Receptor-binding domain of MERS-CoV with optimal immunogen dosage and immunization interval protects human transgenic mice from MERS-CoV infection

ABSTRACT

Middle East respiratory syndrome (MERS) continues to raise worldwide concerns due to its pandemic potential. Increased MERS cases and no licensed MERS vaccines highlight the need to develop safe and effective vaccines against MERS. We have previously demonstrated that a receptor-binding domain (RBD) fragment containing residues 377–588 of MERS-coronavirus (MERS-CoV) spike protein is a critical neutralizing domain and an important vaccine target. Nevertheless, its optimal immunogen dosage and immunization interval, key factors for human-used vaccines that induce protective immunity, have never been investigated. In this study, we optimized these criteria using a recombinant MERS-CoV RBD protein fused with Fc (S377–588-Fc) and utilized the optimal immunization schedule to evaluate the protective efficacy of RBD against MERS-CoV infection in human dipeptidyl peptidase 4 transgenic (hDPP4-Tg) mice. Compared with one dose and 2 doses at 1-, 2-, and 3-week intervals, a regimen of 2 doses of this protein separated by an interval of 4 weeks induced the strongest antibody response and neutralizing antibodies against MERS-CoV infection, and maintained at a high level during the detection period. Notably, RBD protein at the optimal dosage and interval protected hDPP4-Tg mice against lethal MERS-CoV challenge, and the protection was positively correlated with serum neutralizing antibodies. Taken together, the optimal immunogen dosage and immunization interval identified in this study will provide useful guidelines for further development of MERS-CoV RBD-based vaccines for human use.

KEYWORDS:

• hDPP4-transgenic mice
• MERS-CoV
• neutralizing antibodies
• optimal immunogen dosage
• optimal immunization interval
• protection
• receptor-binding domain

Introduction

Middle East respiratory syndrome (MERS) caused by MERS coronavirus (MERS-CoV) continues to raise global concerns due to its pandemic potential. MERS-CoV was first reported in Saudi Arabia in 2012,¹ and it has since infected at least 1,888 individuals worldwide including 670 deaths (mortality rate ∼35%) in a total of 27 countries (http://www.who.int/emergencies/mers-cov/en/). Dromedary camels and bats are believed to be the important sources of zoonotic transmission of MERS-CoV, and the mechanisms driving bat-to-human transmission of MERS-CoV are being extensively explored.^2-7 MERS-CoV causes human infections after close contact with sick patients, resulting in illness in family clusters and healthcare workers,^8-11 and the virus once had significant spread in South Korea in 2015.¹² The continuous threat of MERS-CoV and the absence of licensed vaccines make it a high priority to develop safe and efficacious MERS vaccines.

The spike (S) protein of MERS-CoV plays critical roles in virus infection and pathogenesis. It consists of a globular S1 subunit responsible for cellular tropism and interaction with target cells, and a membrane-proximal S2 subunit which mediates fusion of virus and cell membranes.^13-15 Viral receptor-binding domain (RBD) in the S1 subunit binds the cellular receptor, human dipeptidyl peptidase 4 (hDPP4), mediating virus entry into target cells.^16-19 Therefore, RBD of MERS-CoV is an attractive target for the development of vaccines against MERS.

Indeed, MERS-CoV RBD induced strong neutralizing antibodies in experimental animals.^18,20-22 Several fragments containing different lengths of MERS-CoV S1 subunit are defined as RBD.^16-18,22,23 By comparing these fragments, we identified a fragment containing residues 377–588 of MERS-CoV RBD as a critical neutralizing domain (CND),^22,24-27 and, hence, a promising MERS vaccine candidate.

