NEUTRALISING ANTIBODY AGAINST DENGUE FOR USE IN A METHOD OF PREVENTION AND/OR TREATMENT OF ZIKA INFECTION
20210355167 · 2021-11-18
Inventors
- Felix REY (PARIS, FR)
- Giovanna Barba Spaeth (Paris, FR)
- Marie-Christine Vaney (Paris, FR)
- Alexander Rouvinski (Jerusalem, IL)
- Gavin SCREATON (London, GB)
- Juthathip Mongkolsapaya (London, GB)
Cpc classification
C12N2770/24122
CHEMISTRY; METALLURGY
C12N2770/24134
CHEMISTRY; METALLURGY
C07K16/1081
CHEMISTRY; METALLURGY
C07K14/1825
CHEMISTRY; METALLURGY
Y02A50/30
GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
C07K2317/33
CHEMISTRY; METALLURGY
C07K2317/76
CHEMISTRY; METALLURGY
C07K2317/34
CHEMISTRY; METALLURGY
International classification
Abstract
A flavivirus Envelope Dimer Epitope (EDE) and isolated neutralizing antibody or antigen binding fragment thereof directed against the EDE for use in vaccinating an individual against one or more flaviviruses wherein the EDE is a stabilized recombinant flavivirus are provided. The dimer is: covalently stabilized with at least one disulphide inter-chain bond or one sulfhydryl-reactive crosslinker between the two sE monomers, and/or by being formed as a single polypeptide chain, and/or by linking the two sE monomers through modified sugar, and/or non-covalently stabilized by substituting at least one amino acid residue in the amino acid sequence of at least one sE monomer with at least one bulky side chain amino acid, at the dimer interface or in domain 1 (D1)/domain 3 (D3) linker of each monomer. The dimer is a homodimer or heterodimer of native and/or mutant envelope polypeptides, from DENV-1, DENV-2, DENV-3, DENV-4, Zika and/or other flavivirus.
Claims
1-44. (canceled)
45. An isolated neutralizing antibody or antigen binding fragment thereof directed against a flavivirus envelope dimer epitope (EDE) wherein the antibody or fragment comprises a CDR comprising an amino acid sequence selected from the group consisting of SEQ ID Nos: 15 to 26 and sequences with no more than 30% modificationfrom any one said SEQ ID No. 15 to 26 wherein the one or more flaviviruses is selected from zika virus; zika virus and dengue virus; zika virus and other flaviviruses; flaviviruses other than dengue, wherein: (a) said antibody or fragment thereof binds the five polypeptide segments of the dengue virus glycoprotein E ectodomain (sE) consisting of the residues 67-74, residues 97-106, residues 307-314, residues 148-159 and residues 243-251, or corresponding residues of the flavivirus or Zika virus glycoprotein E ectodomain, or consisting of Zika PF13 residues 67-77, residues 97-106, residues 313-315, residues 243-253 residue K373 or corresponding residues of the flavivirus glycoprotein E ectodomain, (b) binding is unaffected by presence or absence of dengue N153 (Zika N154) glycan or corresponding residue.
46. (canceled)
47 The antibody or fragment thereof of claim 45 wherein it recognizes exclusively virion-dependent (including sub-viral particle or virus-like particle) epitope(s) of a flavivirus, optionally Zika or dengue virus.
48. The fragment of claim 45 wherein it is a Fab fragment.
49. The antibody or fragment thereof of claim 45, comprising an amino acid sequence selected from the group consisting of SEQ ID Nos: 15 to 26 and sequences with no more than 20, 15, or 10% modification from any one said SEQ ID No. 15 to 26.
50. The antibody or fragment thereof of claim 45 wherein the EDE comprises a region centred in a valley lined by the b strand on the domain II side, and the “150 loop” on the domain I side (across from the diener interface), wherein the 150 loop spans residues 148-159, connecting b-strands E0 and F0 of domain I, and carries the N153 glycan, which covers the fusion loop of the partner subunit in the dimer, optionally wherein the region comprises the b strand (residues 67-74 which bear the N67 glycan), the fusion loop and residues immediately upstream (residues 97-106) and the ij loop (residues 246-249) of the reference subunit,erein the reference subunit is the subunit which contributes the fusion loop, optionally wherein the EDE further comprises the 150 loop and the N153 glycan chain of the second subunit, optionally wherein one or both regions is in a substantiallysimilar spatial configuration as the native region; or wherein the EDE comprises the Zika PF13 beta strand b of domain II, bed beta-sheet edge, fusion loop main chain, fusion loop R99 side chain, Q77 side chain, disulphide bond between C74 and C105; beta strand E, K373, charged residues in domain I, kl loop of domain II, or regions corresponding thereto.
51. The antibody or fragment thereof of claim 45, wherein the antibody or fragment thereof neutralises one or more serotypes of Dengue virus and/or Zika virus.
52. The antibody or fragment thereof of claim 45, wherein the antibody or fragment thereof neutralises all serotypes of Dengue virus and Zika virus.
53. The antibody or fragment thereof of claim 45 wherein the antibody or fragmen f neutralises one or more serotypes of Dengue virus and/or Zika virus to 80, 90, 98 or 100% at a concentration of 0.5-0.01 μg/ml.
54. The antibody or fragment thereof of claim 45 wherein the antibody or fragment f neutralises all serotypes of Dengue virus and Zika virus to 80, 90, 98 or 100% at a concentration of 0.5-0.01 ug/ml
55. The antibody or fragment thereof of claim 45 wherein the fragment is a Fv fragment; a Fab-like fragment; or a domain antibody; or wherein the antibody is a monoclonal antibody or a recombinant antibody.
56. The antibody or fragment thereof of claim 45 wherein the antibody is a polyclonal antibody or antigen binding portion thereof.
57. The antibody or fragment thereof of claim 45 wherein the antibody or fragment thereof is part of a composition comprising a mixture or antibodies, optionally: a) a mixture of monoclonal antibodies or antigen binding portion thereof, or b) a mixture of polyclonal antibodies or antigen binding portion thereof, or c) a mixture or monoclonal and polyclonal antibodies or antigen binding portion thereof.
58-64. (canceled)
65. The antibody or fragment thereof of claim 51, wherein the antibody or fragment thereof neutralises one or more serotypes of Dengue virus and/or Zika virus to 90% or 98% or 100%.
66. The antibody or fragment thereof of claim 65, wherein the antibody or fragment thereof neutralises one or more serotypes of Dengue virus and/or Zika virus to 98% or 100%.
67. The antibody or fragment thereof of claim 52, wherein the antibody or fragment thereof neutralises all serotypes of Dengue virus and Zika virus to 90%.
68. The antibody or fragment thereof of claim 52, wherein the antibody or fragment thereof neutralises all serotypes of Dengue virus and Zika virus, 98% or 100%.
69. The antibody or fragment thereof of claim 68 wh erein the antibody or fragment thereof neutralises all serotypes of Dengue virus and Zika virus to 100% at the same concentration of antibody or fragment.
70. The antibody or fragment thereof of claim 55 wherein the Fab-like fragment is a Fab′ fragment or a F(ab)2 fragment.
Description
FIGURE LEGENDS
[0499] Example 1:
[0500]
[0501] (a) Binding titration curves of 6 representative DENV sera against ZIKV strains PF13 and HD78788 and DENV measured by capture ELISA (6-month convalescent plasma using the DENV serotype corresponding to their previous acute infection). (b) End point titers of DENV plasma against ZIKV (strain PF13 and HD78788) and DENV determined by capture ELISA (n=18). Small horizontal lines indicate the median values.
[0502]
[0503] (a) Neutralization of ZIKV determined on Vero cells for 6 representative DENV plasma with 2 ZIKV strains PF13 and HD78788 and DENV (6-month convalescent plasma using the DENV serotype corresponding to their recent infection). Pooled DENV negative serum (PND) was used as negative control. (b) NT50 values for DENV plasma on ZIKV and DENV infection (n=18).
[0504]
[0505] (a) Six representative ADE curves of U937 cells infected with ZIKV strains PF13 and HD78788 and DENV (6-month convalescent plasma using the DENV serotype corresponding to their recent infection) in the presence of serially diluted DENV plasma. Pooled negative serum (PND) was used as negative control. (b) Peak fold enhancement of DENV plasma on ZIKV and DENV (n=18).
[0506]
[0507] (a) Binding of ZIKV strains PF13 and HD78788 and DENV serotype 1 by 33, 17, 45, 37 of anti-EDE1, EDE2, FLE and non-FLE mAbs at lOug/ml, this is representative of three separate experiments. The arrows indicated mAbs used in
[0508]
[0509] Infection enhancement curves of 9 anti-DENV mAbs (3 each for anti-EDE1, EDE2, and FLE mAbs) on ZIKV strains PF13 and HD78788. U937 cells were used as target cells. The data are shown as mean±2SE from 2 independent experiments.
[0510]
[0511] The inhibition curves of 9 anti-DENV mAbs 3 each for anti-EDE1, EDE2, and FLE mAbs) on ZIKV strains PF13 and HD78788. U937 cells were infected with ZIKV in the presence of 1:1000 pooled convalescent dengue serum (the dilution giving peak enhancement) together with serially diluted anti-DENV mAbs. Anti-flu mAb, 28C, was used as a negative control. The data are representative of 3 independent experiments.
