicate for an hour at 37°. The plate was
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TABLE 1. Hemagglutination inhibition (HAI) activity and neutralization titers of 5J8 in representative 20th century influenza virus H1N1 strainsa HAI activity Neut titer (g/ml) (g/ml)
Strain
A/South Carolina/1/1918 A/swine/Iowa/15/1930 A/Weiss/1943 A/L3/1947 A/New Jersey/11/1976 A/USSR/92/1977 A/New Caledonia/20/1999 A/Brisbane/59/2007 A/California/04/2009
0.04 0.04 5 0.08 0.32 0.63 ⬎20 ⬎20 0.09
NT ⬍0.025 10 0.1 NT 0.2 ⬎20 NT 0.0017
TABLE 3. Escape mutations in HA residues of influenza virus H1N1 strains Mutation at indicated positiona
HA residue at indicated positionb
Strain
133A 137 199 222
A/swine/Iowa/15/1930 A/L3/1947 A/California/04/2009
K R R R R R Del Del K
A A A A A A A A A
D D N N D N N N D
K K K K K K K K K
a All human H1N1 HA protein sequences were acquired from the Influenza Research Database for the years 1918 through 2008. The search was performed on 26 March 2011. Sequences that were duplicates or were from viruses generated through genetic manipulation were removed prior to analysis. NT, not tested; Neut, neutralization; Del, deletion. b Based on H3 numbering.
harvested 3 days later and read in an HA assay. The endpoint was the lowest concentration that gave no HA activity. The microneutralization assay generally agreed closely with the HA assay (Table 1). Biosensor studies to determine affinity. The binding affinities of 5J8 Fab to recombinant trimeric His-tagged HA proteins were measured using anti-penta-His tips on an Octet Red instrument (Forte´Bio); 1918 HA variants were created with a QuikChange II XL mutagenesis kit (Agilent). The extracellular domain of full-length 1918 HA cDNA was cloned into a pcDNA3.1(⫹) construct that contained an SGR linker, a thrombin recognition site, a fibritin trimerization domain, and a 6⫻His tag. The protein was expressed in 293F cells and ¨ KTA FPLC instrument purified from the supernatant on an A using Ni2⫹ columns (GE). The binding affinity of 5J8 Fab to 2009 H1 HA protein was 2.6 ⫻ 10⫺8 M (Table 4). Animal studies. We tested 5J8 for therapeutic efficacy in a lethal animal model of 1918 virus infection (25). Female BALB/c mice (8 weeks old, weighing approximately 20 g) were inoculated intranasally with five times the 50% lethal dose (LD50) in a 50-l volume of the virulent reconstituted virus. At 24 h after inoculation, mice were administered 200, 20, or 2 g of 5J8 or an equal amount of human IgG (Sigma), each by the intraperitoneal route, in groups of 10 mice. Mice were ob-
TABLE 2. Amino acid point mutations in naturally occurring field strains of influenza virus H1N1a Amino No. of acid deletions positionb
133A 137 199 222 a
1,665 1 1 1
No. of mutations of indicated amino acid A
D E H I
K
N
P R S T V X
137 1,861 19
1 1 2
1 1,811 1,813 1
45
31 2 2 11 6 1 4
All human H1N1 HA protein sequences were acquired from the Influenza Research Database for the years 1918 through 2008. The search was performed on 26 March 2011. Sequences that were duplicates or were from viruses generated through genetic manipulation were removed prior to analysis. b Based on H3 numbering.
133A
a
137
199
222
A3T R3I K3Q
K3Q D3H
Based on H3 numbering.