Since MERS-CoV does not infect wild-type mice, we have developed a transgenic mouse model expressing MERS-CoV's receptor hDPP4 (hDPP4-Tg).²⁸ The codon-optimized hDPP4 gene is transferred into the wild-type mice, and the generated Tg mice show global expression of hDPP4, including lung, trachea, spleen, kidney, and brain. The Tg mice are fully permissive to MERS-CoV infection, resulting in severe pneumonia and multi-organ damage, with viral replication being detected in lung, kidney, and brain. The MERS-CoV-infected Tg mice also exhibit constant weight loss and death. Therefore, the established hDPP4-expressing Tg mouse model has become an evaluable and convenient tool to evaluate the efficacy of MERS-CoV vaccines and anti-MERS-CoV therapeutic agents.^27,29,30

Adjuvants are widely used to improve the efficacy of subunit vaccines. After comparing several commercially available adjuvants, such as Freund's adjuvant, aluminum (alum), Monophosphoryl lipid A, Montanide ISA51 and MF59, we identify MF59 as a potent adjuvant to promote MERS-CoV RBD to induce higher titers of antibody responses, neutralizing antibodies, and protective immunity against MERS-CoV infection.²⁵ MF59 adjuvant has been approved for human use with influenza vaccines (https://www.cdc.gov/flu/protect/vaccine/adjuvant.htm),^31-33 and is evaluated in clinical trails with other vaccines against viruses such as HSV, HIV, HBV, and cytomegalovirus (CMV).^34-36 In addition to MF59, alum is also effective to trigger MERS-CoV RBD protein to elicit sufficient antibody responses and neutralizing antibodies against MERS-CoV.²⁵ Compared with MF59, alum is used in >80% of licensed human vaccines with a long-term history. Alum has high safety, strong antigen stabilization, and significant augmentation of high and long-lasting antibody titer.³⁴ Thus, MF59 and alum adjuvants are applicable for MERS-CoV RBD vaccines. Since MF59 and alum have been optimized for RBD proteins in wild-type mice and hDPP4-Tg mice, respectively,^25,27 they are used in the present study.

To achieve the best effect, optimization of vaccine immunogen dosage is a critical step to reduce dosage and increase population coverage, which will help determine the stockpile size of pandemic vaccines.³⁷ Optimization of immunization schedule is another crucial step since traditional immunization pathways require booster doses to maintain protective antibody concentrations, reinforcing the significance of optimizing immunization dose spacing. In this study, we aimed to optimize the immunization dosage and interval of a MERS-CoV RBD-based subunit vaccine (S377-588-Fc), and then utilize the optimal schedule to evaluate its protective efficacy in our established hDPP4-Tg mouse model.

Results

Two doses of S377-588-Fc separated by an interval of 4 weeks induced the highest IgG antibody responses when compared to mice receiving one or two doses at 1-, 2- or 3-week intervals

To optimize the dosage of immunogen, S377-588-Fc protein, and the interval between two immunizations, we immunized mice as described in Methods (Fig. 1A). Mice were boosted at 1-, 2-, 3-, and 4-week intervals, respectively, or not boosted, and sera were collected at 10 and 40 days post-last vaccination to detect IgG antibody responses.

Figure 1. Schematic diagrams of optimization and challenge schedules. (A) Optimization schedule. BALB/c mice (5 groups) were immunized with S377-588-Fc protein and either boosted once at 1-, 2-, 3-, or 4-week intervals, respectively, or not boosted, as described in Materials and Methods. Sera were collected from the immunized mice at 10 and 40 days after the last immunization and tested for IgG, IgG1 and Ig2a, as well as neutralizing antibodies. (B) Challenge schedule. The hDPP4-Tg mice were first immunized with S377-588-Fc protein, or PBS control, at the optimal doses and interval. Next, sera were collected before challenge for testing neutralizing antibodies and the challenged mice were evaluated for pathological effects and survival, as described in Materials and Methods.

In general, two doses of S377-588-Fc induced significantly higher titers of IgG antibodies than those induced with one dose (Fig. 2A–B). When two doses were administered, IgG antibodies elicited at an interval of 1 week were lower, or significantly lower, than those elicited at an interval of 2, 3 or 4 weeks, respectively. Correspondingly, IgG antibodies elicited at an interval of two weeks were significantly lower than those elicited at an interval of four weeks. Importantly, the 4-week interval induced the highest IgG titers in the sera collected at 10 days post-last immunization (Fig. 2A). Under this regimen, the potency of MERS-CoV S1-specific IgG antibody continuously increased and was maintained at high levels for at least 40 days post-last immunization. Among all groups, IgG antibodies elicited at an interval of 4 weeks were the highest and significantly higher than those induced at intervals of 1 and 2 weeks (Fig. 2B). Therefore, induction of the highest antibody responses in mice can be achieved by immunization consisting of two doses of S377-588-Fc protein spaced four weeks apart.