[0512]
[0513] Example 2 Figure Legends:
[0514]
[0515]
[0516]
[0517]
[0518] ED
[0519] ED
[0520] ED
[0521] ED
[0522] ED
[0523] ED
[0524] ED
[0525] Example 3 Figure Legends:
[0526]
[0527]
[0528]
[0529]
[0530]
[0531]
[0532]
[0533] Example 4
[0534]
[0535] In vivo efficacy of C10 in the AG129 mouse model. AG129 mice (female, 8-10 weeks of age; n=3) were treated with 50 or 200 μg purified C10 or 2-8C intraperitoneally as described in the Methods section. Mice were infected intraperitoneally with 200 μL of a 1.2×10.sup.2 FFU/mouse with ZIKV PE243; 24 h post antibody treatment. (A) Percentage original body weight curves of ZIKV-infected mice treated with C10 antibody (red and blue symbols) or 2-8C isotype control (green and purple symbols) were plotted compared to PBS treated uninfected mice (black symbols). Data represent results from one experiment and are plotted as average +/− weight measurements from 3 mice per infected group. (B) Viral titres were determined from plasma samples isolated from individual mice at day 2 and day 4 post infection. Viral titres calculated as foci forming units per ml plasma have been represented as mean +/− SEM of plasma viral titres in individual mice.
EXAMPLE 1
Dengue Serocrossreactivity Drives Antibody Dependent Enhancement of Zika virus Infection
[0536] Zika virus was discovered in 1947 and was thought to lead to relatively mild disease. The recent explosive outbreak of Zika in South America has led to widespread concern with reports of neurological sequelae ranging from Guillain Bane syndrome to microcephaly. Zika has followed in the path of dengue a flavivirus closely related to Zika. Here we investigate the serological crossreaction between the two viruses. Dengue immune plasma substantially crossreacts with Zika and can drive antibody dependent enhancement of Zika infection. Using a panel of human anti-dengue monoclonal antibodies we show that most antibodies reacting to dengue envelope protein also react to Zika and antibodies to linear epitopes including the immunodominant fusion loop epitope whilst binding to Zika cannot neutralize the virus but promote ADE. These data indicate that dengue immunity may drive higher Zika replication and have implications for disease pathogenesis and future Zika and dengue vaccine programmes.
[0537] Zika virus (ZIKV) is an arbovirus belonging to the family flaviviridae and is transmitted to man by Aedes mosquitos'. ZIKV was first isolated from a sentinel rhesus monkey in the Zika forest of Uganda in 1947 and has subsequently been found in mosquitos and humans.sup.2,3. Until recently ZIKV has not been viewed as a particularly important pathogen as the majority of infections are asymptomatic.sup.4. Symptomatic cases of ZIKV resemble mild cases of dengue fever with fever, myalgia, arthralgia, headache, conjunctivitis and rash.sup.1, 7, 12, 13, 14.
[0538] Until recently cases were sporadic largely in Africa and South East Asia and epidemic activity had not been observed.sup.1, 8, 9, 10, 11. A large outbreak of ZIKV occurred on Yap island in the Western Pacific in 2007 and spread through Oceania and reached Brazil in 2015 where it rapidly spread to involve other South American countries.sup.1, 7, 12, 13, 14.
[0539] It is now apparent that ZIKV infection can case significant neurological complications; increased cases of Guillain Bane syndrome were first reported following the outbreak in French Polynesia in 2013 .sup.15. Dramatic increases in the incidence of microcephaly originating in North Eastern Brazil were reported in late 2015 coincident with a large increase in ZIKV infection 16, 17. These increases in Guillain Barre syndrome and microcephaly led the World Health Organization to declare ZIKV a public health emergency in February 2016 .sup.18.
[0540] ZIKV can be carried by a variety of Aedes mosquitos but the principal species responsible for the current outbreaks is thought to be Aedes aegypti.sup.1, 5. In parts of Brazil Aedes aegypti is also spreading DENV and chikungunya viruses concurrently with ZIKV.sup.19, 20, 21, 22, 23, 24. In the last 20 years DENV has spread through areas of South America and the seroprevalence of DENV in some areas affected by ZIKV exceeds 90%.sup.25, 26, 27.
[0541] DENV exists as four serotypes which differ in amino acid sequence by 30-35% and the DENV serocomplex in turn differs from ZIKV by 41-46%(E protein).sup.28. Recent reports have shown difficulty in distinguishing DENV and ZIKV infections serologically implying a degree of antigenic similarity between the viruses.sup.7, 29, 30.
[0542] Following a primary DENV infection an individual develops life long immunity to the infecting serotype but not to the other serotypes.sup.31, 32. In DENV endemic areas all four viruses frequently co-circulate or cyclically replace each other meaning that multiple sequential infections are common.sup.33. One of the interesting features of DENV infection is that the life threatening complications, leading to dengue haemorrhagic fever, are more common following secondary rather than primary infections.sup.28. One theory to explain this is antibody dependent enhancement (ADE).sup.28. The ADE hypothesis suggests that antibodies generated to a primary infection will not be of sufficient concentration or avidity to neutralize a secondary infecting DENV, which differs in amino acid sequence by 30-35%. However, they may still opsonize the secondary virus and target it for Fc receptor mediated endocytosis into myeloid cells, such as monocytes and macrophages, which are the principal site for DENV replication, thus driving higher virus loads. ADE can be readily demonstrated in vitro and has also be shown to drive higher dengue virus loads in animal models.sup.34, 35, 36, 37.
[0543] Here we take advantage of panel of 132 human monoclonal antibodies generated from DENV infected individuals to demonstrate substantial crossreactivity between DENV and ZIKV. Most anti-DENV monoclonal antibodies also bind to ZIKV but those recognizing the major linear fusion loop epitope (FLE) are non-neutralizing. DENV plasma and mAb can potently enhance ZIKV infection suggesting the possibility that preexisting DENV immunity may increase ZIKV replication.
[0544] Results
[0545] DENV Plasma Crossreacts with ZIKV
[0546] Plasma from individuals taken 6 months following secondary DENV infection with serotypes 1-4 was tested for binding to ZIKV and DENVs by capture ELISA. In all cases DENV immune plasma bound to both DENV and ZIKV (
[0547] Next we tested neutralization of ZIKV by convalescent DENV plasma. All convalescent DENV plasma could neutralize DENV infection to nearly 100% at the lowest dilution used of 1: 50 (
[0548] DENV Plasma Potently Induces ADE
[0549] One of the hallmarks of DENV infection is the increase in severity of illness during secondary infections. One of the explanations of this is antibody dependent enhancement, whereby preexisting antibodies directed to a previous DENV infection, opsonize but do not neutralize a secondary infection. Opsonized virus is targeted for uptake by Fc receptor expressing myeloid cells such as monocytes and macrophages driving higher virus replication.
[0550] We tested the ability of DENV plasma to promote ADE in the myeloid cell line U937 which is relatively resistant to infection by DENV in the absence of ADE and here we show U937 is also poorly permissive to ZIKV infection in the absence of ADE. ZIKV was preincubated with a titration of pooled convalescent anti-dengue plasma obtained at 2 weeks and then used to infect U937 cells. Pooled convalescent plasma led to substantial enhancement of infection>100 fold to both Zika viruses and as expected pooled control non-dengue serum did not enhance infection (
[0551] Cross Reaction of Anti DENV Monoclonal Antibodies
[0552] We have previously created a pool of 145 human monoclonal antibodies reacting to the DENV envelope protein, generated from plasmablasts isolated from DENV infected patients.sup.34. Detailed epitope mapping of these antibodies demonstrated three broad reactivities. Around ⅓ of the antibodies reacted to the well described fusion loop epitope (FLE), ⅓ were not definitively mapped, but like the fusion loop antibodies they reacted to envelope protein by Western Blot (these are termed non-FLE as they were not sensitive to mutation of envelope residue W101). Finally, a group of around 40 antibodies did not react to envelope protein by western blot and only bound to intact virus particles. These antibodies were shown by cryo electron microscopy and X-ray crystallography to bind to a conformational quaternary epitope formed at the interface of two envelope protein monomers making up the basic head to tail dimer, 90 of which are arranged in icosahedral symmetry into the DENV glycoprotein shell .sup.34, 38. We termed this new epitope the E dimer epitope (EDE), which were subdivided into two groups EDE1 and EDE2 based on the sensitivity to the removal of the N-linked glycan N153 in E (EDE2 binding was reduced by removal of N153, EDE1 not). Some EDE antibodies were fully crossreactive to all four DENV serotypes and could neutralize infection in the picomolar range.