served for weight loss for 14 days (Fig. 1B). Subsets of four animals treated with the MAbs were euthanized on day four after infection, and whole lungs were homogenized in 1 ml of sterile phosphate-buffered saline (PBS). Virus titers in lung tissue homogenates were determined by plaque titration in MDCK cell monolayer cultures. MAb 5J8 protected all animals at the high and medium dose from lethal challenge (Fig. 1A) and reduced lung virus titers compared to the IgG control by 2.6 log10 PFU/ml at the 200-g-dose level, by 2.0 log10 PFU/ml at the 20-g-dose level, and by 0.7 log10 PFU/ml at the 2-g-dose level (Table 5). Isolation and characterization of antibody escape mutant viruses. We selected and sequenced the HA gene of the new antibody escape mutant viruses (4, 23). Following MAb 5J8 selection, HA mutations were identified in positions 133A, 137, 199, and 222 (based on H3 numbering [16]) in some of these strains (Table 3). We then introduced these naturally occurring mutations into the soluble 1918 HA protein for in vitro binding studies to validate the effect of the putative escape mutations. 133A K3I, A137T, and K222Q each eliminated binding of MAb 5J8 to the mutant protein (Table 4). However, binding of MAb 5J8 to the D199H mutant (which we detected only in concert with a 133A K3Q mutant) was retained (Table 4). We were technically unable to create the 133A K3Q mutant construct, but this residue would likely mediate escape, based on the effect of the validated 133A K3I escape mutant. Of note, all of these escape mutations are outside conventionally defined antigenic sites (3, 4) and are located between the receptor-binding pocket and the Ca2 antigenic site (Fig. 2). Review of H1N1 sequences. We then queried the Influenza Research Database (IRD; www.fludb.org) for all naturally oc-
TABLE 4. Binding of Fab 5J8 to soluble influenza HA proteins by biolayer interferometrya HA protein
KD (M)
kon (1/Ms)
koff (1/s)
SC/18 wt del133A 133A K3I 133A K3T A137T D199H K222Q
1.9 ⫻ 10⫺7 ND ND ND ND 1.7 ⫻ 10⫺7 ND
2.0 ⫻ 105 ND ND ND ND 1.9 ⫻ 105 ND
3.6 ⫻ 10⫺2 ND ND ND ND 3.1 ⫻ 10⫺2 ND
Ca/04 wt
2.6 ⫻ 10⫺8
2.5 ⫻ 105
6.5 ⫻ 10⫺3
a SC/18, A/South Carolina/1/1918; Ca/04, A/California/04/2009; KD, equilibrium dissociation constant; kon, association rate; koff, dissociation rate; ND, not detectable.
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FIG. 1. Therapeutic efficacy of MAb 5J8 against disease caused by the 1918 influenza A(H1N1) virus in mice. Mice were inoculated on day 0 and treated on day 1 with the indicated antibody and dose. In each group, six mice were monitored every other day for survival (A) and weight (B). At the high-dose level and the intermediate-dose level, the differences in survival distribution between the MAb 5J8 and the human IgG control groups were significant by log-rank test (P ⫽ 0.001 for the high-dose level, P ⬍ 0.001 for the intermediate-dose level, P ⫽ 0.238 for the low-dose level).
curring, nonredundant human H1N1 viruses between 1918 and 2008 to identify the frequency of sequence variation in the residues mediating escape from MAb 5J8. Of note, residues 137 (alanine) and 222 (lysine) were each found in greater than 98% of all the sequences (Table 2). There was greater sequence variability at the position of the critical residue 133A: the lysine from the 1918 virus was replaced by arginine (a conservative mutation to a similarly positively charged residue)
TABLE 5. Therapeutic efficacy of MAb 5J8 against virus replication in mice inoculated with 1918 influenza A virus Dose (g/mouse)
Mean lung virus titer (log10 PFU/ml ⫾ SD)a
MAb 5J8
200 20 2
4.1 ⫾ 0.2 4.9 ⫾ 0.7 5.9 ⫾ 0.1
Human IgG control
200 20 2
6.7 ⫾ 0.1 6.9 ⫾ 0.0 6.6 ⫾ 0.4
Antibody
a At the ␣ ⫽ 0.025 level controlling the overall type I error at 7.5%, the lung homogenates of the MAb 5J8 groups had lower virus titers than the human IgG control groups for all dose levels, as determined by the Wilcoxon rank sum test (P ⫽ 0.01429 for the high-dose groups, P ⫽ 0.01429 for the intermediate-dose groups, P ⫽ 0.0147 for the low-dose groups). SD, standard deviation.