Figure 2. Antibody responses induced by S377-588-Fc at different doses and immunization intervals. Sera of mice immunized with S377-588-Fc were collected at 10 (A, C, E) and 40 (B, D, F) days post-last immunization and detected for MERS-CoV S1-specific IgG (A, B), IgG1 (C, D), and IgG2a (E, F) antibodies by ELISA. The antibody titers are presented as the endpoint dilutions that remain positively detectable, and the data are shown as mean titers ± standard deviation (SD) from five mice in each group. For (A) and (B), significant differences were revealed between 1-dose and four 2-dose groups, respectively (*); between 2-dose-1-week and 2-dose-3- or 4-week, respectively (^#); or between 2-dose-2-week and 2-dose-4-week (^▪). For (C), significant differences were observed between 1-dose or 2-dose-1-week and 2-dose-2-, 3- or 4-week, respectively (*); between 2-dose-2-week and 2-dose-3- or 4-week, respectively (^#); or between 2-dose-3-week and 2-dose-4-week (^▪). For (D) and (F), significant differences were revealed between 1-dose and the four 2-dose groups, respectively (*); between 2-dose-1-week and 2-dose-2-, 3- or 4-week, respectively (^#); or between 2-dose-2-week and 2-dose-3- or 4-week, respectively (^▪). For (E), significant differences were observed between 1-dose or 2-dose-1-week and 2-dose-2-, 3- or 4-week, respectively (*); or between 2-dose-2- or 3-week and 2-dose-4-week, respectively (^#). For (F), significant differences were also observed between 2-dose-3-week and 2-dose-4-week (^•).

Two doses of S377-588-Fc spaced 4 weeks apart induced the highest subtype antibody response among the groups receiving one and two doses at intervals of 1, 2, or 3 weeks

We tested IgG1 (T-helper 2: Th2) and IgG2a (Th1) subtypes in mouse sera. S377-588-Fc-induced IgG1 antibodies were higher, or significantly higher, when immunization was carried out in two doses, rather than one, reaching the highest level at an interval of 4 weeks. Additionally, significantly higher titers of IgG1 were revealed in sera when two doses were administered 3 or 4 weeks apart compared to those detected when two doses were administered 1 or 2 weeks apart. At an interval of 4 weeks, IgG1 titers were significantly higher than those detected when two doses were administered after 3 weeks (Fig. 2C). MERS-CoV S1-specific IgG2a antibodies raised against S377-588-Fc basically followed a similar trend. Two doses of S377-588-Fc administered 4 weeks apart elicited the highest titer of IgG2a antibody compared to the other groups administered one dose, or two doses 1, 2, or 3 weeks apart, respectively (Fig. 2E). High subtype antibodies were maintained for at least 40 days after the last immunization (Fig. 2D, F). Thus, a S377-588-Fc booster at 4 weeks is ideal since it allows the protein to induce strong Th1 (IgG2a)- and Th2 (IgG1)-mediated antibody responses.

Two doses of S377-588-Fc spaced 4 weeks apart induced the highest neutralizing antibodies among the groups receiving one or two doses at intervals of 1, 2, or 3 weeks

We first performed a MERS pseudovirus neutralization assay in hDPP4-expressing Huh-7 cells using sera collected at 10 and 40 days, respectively, post-last immunization. This method is safe, rapid and efficient, and does not require live MERS-CoV or BSL-3 laboratories.³⁸ As shown in Fig. 3A–B, S377-588-Fc at one dose failed to induce sufficient neutralizing antibodies against pseudotyped MERS-CoV expressing S protein, whereas such antibodies induced at two doses 4 weeks apart were the highest against infection of MERS pseudovirus, and significantly higher than those detected in all other groups. Significantly higher pseudovirus neutralizing antibodies were also manifested in mouse sera at an interval of 3 weeks with two doses when compared to one or two doses at intervals of 1 or 2 weeks, respectively, in the sera of mice collected at 10 days post-last immunization (Fig. 3A). Neutralizing antibodies increased dramatically following the regimen of two doses spaced 3 weeks apart, reached the highest level at 4 weeks apart, and maintained this level for at least 40 days post-last immunization (Fig. 3B).