[0553] Binding of the panel of anti-DENV monoclonal antibodies to ZIKV was tested by capture ELISA and compared to binding to DENV (
[0554] It has previously been demonstrated that almost all mAb generated against DENV promote ADE, which includes all of the 145 human monoclonal antibodies we generated in our previous studies'. Because of the crossreactivity of the DENV mAb to ZIKV we next tested the ability of anti-DENV monoclonal antibodies to promote ADE of ZIKV virus infection (
[0555] EDE mAb can Inhibit ADE of DENV Plasma
[0556] Fusion loop and EDE mAb have overlapping epitopes as the footprint of the EDE also covers the fusion loop region. To test whether EDE antibodies could overcome ADE induced by polyclonal anti-DENV plasma we added a titration of anti-DENV EDE1 mAb to ZIKV incubated with an enhancing concentration of anti-DENV plasma (
[0557] Discussion
[0558] The recent explosion of ZIKV virus infection in South America with associated Guillain Barre syndrome and microcephaly are of great concern.sup.15, 16, 17. Guillain Bane Syndrome, is a relatively rare complication, estimated to affect 0.024% of ZIKV infected individuals, but owing to the scale of the ZIKV epidemic this still translates to large number of cases.sup.15. Much work still needs to be performed to understand the exact causes of microcephaly, however, it is becoming increasingly clear that this is caused by intrauterine infection of the developing brain.sup.17, 39, 40, 41, 42. Zika has been shown in animal models to infect the placenta and stunt growth and also to be able to cross the placenta and infect the brain.sup.43, 44, 45. Furthermore in vitro ZIKV can infect neural cell cultures and disrupt development in neurospheres.sup.46, 47. The exact risk of neurological damage following maternal infection remains to be determined, but early studies suggest that this may be up to 22% in the first trimester although other reports from French Polynesia put the risk at around 1%.sup.48, 49.
[0559] ZIKV is spread by Aedes mosquitos and currently in South America these mosquitos are also promoting epidemic spread of DENV and Chikungunya viruses.sup.19. In many areas affected by ZIKV the seropositivity to DENV is very high and in such areas there is great difficulty in distinguishing ZIKV and DENV infection serologically.sup.26, 27, 30. In this paper we have demonstrated substantial crossreactivity of the anti-DENV serological response towards ZIKV. Most anti-DENV plasma poorly neutralizes ZIKV yet can potently induce ADE.
[0560] In a related Example we have studied neutralization of ZIKV by anti-DENV human monoclonal antibodies. Interestingly, anti-fusion loop antibodies, which form a major part of the antibody response in DENV infection.sup.28 and which we show here promote ADE, fail to neutralize infection. Antibodies reacting to the fusion loop are known to be broadly reactive across a number of flaviviruses but despite often strong crossreaction by ELISA methods rarely show crossneutralizing activity perhaps because their epitopes are poorly exposed on native virus particles.sup.50. In addition we show that EDE1 mAb showed potent neutralization in a similar picomolar range to their neutralization of DENV whilst EDE2 mAb also neutralize ZIKV but not as potently as EDE1 mAb. These results are presented together with X-ray crystallographic structures of EDE1 and EDE2 Fab in complex with the ZIKV envelope.
[0561] Antibody dependent enhancement was first recognized nearly 50 years ago in DENV infection and is believed to be one of the factors driving increased severity of secondary infections which is a hallmark of DENV.sup.36. The risk of ADE has made the development of DENV vaccines particularly difficult. The most advanced DENV vaccine Dengvaxia (CYD-TDV) produced by Sanofi Pasteur has just been licensed in several countries and gives some protection from infection; it is estimated that it will reduce the burden of disease by 10-30% over a 30 year period if deployed in endemic countries.sup.51.
[0562] Dengvaxia is a tetravalent live attenuated vaccine where the sequences encoding the precursor membrane protein and envelope proteins that make up the glycoprotein shell of the DENV are combined with the non-structural sequences from the attenuated 17D yellow fever vaccine strain.sup.28. Dengvaxia seems to give protection to individuals who have been previously infected with DENV but efficacy when given to DENV naive vaccinees is less.sup.28, 51.
[0563] A recent longer term analysis of the vaccine trials of Dengvaxia has raised some safety concerns.sup.28. In the under 9 age groups hospitalization from DENV infection was higher in vaccinated children than in the non-vaccinated control group. This may represent antibody dependent enhancement in children who at entry to the study trial were DENV naive and have been primed but not protected by the vaccine. For this reason the vaccine is not licensed for use in children<9 years and furthermore it is recommended to be used only in populations where the seroprevalence of prior DENV exposure in the age group to be vaccinated is 70% or greater.sup.51.
[0564] There is now great pressure to produce a vaccine against ZIKV, the extensive crossreaction between DENV and ZIKV serologically must be considered in this regard. It is likely that the vaccine will need to be deployed in areas with high DENV seroprevalence and raising de novo ZIKV neutralizing responses in such a setting may be challenging. There is also the possibility that ZIKV vaccination in DENV naive subjects may promote ADE of DENV and conversely that DENV vaccination may promote ADE of ZIKV infection.
[0565] The results described here show a complex serological interaction between DENV and ZIKV. The precise reason for the explosion of ZIKV infection and its complications in Brazil will need to be fully determined but it is possible that the preexisting DENV immunity is driving higher virus replication in infected individuals which may in turn may drive higher mosquito infection and spread and greater risk of complications. The possibility that ADE may aid transplacental transfer of ZIKV also needs to be investigated. The timings of DENV versus ZIKV infection may also be important as cross reacting protective and enhancing immunity may change over time following DENV infection.
[0566] In summary, although ZIKV differs in sequence by around 41-46% (E protein) from DENVs the similarities are sufficient to allow crossreaction of anti-DENV antibodies with the ZIKV and to drive antibody dependent enhancement. In this respect ZIKV could be considered as a fifth member of the DENV serocomplex, a factor which must be considered in vaccine approaches to these two viruses.
[0567] Methods
[0568] Samples
[0569] Blood samples were collected after written informed consent and the approval of the ethical committee of the Khon Kaen and Siriraj Hospitals in Thailand and the Riverside Ethics Committee in UK. The serotypes of DENV infection was determined by RT-PCR detection of the viral genome. Samples were collected 6 months after recovery from dengue illness.
[0570] Cells, Reagents and Antibodies Vero cells (a gift from AFRIMS), 293T, and U937 cells were cultured at 37° C. in MEM, DMEM and RPMI-1640, respectively. C6/36 cells (a gift from AFRIMS) were grown in Leibovitz L-15 at 28° C. All media contained 10% heat-inactivated foetal bovine serum, 100 units/ml penicillin and 100 ug/ml streptomycin. All cell lines were free from mycoplasma contamination.
[0571] Alkaline phosphatase (ALP)-conjugated anti-human IgG (A9544) and horseradish peroxidase-conjugated anti-human IgG (P0214) were purchased from Sigma and Dako, respectively. Mouse monoclonal anti-DENV E, 4G2, was a gift from AFRIMS. RPMI-1640 (R8758), DMEM (D5046), p-nitrophenylphosphate (PNPP, N2770-50), Bovine serum albumin (BSA, A7030), diaminobenzidine (D5905), and polyethylenimine (408727; Sigma) were from Sigma. MEM (31095) and Leibovitz L-15 (11415) were from Gibco and UltraDOMA-PF (12-727F) was from Lonza.
[0572] Viral stocks.
[0573] All viruses were grown in C6/36 cells. ZIKV strain HD78788 (African strain) was provided by Anavaj Sakuntabhai. ZIKV strain PF13/251013-18 (PF13) was isolated from a patient during ZIKV outbreak in French Polynesia 2013. DENV-1 (Hawaii), DENV-2 (16681), DENV-3 (H87) and DENV-4 (1-0093) were gifts from AFRIMS. Virus containing supernatants were clarified by centrifugation at 2000 rpm at 4° C. before being stored at −80° C. Viral titres were determined by a focus-forming assay on Vero cells.sup.34. All virus stocks were free from mycoplasma contamination.
[0574] Expression of Human Monoclonal Anti-DENV E Antibodies
[0575] A pair of plasmids containing heavy and light chains of immunoglobulin G1 were co-transfected into 293T cells by a polyethylenimine method and cultured in protein-free media. Culture supernatant containing antibodies was harvested after 5 days.
[0576] Determination of ZIKV Crossreactivity of anti-DENV Antibodies by ELISA
[0577] A MAXISORP immunoplate (442404; NUNC) was coated with mouse anti-E protein, 4G2 (a fusion loop murine Ab which crossreacts to ZIKV). Plates were blocked with 3% BSA for one hour followed by incubation with viral supernatant. After one hour, 10 ug/ml anti-DENV humAbs or serially diluted plasma was added. The reaction was visualized by ALP-conjugated anti-human IgG antibody (A9544; Sigma) and PNPP substrate. Reactions were stopped with NaOH and the absorbance measured at 405 nm. Endpoint titers (EPTs) were defined as reciprocal plasma dilutions that corresponded to 2 times the average OD values obtained with mock antigen.
[0578] Neutralization Assay
[0579] The focus reduction neutralization assay (FRNT) was employed to determine the neutralizing potential of antibodies. Virus was incubated with serial dilutions of antibodies or plasma samples for an hour at 37° c. The mixtures were then added to Vero cells and incubated for 2 (for ZIKV) or 3 days (for DENV). Focus forming assays were then performed as described'. Briefly, Vero cells were stained with anti-E mAb 4G2 followed by peroxidase-conjugated goat anti-mouse Ig (P0047; Sigma). The foci (infected cells) were visualized by adding peroxidase substrate, DAB. The percentage focus reduction was calculated and 50% FRNT was calculated using the probit program from the SPSS package.