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FIG. 2. Space-filling model of 1918 influenza virus HA (PDB ID, 1RD8) (16); view onto the membrane-distal globular head. The three HA monomer subunits are colored white, gray, or black. The conventionally defined antigenic sites on HA are colored blue (site Sa), yellow (site Sb), or green (site Ca2). MAb 5J8 selected for mutations in residues 133A, 137, 199, or 222 in certain H1N1 viruses (magenta). Residues 133A, 137, and 222 are situated between the receptor-binding pocket and the Ca2 antigenic site but are not themselves part of a conventionally defined antigenic site.
as early as in the 1930 virus without a subsequent change in HAI activity. A lysine was seen again in the 2009 H1N1 virus, but residue 133A was deleted in previous seasonal strains. When built back into 1918 HA, this deletion by itself did lead to loss of binding (Table 4). In fact, over 80% of all strains in the data set showed this deletion, but this high percentage is biased because most sequences in the IRD are recent. The 133A deletion was rarely observed prior to 1997 but has been dominant since then. Thus, MAb 5J8 recognizes a novel, conserved epitope on the globular head of H1N1 HA that is characterized by residues 133A, 137, and 222 in close proximity to the RBD, which illuminates the molecular basis for the breadth of neutralization of MAb 5J8. There likely are very strong structural constraints on this epitope. It is certainly possible that MAb 5J8 makes contacts deeper within this pocket that H1N1 cannot change without loss of replicative capacity and that would not be revealed by escape mutations. In conclusion, MAb 5J8 helps paint a more detailed picture of why older human subjects possessed 2009 H1N1 cross-reactive HA antibodies prior to exposure to the 2009 H1N1 virus (2, 9, 10), since epitopes other than the Sa site (11, 13, 19, 22) or stem epitopes (6, 8, 17, 18, 21) contributed to this cross-protective effect. The relative importance of these epitopes for the antiviral humoral response may vary from person to person. The fact that it took decades for H1N1 viruses to escape MAb 5J8-like antibodies may reflect the fact that such antibodies are relatively rare and thus pose little evolutionary pressure on H1N1. We propose that presenting the RBD epitope in a more exposed way without surrounding hypervariable loops might make it more immunogenic and thus
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might contribute to a universal influenza virus vaccine design strategy. Nucleotide sequence accession numbers. Antibody nucleotide sequences have been deposited in GenBank under accession numbers JF791168 and JF791169. We thank the anonymous blood donor; Jose A. Archuleta, Jr., and Cheryl Kinnard of the Vanderbilt Clinical Trials Center; M. Posner and L. Cavacini for the HMMA2.5 cell line; Patricia B. Smith for technical assistance; Kimberly S. Crimin for statistical help; and Cyrille Dreyfus, Damian C. Ekiert, Ian A. Wilson, and Scott A. Smith for helpful discussions. J.E.C. is a Burroughs Wellcome Fund Clinical Scientist in Translational Research. This work was supported by NIH grants P01 AI058113 and R21 AI085306, by NIH contract HHSN272200900047C, and by DOD grant HDTRA1-08-10-BRCWMD-BAA. The findings and conclusions in this report are those of the authors and do not necessarily reflect the views of the funding agency. Vanderbilt University submitted a patent covering the diagnostic and therapeutic use of the antibody described in this paper. J.E.C. is the founder of Corbeau Biotech LLC.
5.
6.
7. 8. 9. 10. 11.
12. 13.
14.
ADDENDUM IN PROOF 15.
After this manuscript was accepted for publication, data on a similar H1N1 antibody, MAb CH65, were published (J. R. Whittle et al., Proc. Natl. Acad. Sci. U.S.A. 108:14216–14221, 2011). MAb 5J8 did not inhibit HA with deletions in position 133A, MAb CH65 needs a deletion in position 133A of HA for inhibition of virus. Strikingly, these two antibodies complement each other in their breadth, and a cocktail of CH65 and 5J8 might inhibit most human H1N1 strains. Furthermore, the fact that CH65 makes contacts deeply within the receptorbinding pocket would suggest that 5J8 has a similar mode of binding.
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