Figure 3. Neutralizing antibodies induced by S377-588-Fc at different immunization doses and intervals. Neutralizing antibodies against MERS pseudovirus (A, B) and live MERS-CoV (C, D) infection in Huh-7 and Vero E6 cells, respectively, in mouse sera from 10 (A, C) and 40 (B, D) days post-last immunization. Pseudovirus neutralizing antibodies are expressed as 50% neutralizing antibody titers (NT₅₀), and live neutralizing antibody titers are expressed as the reciprocal of the highest dilution of sera that resulted in complete inhibition of virus-induced cytopathic effect (CPE) in at least 50% of the wells (NT₅₀). The data are presented as mean ± SD from five mice in each group. For (A) and (C), significant differences were revealed between 1-dose and 2-dose-2-, 3- or 4-week, or 2-dose-1-week and 2-dose-3- or 4-week, respectively (*); or between 2-dose-2-week and 2-dose-3- or 4-week, respectively (^#). For (A), significant differences were also revealed between 2-dose-3-week and 2-dose-4-week (^▪). For (B), significant differences were observed between 1-dose and the four 2-dose groups, respectively (*); between 2-dose-1-week and 2-dose-2-, 3- or 4-week, respectively (^#); between 2-dose-2-week and 2-dose-3- or 4-week, respectively (^▪); or between 2-dose-3-week and 2-dose-4-week (^•). For (D), significant differences were observed between 1-dose and the four 2-dose groups, respectively (*); or between 2-dose-1-week or 2-dose-2-week and 2-dose-3- or 4-week, respectively (^#).

A live MERS-CoV-based neutralization assay was then performed in Vero E6 cells to confirm the above results. As shown in Fig. 3C, while one dose of S377-588-Fc was unable to induce MERS-CoV-specific neutralizing antibodies, two doses of this protein spaced 4 weeks apart elicited the highest titer of such antibodies against MERS-CoV infection, which were significantly higher than those induced at one or two doses at intervals of 1 or 2 weeks, respectively. Significant differences were also revealed among the groups receiving 2 doses spaced 3 weeks apart and groups receiving one or two doses at intervals of 1 or 2 weeks, respectively, in the sera at 10 days post-last immunization (Fig. 3C). As expected, for an immunization regimen consisting of 2 doses spaced 3 or 4 weeks apart, high titers of neutralizing antibodies were maintained for at least 40 days post-last immunization (Fig. 3D).

Therefore, two doses of S377-588-Fc boosted at a 4-week interval induced the highest titer of neutralizing antibodies against MERS-CoV infection.

Two doses of S377-588-Fc spaced 4 weeks apart protected hDPP4-Tg mice from lethal challenge of MERS-CoV

We therefore established the 2-dose-4-week interval of RBD immunization as an optimal regimen inducing the highest neutralizing antibody responses. We then tested this schedule using hDPP4-Tg mice (Fig. 1B). As expected, S377-588-Fc protected mice against MERS-CoV infection, with slightly thickened alveolar walls in the lung tissues (Fig. 4Aa), as compared with those of normal control mice (Fig. 4Ab). In contrast, after MERS-CoV challenge, mice in the PBS control had significant lung damage with serious inflammatory cell infiltration, focal exudation and hemorrhage, as well as severe tissue edema (Fig. 4Ac).

Figure 4. Protective efficacy of S377-588-Fc in hDPP4-Tg mice with optimal immunization doses and intervals. (A) Detection of pathological changes in challenged mice. Mice were immunized with S377-588-Fc protein or PBS control, boosted once at a 4-week interval, challenged with MERS-CoV at 12 weeks post-boost, and then observed for pathological changes in lung tissues (Aa). Lung tissues from normal mice (Ab) and those injected with PBS and challenged with MERS-CoV (Ac) were included as controls. Shown are representative images of lung tissue sections stained with hematoxylin and eosin (H&E) and observed under light microscopy (100× magnification). (B) Detection of IgG antibody titers of mouse sera before MERS-CoV challenge. The antibody titers are presented as the endpoint dilutions that remain positively detectable. (C) Detection of neutralizing antibody titers (NT₅₀) of mouse sera against live MERS-CoV infection before MERS-CoV challenge. (D) Calculation of mouse survivals at 21 days after MERS-CoV challenge. For (B)-(D), M1-M6 indicates mouse numbers in each group.