[0580] Antibody-Dependent Enhancement Assay
[0581] Serially diluted antibody or plasma samples were incubated with virus for one hour at 37° C. before adding to U937 cells. After incubation 2 (for ZIKV) or 3 days (for DENV), supernatants were harvested and viral titres determined by focus forming assay. Fold enhancement was calculated by comparison to viral titres in the presence/absence of antibody.
[0582] The ADE inhibition by human mAbs was performed by premixing pooled convalescent dengue hyper immune serum at 1:10,000 dilution (a peak enhancing dilution) with serially diluted antibody before performing the ADE assay as described above.
REFERENCES
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EXAMPLE 2
Structural Basis of Potent Cross-Neutralization between Zika and Dengue Viruses
[0636] Zika virus is a member of the flavivirus genus that had not been associated with severe disease in humans until the recent outbreaks, when it was linked to microcephaly in newborns in Brazil and to Guillain-Barré Syndrome in adults in French Polynesia. Zika virus is related to dengue virus, and we report here that a category of antibodies isolated from dengue patients and targeting a conformational epitope potently neutralize Zika virus. The crystal structure of two of these antibodies in complex with the envelope protein of Zika virus reveals the details of a conserved epitope, which is also the site of interaction of the envelope protein dimer with the precursor prM protein during virus maturation. Comparison of the Zika and dengue virus immunocomplexes lays the foundation for a rational, epitope-focused design of a universal vaccine capable of eliciting potently neutralizing antibodies to protect against Zika and dengue viruses simultaneously.
[0637] The explosive spread of Zika Virus (ZIKV) in Brazil and other South and Central American countries upon its recent introduction was linked with increasing numbers of microcephaly cases.sup.1-4. There have also been cases of Guillain Barré syndrome linked to ZIKV infections in the 2013-2014 French Polynesian outbreak.sup.5, leading the World Health Organization to declare these neurological disorders a Public Health Emergency of International Concern on Feb. 1, 2016.sup.6. Prior to the epidemics of recent years, ZIKV was thought to cause only mild or self-limiting disease.sup.7. The physiological processes leading to fetal infections and neurological complications are unresolved and specific therapeutic or prophylactic interventions are currently not available. In order to obtain insight into ZIKV pathogenesis and especially for developing safe and protective vaccines it is essential to understand the structural basis of virus neutralization and cross-reactivity with other flaviviruses. ZIKV transmission among humans and epidemic spread is primarily maintained by Aedes mosquitoes, but there are reports of sexual transmission as well.sup.8-10. ZIKV is an arthropod-borne enveloped virus belonging to the flavivirus genus in the family Flaviviridae, which also includes the human pathogenic yellow fever, dengue, West Nile and tick-borne encephalitis viruses.sup.11. Flaviviruses have two structural glycoproteins, prM and E (for precursor Membrane and Envelope proteins, respectively), which form a heterodimer in the endoplasmic reticulum (ER) of the infected cell and drive the budding of spiky immature virions into the ER lumen. The budded particles are subsequently transported across the secretory pathway of the cell, a process during which prM undergoes proteolytic maturation by the trans-Golgi resident furin protease.sup.12-14. This maturation process is required for infectivity and results in the reorganization on E at the virion surface. The mature particles released from the infected cell have a smooth aspect, with 90 E dimers coating the external surface of the virion, organized with icosahedral symmetry in a “herringbone” pattern.sup.15,16. Three-dimensional cryo-EM structures of the mature ZIKV particles have recently been reported to near atomic resolution (3.8Å).sup.17,18, showing that it has essentially the same organization as the other flaviviruses of known structure, such as dengue virus (DENV), for which a 3.5Å cryo-EM reconstruction was reported previouslyl.sup.16 and also West Nile virus.sup.19,20. The E protein is about 500 amino acids long, with the 400 N-terminal residues forming the ectodomain, essentially folded as β-sheet with three domains named I, II and III, aligned in a row with domain I at the center. The highly conserved fusion loop is at the distal end of the rod in domain II, buried at the E dimer interface. At the C-terminus, the E ectodomain is followed by the so-called “stem”, featuring two a-helices lying flat on the viral membrane (the “stem” helices), which link to two C-terminal trans-membrane α-helices. The main distinguishing feature of the ZIKV virion is an insertion in a glycosylated loop of E (the “150” loop), which protrudes from the virion surface.sup.17,18.
[0638] Flaviviruses have been grouped into serocomplexes based on cross-neutralization studies with polyclonal immune sera.sup.21. The E protein is the main target of neutralizing antibodies. Because E is responsible for membrane fusion during virus entry, it is maintained in a metastable conformation such that it can be triggered to undergo a conformational change to induce fusion of the viral envelope with an endosomal membrane, thereby releasing the viral genetic material into the cytoplasm. One consequence of this metastability of the E dimer is that it displays a dynamic behavior, termed “breathing”.sup.22, such that it exposes regions normally buried within the dimer interface. One such region is the fusion loop epitope (FLE), which is a dominant cross-reactive antigenic site.sup.23. Although antibodies to this site can be protective by complement-mediated mechanisms, as shown for West Nile virus in a mouse model.sup.24, they are poorly neutralizing and have been shown to lead to antibody-dependent enhancement (ADE).sup.25-29, thereby aggravating flavivirus pathogenesis and complicating the development of safe and effective vaccines.
[0639] We recently reported the isolation and structural characterization of a panel of antibodies isolated from dengue patients.sup.27,30. A majority of these antibodies targeted the FLE, but others targeted a quaternary site readily accessible at the exposed surface of the E protein on the virion, at the interface between the two E subunits in the dimer. These broadly neutralizing antibodies (bnAbs), termed EDE for “E-dimer epitope”, potently neutralize all four serotypes of DENV. Their binding site is conserved across serotypes because it is also the interaction site of prM with E dimers during transport of the immature virus particles through the Golgi apparatus of the cell. There were two subsets of EDE Mabs, characterized by a differential requirement for glycosylation on the 150 loop for binding. The EDE1 bnAbs bind better in the absence of glycan, whereas EDE2 bnAbs bind better when the glycan is present.
[0640] In this Example we identified that the EDE Mabs neutralize ZIKV as potently as they neutralize DENY. We also found that the FLE antibodies, which neutralize DENV—although not as potently as the EDE Mabs—do not neutralize ZIKV at concentrations up to 1 μM in spite of a very high affinity for the recombinant ZIKV E protein. We further describe the crystal structure of the ZIKV E protein dimer in complex with EDE1 C8 and EDE2 A11, identifying their binding determinants. We show that EDE2 A11, which requires the glycosylation site at position 153 in DENV for binding, cannot make the same interactions with the 154 gly can on ZIKV sE, which strongly reduces its binding potential such that despite its nM IC50, it displays increased ADE as described in Example 1.
[0641] A ZIKV-DENV super serogroup
[0642] Phylogenetic analyses of the main human pathogenic flaviviruses using the amino acid sequences of the viral RNA polymerase NS5 indicate a clustering of ZIKV with the group of mosquito-borne encephalitic viruses (
[0643] The BLI experiments were done using three antibodies, EDE1 C8, EDE2 A11 and a representative FLE antibody, P6B10. The FLE Mab bound with almost one log higher affinity with respect to EDE1 C8 (1.5nM vs 9nM), and about 3 logs higher than EDE2 A11, which had a dissociation constant close to the μM range (
[0644] The immune complexes of ZIKV with EDE bnAbs
[0645] The crystallization conditions, the crystals obtained and the structure determination are described in the Online Methods section and are summarized in ED Table 1 (ED
[0646] As expected, the antibodies recognize a quaternary epitope in the ZIKV sE dimer in the same way they recognize the DENY serotype 2 (DENV-2) sE dimer described earlier.sup.30.
[0647] The antibody contacts per E amino acid on the ZIKV and DENV-2 sE alignment are displayed in ED
[0648] The binding angles of the MAbs to ZIKV sE are different compared to DENV-2 sE (see insets in
[0649] For comparison, superposition of the ectodomain of virion E from ZIKV and DENV-2 results in a similar 1.5 Å RMSD, indicating that they are presented roughly in the same way, but that DENV sE is more deformable in solution. This malleability may reflect the high conformational breathing reported for DENV E.sup.22. In contrast, the conformation of the E ectodomain in ZIKV seems to be more stable, remaining the same in the absence of additional interactions on the virion. This feature may be linked to the higher stability of the ZIKV virion described recentlyl.sup.17.
