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. 2018 Aug 8;4(8):eaas9667.
doi: 10.1126/sciadv.aas9667. eCollection 2018 Aug.

Chimeric camel/human heavy-chain antibodies protect against MERS-CoV infection

V Stalin Raj  1 , Nisreen M A Okba  1 , Javier Gutierrez-Alvarez  2 , Dubravka Drabek  3 , Brenda van Dieren  4 , W Widagdo  1 , Mart M Lamers  1 , Ivy Widjaja  4 , Raul Fernandez-Delgado  2 , Isabel Sola  2 , Albert Bensaid  5 , Marion P Koopmans  1 , Joaquim Segalés  6   7 , Albert D M E Osterhaus  8   9 , Berend Jan Bosch  4 , Luis Enjuanes  2 , Bart L Haagmans  1
Affiliations

Chimeric camel/human heavy-chain antibodies protect against MERS-CoV infection

V Stalin Raj et al. Sci Adv. .

Abstract

Middle East respiratory syndrome coronavirus (MERS-CoV) continues to cause outbreaks in humans as a result of spillover events from dromedaries. In contrast to humans, MERS-CoV-exposed dromedaries develop only very mild infections and exceptionally potent virus-neutralizing antibody responses. These strong antibody responses may be caused by affinity maturation as a result of repeated exposure to the virus or by the fact that dromedaries-apart from conventional antibodies-have relatively unique, heavy chain-only antibodies (HCAbs). These HCAbs are devoid of light chains and have long complementarity-determining regions with unique epitope binding properties, allowing them to recognize and bind with high affinity to epitopes not recognized by conventional antibodies. Through direct cloning and expression of the variable heavy chains (VHHs) of HCAbs from the bone marrow of MERS-CoV-infected dromedaries, we identified several MERS-CoV-specific VHHs or nanobodies. In vitro, these VHHs efficiently blocked virus entry at picomolar concentrations. The selected VHHs bind with exceptionally high affinity to the receptor binding domain of the viral spike protein. Furthermore, camel/human chimeric HCAbs-composed of the camel VHH linked to a human Fc domain lacking the CH1 exon-had an extended half-life in the serum and protected mice against a lethal MERS-CoV challenge. HCAbs represent a promising alternative strategy to develop novel interventions not only for MERS-CoV but also for other emerging pathogens.

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Figures

Fig. 1
Fig. 1. Schematic overview of VHH identification by direct cloning using bone marrow from immunized dromedary camels.
Immunized dromedary camels were anesthetized, and bone marrow aspirations were performed. After total RNA isolation and first-strand cDNA synthesis, VHH genes were amplified and cloned into a prokaryotic expression vector (pMES4) and transformed into E. coli WK6. Individual clones were grown overnight in 96-deep-well plates, during which they expressed the VHHs in the periplasm. Next, crude VHHs were released from the periplasm by freeze-thawing the bacterial pellet. Crude VHHs were used for immunofluorescent staining on virus-infected cells. Immunofluorescent positive clones were further characterized for their genetic makeup, specificity, and potency by sequencing, antigen-specific enzyme-linked immunosorbent assay (ELISA), virus neutralization assay, epitope mapping, and structural analysis. Finally, potent VHHs were produced as camel/human chimeric single chain–only antibodies.
Fig. 2
Fig. 2. Identification of VHHs directed against the spike (S) protein of MERS-CoV.
(A) Virus-neutralizing antibody titers (VNT) of sera from two dromedary camels immunized with MVA expressing the MERS-CoV-S (MVA-S) and challenged with MERS-CoV. (B) Immunofluorescent staining of MERS-CoV–infected Huh-7 cells with crude VHHs. Each square represents staining of an individual VHH. (C) Immunofluorescent staining of MERS-CoV–infected Huh-7 cells with rabbit serum (anti–MERS-CoV) or crude VHHs and overlay. (D) Correlation of the S1-specific ELISA and the RBD-specific ELISA for the 46 MERS-CoV–neutralizing VHHs (red dots) and control VHHs indicated as blue dots (Spearman correlation r = 0.9258; P < 0.0001; 95% confidence interval, 0.8677 to 0.9589). OD, optical density.
Fig. 3
Fig. 3. MERS-CoV–neutralizing efficacy of monomeric VHHs and chimeric antibodies on Huh-7 cells.
MERS-CoV (EMC isolate) was incubated with either VHHs (monomer), chimeric antibodies, or controls at various concentrations for 1 hour and then the mix was transferred on Huh-7 cells. Cells were fixed 8 hours after infection and stained using rabbit polyclonal antibodies. The PRNT titer was calculated on the basis of a 50% or greater reduction of infected cells (PRNT50). (A) PRNT assay for VHH monomer. (B) PRNT for camel/human chimeric heavy-chain antibodies. Experiments were performed at least two times in triplicate, data from an experiment were presented, and error bars show SEM. (C) MERS-CoV S1 ELISA using different HCAbs. The optical density at 450 nm was presented in triplicate, with error bars showing SEM.
Fig. 4
Fig. 4. Effect of MERS-CoV RBD residue substitution on VHH binding.
Binding efficiency of VHHs to the wild-type and mutant forms of viral spike glycoprotein was analyzed by ELISA. The binding efficiency was calculated on the basis of optical density (OD450) of mutant protein versus that of the wild-type spike. (A) Anti-human IgG polyclonal antibodies were used to corroborate equivalent coating of the S1-hFc variants. (B) One irrelevant VHH (VHH-p2E6) lacked binding to wild-type and mutant proteins. (C) VHH-1. (D) VHH-4. (E) VHH-83. (F) VHH-101.
Fig. 5
Fig. 5. Prophylactic efficacy of HCAb-83 in K18 mice challenged with a lethal dose of MERS-CoV.
K18 mice (n = 9 per group) were injected intraperitoneally with HCAb-83 (20 or 200 μg per mouse) 6 hours before challenge with 105 TCID50 (median tissue culture infectious dose) of MERS-CoV (EMC isolate). HCAb-p2E6 was injected as a negative control (n = 9). Mice were monitored daily for (A) weight loss and (B) mortality. Weight loss is expressed as a percentage of the initial weight. Lungs were collected at days 2, 4, and 8 (n = 3 per time point) or from mice that died in between and were processed to asses gross pathology (C and D) and histopathological changes (E to G). Gross pathology of one representative animal that died at day 7 when treated with HCAb-p2E6 is indicated by a green arrow (C, right). Lung sections were stained with hematoxylin and eosin. Asterisk indicates alveolar edema. (H) MERS-CoV viral titer quantitation of infected lungs at days 2, 4, and 8 (n = 3 per time point) after infection (n = 3 mice per time point); one-way ANOVA. *P < 0.05. ns, not significant.
Fig. 6
Fig. 6. Pharmacokinetics of HCAb-83 in K18 transgenic mice.
(A) MERS-CoV PRNT performed on sera of mice collected 2 days after treatment with VHH-83, HCAb-83, or controls. The PRNT titer was calculated on the basis of a 90% reduction in the infected cell counts. Statistically significant differences were observed between groups HCAb-p2E6 200 μg, HCAb-83 200 μg, and HCAb-83 20 μg (one-way ANOVA test, *P < 0.05). (B) Detection of HCAbs in the sera of HCAb-83–treated mice at various time points using ELISA. N.D., not determined.
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