Neutralizing antibodies induced by severe acute respiratory syndrome coronavirus (SARS-CoV) RBD protein were shown to protect against SARS-CoV infection.^39-41 Here we tested the association between MERS-CoV RBD-induced neutralizing antibodies and protection (Fig. 4B–D). Four S377-588-Fc-immunized mice that had higher IgG (Fig. 4B) and neutralizing antibody (Fig. 4C) titers (≥ 1:141) against MERS-CoV infection in their sera survived MERS-CoV challenge (Fig. 4D), while two of these mice with lower levels of IgG and neutralizing antibodies died (Fig. 4B–D). In contrast, control mice having only a background level of IgG and neutralizing antibodies did not survive (Fig. 4D). Therefore, MERS-CoV RBD-induced neutralizing antibodies are positively correlated with protection against MERS-CoV infection.

Discussion

MERS-CoV is an emerging infectious respiratory tract disease causing serious public health problems. Since vaccination is one of the most effective strategies to prevent and treat virus infection, exploration of a safe and efficacious vaccine is required to prevent further spread of MERS-CoV and its potential pandemic. We and others have proven that MERS-CoV RBD is an important target for vaccine development.^20-24 Our previous studies have demonstrated that a recombinant protein containing residues 377–588 of MERS-CoV RBD fused with human IgG-Fc (S377-588-Fc) induced potent neutralizing antibodies against MERS-CoV infection,^22,24-26 suggesting that vaccines based on this critical neutralizing domain fragment could be further developed as an ideal candidate to prevent MERS-CoV infection.

A key step in vaccine development involves the identification of an ideal immunogen dosage and immunization interval. A vaccine candidate capable of inducing high titers of antibody responses with minimal dosage is preferable for further development as it will significantly reduce the total cost. In addition, certain vaccines, such as adult tetanus-diphtheria toxoid [Td], pediatric diphtheria-tetanus toxoid [DT], and tetanus toxoid, may produce increased local or systemic reactions in some recipients when administered too frequently by the formation of antigen-antibody complexes.^42,43 Thus, administration based on an optimal schedule can decrease the incidence of such reactions without adversely affecting immune responses elicited by vaccines. According to the Advisory Committee for Immunization Practices (ACIP) General Recommendations on Immunization, most vaccines, such as hepatitis B, DTap, Hib, and influenza, have recommended vaccination intervals. Generally speaking, an immunization interval of 4 weeks is widely used in clinical trials of vaccines to prevent diseases. For example, individuals with measles usually receive an extra administration of MMR (measles, mumps and rubella) vaccine, separated by at least 28 days (http://www.cdc.gov/mmwr/preview/mmwrhtml/rr5102a1.htm). We have previously demonstrated the immunogenicity and/or protection of MERS-CoV RBD protein in mice and/or rabbits after three doses of immunization at a 3-week interval.^24-27 The present study further identified the immunogenicity of RBD vaccine in one dose and two doses at intervals of 1-, 2-, 3-, and 4-weeks. Here we aimed to develop a MERS vaccine able to elicit strong neutralizing antibody responses with minimal dosage (2 doses), long interval (4-weeks), yet be safe for individuals in all age groups who are susceptible to MERS-CoV infection. In the current status of no approved MERS vaccines for human use and the high cost and impracticality for conducting such optimization directly in humans, the optimized schedule in mice will be helpful for future clinical trials of MERS vaccines in terms of selection of appropriate antigen doses and immunization intervals, thus saving time and antigens for the vaccination and selection processes.