[0650] EDE1 C8 complex
[0651] The total buried surface area (BSA) of EDE1 in the complex with ZIKV sE is about 900 Å.sup.2, compared to about 1300 Å.sup.2 in the DENV-2 sE complex (ED Table 3) (ED
[0652] EDE2 A11 complex
[0653] The A11 antibody binds at a very different angle than seen with DENV-2 sE, even accounting for the difference in sE dimer curvature. The contacts along the b-strand are preserved, but the antibody makes a different angle the strand (
[0654] Discussion
[0655] Our results identify the structural details of a quaternary epitope that provides a previously unrecognized link of potent cross-neutralization between Zika and dengue viruses, and thus identifies an antigenic flavivirus cluster beyond the traditional serocomplexes. This relationship defines a super serogroup on the basis of strong cross-neutralization through a conserved epitope that had not been recognized using polyclonal sera.sup.21. This work thus lays the foundation for the rational design of a universal vaccine that can protect against all the viruses from this group.
[0656] Vaccine design against dengue virus has been hampered by the heterogeneity of DENV particles and the need to use polyvalent formulas to immunize against all four serotypes.sup.32,33. One feature of DENV is that it undergoes incomplete furin maturation cleavage of prM in many cell types, giving rise to heterogeneous mosaic particles with an immature-like spiky patch on one side and a smooth mature-like region on the opposite side.sup.34. These particles are infectious, as they can fuse with the cellular membrane through the smooth, mature side. Because the FLE is exposed in immature regions.sup.35, an overwhelming antibody response in DENV infected patients is directed against it.sup.36. These highly cross-reactive antibodies coat the particles essentially on the “immature side”.sup.35, and therefore are weakly neutralizing, relying on the “breathing” effect of the E dimers to bind and neutralize on the mature, infectious side.sup.37-39. The high avidity of the FLE antibody for the E protein, as exemplified by Mab P6B10 (
[0657] Our data suggest that developing an epitope-focused vaccine against the ZIKV/DENV super-serogroup is a viable approach. It is clear from our results that the epitope targeted by the EDE1 bnAbs is best suited for this purpose, in stark contrast with the FLE, which induces poorly neutralizing and strong infection enhancing antibodies.sup.26-28. The EDE2 antibodies were also shown to induce ADE.sup.26, in line with their poor avidity for the sE dimer (
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[0697] Methods
[0698] Recombinant production of ZIKV sE protein. Recombinant Zika virus sE protein (strain H/PF/2013, GenBank accession no. KJ776791) was produced with a tandem strep-tag in the Drosophila Expression System (Invitrogen) as described previously.sup.42,43. A chemically synthesized DNA fragment (GeneArt) containing the Zika sE sequence (amino acid 1-408) was cloned into the expression vector pT389.sup.44 that encodes the export signal sequence BIP, an enterokinase cleavage site and the strep-tag. Drosophila Schneider 2 cells were stably transfected using blasticidin for selection. Protein expression was induced by the addition of CuSO4 and supernatants were harvested 7-10 days after induction. Antigens were purified via affinity chromatography with Streptactin columns (IBA) according to the manufacturer's instructions. A final purification gel filtration step used a Superdex increase 200 10/300 GL column equilibrated in 50 mM Tris pH8, 500 mM NaCl.
[0699] Production of antigen-binding (Fab) and single-chain Fv (scFv) fragments of the bnAbs. The bnAb fragments were cloned into plasmids for expression as Fab.sup.45 and scFv.sup.46 in Drosophila S2 cells. The constructs contain a tandem strep tag fused at the C terminus (only of the heavy chain in the case of the Fab) for affinity purification. The purification protocol included a Streptactin affinity column followed by gel filtration as described above.
[0700] Immune complex formation and isolation. The purified ZIKV sE protein was mixed with Fab A11 or scFv C8 (in approximately twofold molar excess) in standard buffer (500 mM NaCl, Tris 50 mM pH 8.0). The volume was brought to 0.5 ml by centrifugation in a Vivaspin 10 kDa cutoff; after 30 min incubation at 4° C., the complex was separated from excess Fab or scFv by size-exclusion chromatography (SEC) for ZIKV sE and scFv C8. For ZIKV sE and Fab A11 no apparent complex formation could be seen in SEC; therefore a solution containing sE at a concentration of 1.5 mg/ml and Fab A11 at a concentration of 3 mg/ml (corresponding to a molar ratio ˜1:2 antigen:antibody) was directly used for crystallization. In all cases, the buffer was exchanged to 150 mM NaCl, 15 mM Tris, pH 8 for crystallization trials. The protein concentrations used for crystallization, determined by measuring the absorbance at 280 nm and using an extinction coefficient estimated from the amino-acid sequences, are listed in Extended Data Table 1.
[0701] Real-time biolayer interferometry binding assays. The interactions of purified ZIKV E protein with Mabs IgG FLE P6B10, IgG EDE1 C8, IgG EDE2 A11, and control Mabs IgG 28C (an anti-Influenza virus) and IgG K9 (an anti-Chikungunya virus) were monitored in real-time using a Bio-layer interferometry Octet-Red384 device (Pall ForteBio). Anti-human IgG Fc capture biosensors (Pall ForteBio) were loaded for 10min at 1000 rpm shaking speed using antibodies at 5 μg/ml in assay buffer (PBS+0.2 mg/ml BSA+tween 0.01%). Unbound antibodies were washed away for 1 min in assay buffer. IgG-loaded sensors were then incubated for 15 min at 1200 rpm in the absence and presence of two fold serially diluted ZIKV sE protein concentrations in assay buffer. Molar concentrations were calculated for the sE protein in a dimeric form. For Mabs FLE P6B10, EDE1 C8 and EDE2 A11, the following ZIKV sE concentration ranges: 50-0.78 nM, 200-3.125 nM and 3200-50 nM, were respectively used. Reference binding experiments were carried out in parallel on sensors loaded with control IgGs (28C and K9). Dissociation of the complexes formed was then monitored for 10 min by dipping sensors in assay buffer alone. Operating temperature was maintained at 25° C. The real-time data was analyzed using Scrubber 2.0 (Biologic Software) and Biaevaluation 4.1 (GE Healthcare). Specific signals were obtained by double-referencing, ie subtracting non-specific signals measured on non-specific IgG-loaded sensors and buffer signals on specific IgG-loaded sensors. Association and dissociation profiles, as well as steady-state signal vs concentration curves, were fitted assuming a 1 :1 binding model.
[0702] Crystallization and X-ray structure determinations. The crystallization and cryo-cooling conditions for diffraction data collection are listed in Extended Data Table 1 (Example 2 ED
[0703] Because of the strong anisotropy of the crystals (see results for anisotropy in Extended Data Table 1), an important number of crystals was tested at several beam lines at different synchrotrons (SOLEIL, St Aubin, France; ESRF, Grenoble, France; SLS, Villigen, Switzerland). The crystals having the less anisotropic diffraction data and used to solve the structures were collected at the beam lines PROXIMA-1 and PROXIMA-2 at the SOLEIL synchrotron and beam line ID23-2 at ESRF. The datasets were indexed, integrated, scaled and merged using XDS.sup.47 and AIMLESS.sup.48. A preliminary model of ZIKV sE protein was built from the DENV-2 sE (4UTA) structure using the structure homology-modeling server SWISS-MODEL.sup.49. The structures of the complexes were then determined by molecular replacement with PHASER.sup.5° using the search models listed in Extended Data Table 1. AIMLESS and PHASER programs were used within the CCP4 suite.sup.51.
[0704] The DEBYE and STARANISO programs developed by Global Phasing Ltd. were applied to the AIMLESS scaled data without truncation of the resolution, using the STARANISO server (http://staraniso.globalphasing.org/). These softwares perform an anisotropic cut-off of merged intensity data with a Bayesian estimation of the structure amplitudes, and apply an anisotropic correction to the data. These corrected anisotropic amplitudes were then used for further refinement of both structures with BUSTER/TNT.sup.52. Please note that the Extended Data Table 1 shows the refinement statistics for the full sets of reflections truncated at the best high-resolution along h, k or 1 axis, values output from AIMLESS without the anisotropic corrections computed by the STARANISO server.
[0705] The models were then alternatively manually corrected and completed using COOT.sup.53 and refined using BUSTER/TNT against the amplitudes corrected for anisotropy. Refinements were constrained using non-crystallographic symmetry (see Extended Data Table 1). The refined structures ZIKV sE/EDE2 A11 Fab, ZIKV sE/EDE1 C8 scFv and ZIKV sE have a final Rwork/Rfree (in %) of 21.8/23.8 and of 18.7/22.0 and of 22.9/27.5, respectively.
[0706] Analysis of the atomic models and illustrations. Each complex was analyzed with the CCP4 suite of programs and the polar contacts were computed with the PISA website.sup.54.
[0707] For the intermolecular interactions shown in Extended Data
[0708] Phylogenic trees. The Maximum likelihood phylogenetic trees were inferred using 12 representative amino-acid sequences of flaviviruses envelope protein E or RNA-polymerase NS5 proteins, utilizing the LG model available in PhyML.sup.58 and a combination of SPR+NNI branch-swapping. Bootstrap values were calculated from 100 bootstrap replicates. Trees were visualized using Figtree (http://tree.bio.ed.ac.uk/software/figtree/). The accession codes of sequences used in the tree : Zika virus (ZIKV, KJ776791, strain H-PF-2013_French_Polynesia); dengue virus serotype 1 (DENV-1, NC_001477); dengue virus serotype 2 (DENV-2, NC_001474); dengue virus serotype 3 (DENV-3, NC_001475); dengue virus serotype 4 (DENV-4, NC_002640); Saint Louis encephalitis virus (SLEV, NC 007580); Japanese encephalitis virus (JEV, NC_001437; Murray Valley encephalitis virus (MVEV, NC_000943); West Nile virus (WNV, NC_001563); yellow fever virus (YFV, NC_002031); tick-borne encephalitis virus (TBEV, NC_001672); Powassan virus (POWV, NC_003687).
[0709] Virus stocks. The African strain Zika HD78788 was obtained from the Institut Pasteur collection and the Asian strain Zika PF13, isolated from a patient during ZIKV outbreak in French Polynesia in 2013, was obtained through the DENFREE (FP7/2007-2013) consortium. Viral stocks were prepared from supernatant of infected C6/36 cells clarified by centrifugation at 3000 g at 4° C. and titrated on Vero cells by a focus-forming assay. Stocks were kept at −80° C. until use.
[0710] Neutralization Assays. Virus neutralization by the tested human antibodies was evaluated using a focus reduction neutralization test (FRNT). About 100 ffu (focus forming unit) from virus stocks were incubated with a serial dilution of antibody for lh at 37° C. The mixture was then added to Vero cells and foci were let to develop in presence of 1.5% methylcellulose for two days. Foci were then stained after fixation with 4% formaldehyde using anti-E 4G2 antibody and anti-mouse HRP-conjugated secondary antibody. The foci were visualized by DAB staining and plates were counted using the ImmunoSpot S6 Analyser (Cellular Technology Limited, CTL). Neutralization curves and 50% FRNT were calculated using GraphPad Prism software.
[0711] Methods References [0712] 42 Vratskikh, O. et al. Dissection of antibody specificities induced by yellow fever vaccination. PLoS Pathog 9, e1003458, doi: 10.1371/j ournal.ppat.1003458 (2013). [0713] 43 Jarmer, J. et al. Variation of the specificity of the human antibody responses after tick-borne encephalitis virus infection and vaccination. J Virol 88, 13845-13857, doi:10.1128/JVI.02086-14 (2014). [0714] 44 DuBois, R. M. et al. Functional and evolutionary insight from the crystal structure of rubella virus protein E1. Nature 493, 552-556, doi:10.1038/nature11741 (2013). [0715] 45 Backovic, M. et al. Efficient method for production of high yields of Fab fragments in Drosophila S2 cells. Protein Eng Des Sel 23, 169-174, doi:10.1093/protein/gzp088 (2010). [0716] 46 Gilmartin, A. A. et al. High-level secretion of recombinant monomeric murine and human single-chain Fv antibodies from Drosophila S2 cells. Protein Eng Des Sel 25, 59-66, doi:10.1093/protein/gzr058 (2012). [0717] 47 Kabsch, W. Xds. Acta Crystallogr D Biol Crystallogr 66, 125-132, doi:10.1107/S0907444909047337 (2010). [0718] 48 Evans, P. R. & Murshudov, G. N. How good are my data and what is the resolution? Acta Crystallogr D Biol Crystallogr 69, 1204-1214, doi:10.1107/S0907444913000061 (2013). [0719] 49 Biasini, M. et al. SWISS-MODEL: modelling protein tertiary and quaternary structure using evolutionary information. Nucleic Acids Res 42, W252-258, doi:10.1093/nar/gku340 (2014). [0720] 50 McCoy, A. J. et al. Phaser crystallographic software. Journal of applied crystallography 40, 658-674, doi:10.1107/S0021889807021206 (2007). [0721] 51 Winn, M. D. et al. Overview of the CCP4 suite and current developments. Acta Crystallogr D Biol Crystallogr 67, 235-242, doi:10.1107/50907444910045749 (2011). [0722] 52 Blanc, E. et al. Refinement of severely incomplete structures with maximum likelihood in BUSTER-TNT. Acta Crystallogr D Biol Crystallogr 60, 2210-2221, doi:10.1107/S0907444904016427 (2004). [0723] 53 Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features and development of Coot. Acta Crystallogr D Biol Crystallogr 66, 486-501, doi:10.1107/S0907444910007493 (2010). [0724] 54 Krissinel, E. & Henrick, K. Inference of macromolecular assemblies from crystalline state. J Mol Biol 372, 774-797, doi:10.1016/j.jmb.2007.05.022 (2007). [0725] 55 Larkin, M. A. et al. Clustal W and Clustal X version 2.0. Bioinformatics 23, 2947-2948, doi:10.1093/bioinformatics/btm404 (2007). [0726] 56 Goujon, M. et al. A new bioinformatics analysis tools framework at EMBL-EBI. Nucleic Acids Res 38, W695-699, doi:10.1093/nar/gkq313 (2010). [0727] 57 Gouet, P., Courcelle, E., Stuart, D. I. & Metoz, F. ESPript: analysis of multiple sequence alignments in PostScript. Bioinformatics 15, 305-308 (1999). [0728] 58 Guindon, S. et al. New algorithms and methods to estimate maximum-likelihood phylogenies: assessing the performance of PhyML 3.0. Systematic biology 59, 307-321, doi:10.1093/sysbio/syq010 (2010).
EXAMPLE 3
Increasing the Flavivirus Envelope Glycoprotein Dimer Stability to Elicit Potent and Broadly Neutralizing Antibody Responses
[0729] Potently cross-neutralizing human antibodies against the four serotypes of dengue virus (DENV) have recently been isolated and structurally characterised. See, for example, WO 2016/012800; Rouvinski et al (2015) Nature 520, 109-113; Dejnirattisai et al (2015) Nature Immunol 16, 170-177. These antibodies bind to a highly conserved epitope termed the E-dimer-epitope (EDE), which we have now discovered is also conserved in Zika virus (ZikaV), leading also to potent neutralization of ZikaV. The mature DENV particle is an assembly of metastable E dimers with a strong “breathing” behaviour, meaning that it promotes the generation of many poorly neutralizing, yet disease enhancing antibodies. We describe a reverse vaccinology approach to develop antigens capable of eliciting a protective immune response against flaviviruses, for example zika-dengue group of flaviviruses, based upon the production of stabilized E-protein dimers whilst minimising the production of poorly neutralizing antibody responses.
[0730] The present inventors have studied the immune response to DENV infection to both understand immunopathogenesis and to inform vaccine design. This has included studying the human antibody response to infection.
[0731] These studies have included consideration of antibodies to precursor membrane protein. PrM-specific antibodies are a major component of the memory B cell response to dengue; these antibodies show poor neutralization (maximum 30-50%) even at high concentration.sup.16,34-37 . prM-specific antibodies do not bind to fully mature virions which do not contain prM, whereas many partially mature particles do not contain a high enough density of prM to allow neutralization but yet may be sufficient to promote ADE.sup.16,38. We have speculated that the inefficient cleavage of prM may be an immune evasion/enhancement strategy, leading to the generation of poorly neutralizing antibodies directed to prM. The high frequency, low potency and high ADE potential of antibodies directed to prM has implications for vaccine design; all attenuated vaccines at an advanced stage of development contain prM, the ideal vaccine would focus responses to the E and the prM component of the response be minimized if the potential for ADE in vaccines is to be reduced.
[0732] In a second series of experiments we have recently described the cloning of a large panel of anti-E mAb from dengue infected patients.sup.17. One third of the antibodies do not bind to recombinant E protein, suggesting a conformationally sensitive quaternary epitope and many of these antibodies showed broad neutralization of all four dengue serotypes. The bnAb anti-dengue mAb (bnAb) are amongst the most potent described to date and bind to the basic repeating envelope dimers making up the virion surface lattice, to a site that we termed the E-dimer epitope EDE (
[0733] Structural characterization of these antibodies has shown they bind in a valley formed between the two E subunits of the head to tail dimers present at the surface of the virion.sup.39. The antibodies make contact with a conserved surface patch at the dimer surface, including atoms of the fusion loop main chain but not its side chains (
[0734] The discovery of the EDE opens up a number of interesting future possibilities in dengue vaccine research. Current vaccination strategies use tetravalent formulations with the aim of raising single serotype specific responses against all four serotypes. The demonstration that potent bnAb are produced in dengue infection, which can also potently neutralize at least ZikaV, means that the generation of such antibodies should be a goal for the next generation vaccines. Importantly, as the response is limited to the E-dimer it opens the way for subunit vaccines consisting of E-dimers alone and furthermore, it may be possible to design a single universal immunogen, rather than a multivalent formulation to achieve this response. Alternatively, heterologous prime boost strategies may be used to focus the response to the EDE, potentially following LATV priming.
[0735] Dengue vaccines are now at an important juncture; a large scale Phase III trial has underperformed expectations and given a concerning safety signal of enhanced infection. We consider the E-dimer can be stabilised, removing prM from the immunogen and further reducing the generation of poorly neutralising antibodies such as the immunodominant response to the fusion loop epitope (FLE). We consider a subunit flavivirus (for example Dengue or Zika) vaccine aimed at driving a potent bnAb response to the EDE also has utility against flavivirus infection beyond Dengue, for example against ZikaV infection; or against both Dengue and ZikaV infection; or against Dengue, ZikaV and other flavivirus infection.
[0736] Possible experimental plan
[0737] A reverse vaccinology approach may be taken to design a subunit vaccine to dengue and/or other flaviviruses. This may make use of the generation and structural characterization of the bnAb EDE epitope based on a panel of recombinant antibodies targeting conformational epitopes such as the EDE as well as linear epitopes such as the FLE and prM, for example as described in WO 2016/012800; Rouvinski et al (2015) Nature 520, 109-113; Dejnirattisai et al (2015) Nature Immunol 16, 170-177. The general aim of this plan is to generate a stable version of the E-dimer and then through an iterative structural/modelling informed design process to develop immunogens to specifically target the generation of an anti-EDE response whilst resurfacing non-EDE related areas of the dimer to reduce the generation of less protective but infection enhancing antibodies. Immunogenicity can be tested in human immunoglobulin transgenic mice (for example mice such as those described in Lee et al (2014) Nature Biotechnology Vol 32(4), 356-363; or mice such as those described in EP1360287 or EP2264163) and in vivo neutralization can be tested in murine models of DENV infection, for example.
[0738] 1. Stabilisation of the E-dimer. The E-dimer is the pre-fusion form of E, which is presented at the virion surface in a metastable conformation.sup.40. This meta-stability is important to allow the glycoprotein shell encasing the viral membrane, which is formed by lateral interactions between E-dimers, to dissociate under the mildly acidic environment of the early endosomes. The resulting E-monomers can then insert the fusion loop into the endosomal membrane.sup.7,41. The subsequent acid-triggered irreversible conformational change of E leads to a very stable “post-fusion” E-trimer, which is the ground state of the molecule.sup.41. The energy released in this transition between a high energy, dimeric state of E and its lowest energy conformation—the post-fusion trimer—is used to drive lipid merger and allow the release of the viral genome into the cytosol of the cell. Because of its meta-stability, E has been shown to display considerable “breathing” at the virion surface under standard conditions (neutral pH), exposing to the immune system regions that are not relevant for antibody neutralization.sup.42-44.
[0739] Recombinant DENV sE (i.e., Dengue “soluble-E”, lacking stem and trans-membrane segments) is predominantly monomeric in solution having a dissociation constant in the micromolar range. For immunogen design, the aim is to make the sE-dimer as stable as possible, rendering it inert and not exhibiting the dynamic breathing observed at the virion surface. In addition, the aim is to alter (resurface) the E-dimer surface on regions outside the EDE, to limit the extent of elicitation of serotype specific antibodies. We have now identified that the ZikaV-sE is stable as a dimer in solution, providing us with an important number of mutations that preserve the EDE, yet in a quite different context, since the rest of the glycoprotein is different enough to those of the DENVs such that the cross-reactivity may be limited to the EDE.
[0740] For other viral diseases, capture and stabilization of quaternary structures in the meta-stable, prefusion conformation (i.e., the active form of the virion) is indeed now a key objective of several subunit anti-viral vaccine approaches. In respiratory syncytial virus, potent neutralizing antibodies to the trimeric pre-fusion conformation of the F-protein have led to the design of novel immunogens stabilizing the F-protein pre-fusion trimer.sup.45,46. In HIV, the recent structural determination of mAb bound to the pre-fusion conformation of Env will drive efforts to stabilise pre-fusion viral intermediates for potential HIV subunit vaccines.sup.47. Similar approaches for influenza-HA have shown that a recombinant stabilized trimeric stalk fragment was able to elicit cross-reactive antibodies against the virus.sup.48,49.
[0741] Two main classes of mutants can be developed to stabilize the dimer:
[0742] A. Disulphide stabilized mutants: We used a structure-based approach.sup.50 for triaging possible pairs of mutations for disulphide bond formation to improve sE-dimer stability. Analysis of the crystal structure of the sE-dimer from DENV revealed a number of pairs of residues facing each other with C.sub.β-C.sub.β distances under 4.5 Å across the dimer interface. We have thus identified six locations where substitution by a pair of cysteines (two of which are residues facing each other across the molecular 2-fold axis of the sE-dimer, requiring only a single substitution to cysteine). 3 of the mutants have already led to successful covalent DENV sE-dimer expression, recapitulating the EDE and binding to our panel of EDE-mAbs (
[0743] Rouvinski et al (2017) NATURE COMMUNICATIONS |8:15411|DOI: 10.1038/ncomms15411
[0744] “Covalently linked dengue virus envelope glycoprotein dimers reduce exposure of the immunodominant fusion loop epitope” also reports the inventors' engineering of E dimers locked by inter-subunit disulfide bonds, and shows by X-ray crystallography and by binding to a panel of human antibodies that these engineered dimers do not expose the FLE, while retaining the EDE exposure.
[0745] B. Cavity filling and resurfacing mutants: Using Rosetta software.sup.51 we have identified hydrophobic cavities in the structure of the sE-dimer, and residues that could be substituted in order to fill these cavities to stabilize the dimer. These mutations will be designed manually using the prevalent rotamers looking to minimize clashes or with Rosetta software. Of particular relevance will be the domain I/III interface, which creates a binding pocket for the fusion loop of the partner subunit in the dimer. Release of domain III from the interaction with domain I is key to expose the fusion loop so freezing the domain I/III interaction is therefore an important goal. Alternatively or in parallel, de novo computational resurfacing, for example as described in .sup.52,53 can be used. This de novo approach may allow a greater variety of potential solutions to be tested. Alternatively or in addition, for example if computational approaches are insufficient, mammalian display directed evolution may be used to carry out resurfacing. For a review relating to resurfacing approaches, see, for example, Chapman & McNaughton (2016) Cell Chemical Biology 23, 543-553.
[0746] Further mutagenesis of the selected re-surfaced genes is considered to allow determination of viable substitutions within the area of the EDE that do not interfere with binding to EDE-antibodies. We have information from previous alanine scanning mutagenesis (see for example WO 2016/012800; Rouvinski et al (2015) Nature 520, 109-113; Dejnirattisai et al (2015) Nature Immunol 16, 170-177), and residues that are not binding determinants can be substituted, as long as they do not introduce a bulky side chain that may cause steric clashes with the antibody. Similarly, additional N-linked glycosylation sites can be introduced strategically positioned to mask serotype specific epitopes while not interfering with binding of EDE-mAbs. In total, we estimate that the process of dimer stabilization and resurfacing may entail screening around 100 mutations on the best performing initial resurfaced genes.
[0747] In total, we have identified ˜100 initial individual mutations of sE, which can, for example, be tested both in a DENV2 (for example) serotype and in a ZikaV-sE background (see for example the Mutation section above). Preliminary data suggest that DENV2 has the least stable sE-dimer, and is the most prone to breathing, whereas the ZikaV sE-protein is the most stable. All mutants can be tested for expression, dimerization and antibody reactivity. The mutants performing best can be used as combinations of pairs of mutants, which can be tested iteratively.
[0748] Analytical ultracentrifugation can be used to determine dimerization constants in solution. Thermofluorimetry along with differential scanning calorimetry can be used to determine the denaturation profile of stabilized mutants upon heating or destabilizing chaotropes +/− EDE/Abs. Surface plasmon resonance, Biolayer interferometry as well as isothermal calorimetry can be used to determine Kon and Koff values between a subset of selected mutants and a panel of EDE/FLE-mAbs in different pH conditions. Stabilized sE-mutants can also be tested by flotation assay in presence of liposomes in comparison with wild type sE. We consider that stabilized dimers may be impaired in flotation upon acidification as the fusion loop should not be available to interact with liposomes. Finally, a subset of stabilized dimer mutants showing high thermal chemical stabilities, high affinities to broadly cross-reactive EDE-Abs, low affinities to FLE-mAbs and low affinities to serotype specific EDE-Abs and to other serotype specific Abs may be selected for further structural studies by X-ray crystallography.
[0749] High throughput expression strategy. Recombinant sE can be produced in a Drosophila expression system; this may be useful particularly in characterizing multiple E-mutants. We have previously used 293T to produce virus like particles (VLP) through transient transfection of vectors encoding prM/E. A large panel of >100 alanine substitutions to surface residues on envelope allowed us to produce mutant VLP, which we used to epitope map anti-dengue mAb.sup.17. In addition, we have developed a mammalian system to produce sE or E-dimers in 293T by transient transfection. This, for example, can be used to produce strep-tagged sE-mutants, promising candidates can then be expressed at high levels by transient transfection in Expi293F cells for further characterization.
[0750] We have generated a considerable resource useful in such a plan, namely the panel of around 150 human anti-dengue mAb (see, for example, WO 2016/012800; Rouvinski et al (2015) Nature 520, 109-113; Dejnirattisai et al (2015) Nature Immunol 16, 170-177). Around ⅓ bind to the EDE, 1/3 to the FLE and 1/3 to as yet undetermined epitopes.sup.17. To understand the structural determinants governing the binding of poorly neutralizing anti-dengue mAb, cryo-EM and crystallography can be used to determine the binding determinants of antibodies taken from such a mAb panel. These results can inform further modelling and mutagenesis to engineer out these unwanted epitopes whilst preserving the EDE. Interestingly, our preliminary results with one of our stabilised dimers shows much reduced reactivity to anti-FLE mAb underscoring the feasibility of manipulating recognition of the EDE vs. FLE, which have overlapping epitopes (
[0751] 3. A universal dengue/zika immunogen. Structural characterization can be used to gain insight into the determinants of the bnAbs and their interactions with E from each of the four-dengue serotypes and of ZikaV. X-ray crystallography and cryo-EM can be used to analyse a selected broadly neutralizing anti-EDE mAb in complex with stabilized sE-mutants. Within the repertoire of anti-EDE mAb we have generated, some show restricted serotype cross-reactivity or even mono-specificity and these can be characterized to understand what determines broad specificity. A cryo-EM structure of mAb-2D22 in complex with a Denv2 virion reported by She-Mei Lok.sup.54 is informative in this respect; 2D22 requires an E-dimer to bind, is specific for serotype 2 viruses (i.e. does not show broad specificity) and has a footprint similar to that of the EDE-1 bnAbs that we have reported, except that it appears to contact more residues on domain III of E.
[0752] In summary, the results of this section can guide further mutagenesis for resurfacing the sE-dimer, helping to develop a single immunogen incorporating the identified cross-reactive elements of the EDE and eliminating those that can result in serotype specific reactivity. These resurfaced immunogens are considered to be useful for heterologous prime boost strategies that may be required to focus responses towards the EDE.
[0753] Finally, once an or most appropriate stabilized, resurfaced sE-dimer has been identified, this sequence may be used in attempts to produce VLPs lacking prM but presenting multiple copies of the corresponding E-dimer at the surface, to increase its immunogenicity. As an alternative to the development of E-only VLPs, self-assembling nanoparticles presenting stabilized sE-dimers on their surfaces may be developed, analogous to, for example, nanoparticles developed for HIV and influenza vaccine development.sup.53,55-58,66. Nanoparticles may be produced by either genetic fusion or chemical conjugation of sE-dimers to pre-existing particles, for example. The particles may comprise ferritin, for example. In the case of genetic fusion, a single chain dimer may be created to allow fusion to a wide variety of nanoparticles or fusion could be restricted to particles with suitable 2-fold symmetry axes, for example. In sum, there are numerous options for how to present stable sE-dimers on nanoparticles for improved immunogenicity and epitope-focusing; different potential avenues may be explored.
[0754] 5. Test immunogens in transgenic mice, for example fully human Ig mice. Transgenic mice useful in vaccine assessment have been developed, for example as described in Lee et al (2014) Nature Biotechnology Vol 32(4), 356-363. Such mice may, for example, have a completely normal immune system except the variable regions of the antibodies are human.
[0755] Using such a mouse model system is considered to be useful for a number of reasons: 1) Most importantly, such models, for example as described in Lee et al supra are probably the closest we can get to a preclinical model of human immunization in terms of the antibody response. 2) Primary immunoglobulin repertoires have diverged significantly between species, thus specific antibody responses in one species differ in both variable region usage therefore epitope selection, consequently extrapolating function from one species to another is unreliable. There is already evidence that murine antibody response to dengue differ from human, in particular antibodies to E domain III are quite dominant in the mouse but less so in humans. 3) Repertoires and fully human mAbs can be rapidly generated from immunized mice by deep sequencing, paired single cell cloning, network analysis and high-through-put expression respectively. 4) There is also the potential to generate further potent broadly neutralizing human anti-EDE mAb in the process, which may outperform those currently available.
[0756] Antigen can be delivered in a variety of different formats, which allows a throughput antigen testing far greater than could be justified in humans. The work may proceed via the following three phases:
[0757] a) High Throughput Polyclonal Analysis
[0758] This can involve the analysis of a large number of antigens (for example n=50, batched for operational efficiency) from which a subset can be selected and iterated further. For example, 5 disulphide stabilized mutants, 5 cavity filling mutants and 20 resurfaced mutant sE-dimers and 20 heterologous prime boost combinations can be examined. Since the number of different antigens is large the number of immunized mice may be limited to five per antigen. Antigen priming and two boosts with appropriate serial and terminal bleeds may be performed, for example. For maximum efficiency tissues can be banked from each animal in a form that it can be recovered and examined later, if required. A down-selection process can be followed based on polyclonal serum as follows: [0759] Polyclonal ELISA positive responses in 4/5 or 5/5 mice with titres>10.sup.−4 using native antigen. [0760] In vitro neutralization 50% titres of>10.sup.−3 [0761] Cross-reactivity of the responses between the 4 virus serotypes and Zika, for example [0762] Binding site analysis using mutant antigen VLP's and antibody competition assays.
[0763] b) Deep sequencing of antibody repertoire, mAb expression and functional screening. The 10 most effective immunization conditions may be selected for deep immune repertoire sequencing and mAb production from antigen sorted B-cells. A total Ig-heavy chain immune repertoire may be produced using NGS and high throughput methods may be used to produce approximately 500 mAb per immunogen, which may be tested for binding to sE-dimers and in neutralization assays. Common BCR solutions to dengue EDE binding may be determined by determining Ig-H&L family frequencies in 4/5 or 5/5 animals at frequencies greater than seen in non-immunized animals. A subset of transgenic mouse-generated mAbs, that represent different BCR evolutionary solutions but bind sE-dimer EDE may be produced in larger quantities for characterization in vitro and in vivo.
[0764] 6. In vivo neutralization. Mice deficient in type I and II interferon receptors (AG129) represent an in vivo model for DENV infection and pathogenesis.sup.59-62. Upon infection with DENV animals develop rapid viraemia in multiple organs.sup.63. Infection is associated with weight loss, thrombocytopenia and vascular leakag.sup.64,65. AG129 mice may be used to demonstrate the presence of neutralizing antibodies from the mouse immunizations described above by injecting serum or individual Kymouse mAbs (or cocktails of mAbs) shown to bind and neutralize DENY in vitro into AG129 mice prior to challenge with mouse adapted dengue-2 strain D2S 10.
[0765] 7. Prime boost strategies. Initial studies may inform 1) whether it is possible to attain a focused response to the EDE and 2) can bnAb responses be generated using single immunogens. We have described a number of strategies to achieve this such as the design of a single universal immunogen and the resurfacing of non-EDE related parts of the E-protein dimer to destroy the epitopes for unwanted responses such as those against the FLE. However, the difficulty of focusing a bnAb response to the EDE may mean that heterologous prime boost strategies may be required to achieve this.
[0766] Heterologous prime boost are considered to increase the focusing of responses on the EDE and drive broad reactivity. A variety of different experimental approaches can be used to achieve these objectives, for example: [0767] Use sE dimers from different DenV serotypes and from ZikaV in prime boost combinations to drive broad reactivity [0768] Use a fully resurfaced sE-dimer only containing the EDE in prime boost combination with wild type dimers. [0769] Prime boost strategies using recombinant sE-dimers and VLP's. [0770] Prime boost combinations of attenuated viruses with sE-dimers.
[0771] In conclusion, we have presented an exemplar plan for exploring the feasibility of a novel subunit vaccine for dengue, which is also considered to have utility for other flavivirus disease, for example zika disease. Despite progress with LATV it is not yet clear that this approach will deliver a safe and efficacious product that can be used in all age groups. Until then, preclinical development of alternative and potentially synergistic technologies to LATV should be pursued. A successful conclusion to this program is considered to lead to production of an immunogen which is suitable for use or further evaluation, for example for primate and early phase clinical evaluation.
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EXAMPLE 4
In Vivo Protection
[0838] Anti-EDE1 mAb clone 753(3)C10 (C10) was tested for its ability to confer protection from Zika infection in the AG129 mouse model. AG129 mice were obtained from B&K (Hull, UK) and were bred at the CBS facility at Imperial College. All animal experiments were performed in containment level 3 facilities as per the guidelines of the Ethical Committee of Imperial College, under the UK home office license. Virus stock was produced as described earlier and titrated on Vero cells prior to use in the mouse model. Female 129/Sv mice deficient in both interferon (IFN)-α/β and IFN-γ receptors (AG129 mice; female, 8-10 weeks of age) were administered purified human anti EDE-1 clone CIO or isotype control 2-8C at either 200 or 50 μg/mouse, intra-peritoneally (i.p; 200 μL) 24 h prior to infection with Zika virus (Brazilian strain PE243). Mice were infected intra-peritoneally with 1.2×10.sup.2 FFU/mouse of Zika PE243. Mice administered PBS alone were used as experimental controls. Mice were monitored by daily body weight measurements and development of virus-induced disease. Blood samples were collected at days 2 and 4 post infection. Plasma samples were titrated for viral loads using focus forming assays on Vero cell monolayers. Mice were euthanized prior to body weight loss nearing 20% and/or severe illness specified under the project license as humane endpoints. Percent original body weight was calculated based on the weight at day 0 just prior to infection. The body weight measurements are represented as mean +/− SEM of 3 mice in each experimental group. The experiment was performed once and showed protection afforded by monoclonal antibody C10.