Our data demonstrated that two doses of MERS-CoV RBD (S377-588-Fc), including a boost dose at 4 weeks, induced strong IgG antibody responses. In comparison, a single dose of such vaccine could not elicit sufficient IgG antibodies. It is noted that the MERS-CoV S-specific IgG antibody titer kept increasing from day 10 to day 40 post-boost. Similar to humans, which show four subclasses of IgG (e.g., IgG1, IgG2, IgG3, IgG4), the mouse IgG subclasses are categorized as IgG1, IgG2a, IgG3, and IgG2b, correspondingly.⁴⁴ Both Th1 and Th2 cells promote humoral immune responses to vaccines. In mice, Th1 cells secret IL-2, INF-γ and TNF-β cytokines to produce IgG2a antibody, thus mediating cellular immunity, while Th2 cells secrete IL-4, IL-5, and IL-10 cytokines to facilitate B cells to produce IgG1 antibody, thus eliciting humoral immunity and regulating Th1 responses to restrain cellular immunity.⁴⁵ The IgG subtypes are largely influenced by the Th1/Th2 immune responses, and the ratio of IgG1 and IgG2a may reflect which Th type is predominant. However, it might be not appropriate to simply define each immune response as only one type (Th1 or Th2) or one type being superior to the other since some cytokines associated with Th1 or Th2 cells may function differently in different cases.⁴⁵ Here we noted that in addition to IgG, significantly higher titers of Th2-associated IgG1 and Th1-associated IgG2a antibodies were also induced in mice immunized with two doses of MERS-CoV RBD vaccine at an interval of 4 weeks compared to those subtype titers in mice immunized with one or two doses at intervals of 1, 2 or 3 weeks, respectively (Table 1), suggesting the importance of antibody responses in MERS-CoV RBD-based vaccines.

Table 1. Summary of antibody responses elicited by S377-588-Fc in BALB/c mice at different immunogen doses and immunization intervals.

	Immunogen dosage and immunization interval
Antibody response	1-dose	2-dose- 1-week	2-dose- 2-week	2-dose- 3-week	2-dose- 4-week
Serum samples collected 10 days after the last immunization
IgG	1,540	2,834	51,511	160,123	239,579
IgG1	14,468	29,971	79,198	413,559	500,498
IgG2a	<100	153	3,111	5,556	28,959
PsV-neutralizing Ab^*	35	38	421	1,022	1,358
Live virus-neutralizing Ab	20	20	133	640	1,493
Serum samples collected 40 days after the last immunization
IgG	7,988	32,115	105,042	192,138	387,640
IgG1	7,141	32,125	108,759	195,361	486,383
IgG2a	101	567	1,533	7,061	60,750
PsV-neutralizing Ab^*	22	818	1,291	3,848	7,148
Live virus-neutralizing Ab	27	187	320	1,067	1,067

* PsV, pseudotyped MERS-CoV

Instead of utilizing 3 doses of immunizations,^26,29 our findings revealed that a satisfactory neutralizing antibody response could be achieved for a regimen consisting of 2 doses of MERS-CoV RBD protein at a 4-week interval, similar to the typical regimen of clinically trialed vaccines against other viral diseases.^46,47 These neutralizing antibodies were able to keep at similar, or higher, levels after 40 days post-last immunization to potently neutralize infections from MERS pseudovirus and live MERS-CoV (Table 1). Importantly, the reduction of immunogen doses and extension of vaccination interval have afforded MERS-CoV RBD protein the ability to induce suitable neutralizing antibodies that were maintained for at least 4 months during the detection period to protect aged hDPP4-Tg mice against lethal MERS-CoV infection. This will be particularly beneficial not only to the young and adults but also to the elders who are infected with MERS-CoV. Currently, there is no defined threshold of neutralizing antibody titers in protection against MERS-CoV infection. While neutralizing antibody titers (NT₅₀) at 1:40 or 1:119 can be protective (unpublished data),²⁶ here we found that two mice at NT₅₀ ≤ 1:141 did not survive from lethal MERS-CoV challenge. This could be due partially to the different doses of infectious viruses used for the challenge as well as other factors such as the role of cellular immune responses in the protection. Nevertheless, data from this study demonstrate that neutralizing antibody titers are, in general, positively correlated with the protection against MERS-CoV infection, a phenomenon also revealed in other MERS-CoV vaccines,^27,48 and SARS-CoV RBD-based subunit vaccines.^49,50 Future studies should be carried out to increase RBD's neutralizing ability to improve protective efficacy by modifying its sequences using structure-based vaccine design,³⁰ or adjusting the RBD to form native conformational structures, as in other viral glycoproteins.⁵¹ Further studies could also be conducted to investigate RBD's protective efficacy after a boost dose of longer than 4 weeks.

MERS-CoV infects cells through two major steps: receptor binding and viral entry. We have demonstrated that MERS-CoV RBD-induced antibodies may efficiently block the binding of MERS-CoV RBD to the viral receptor DPP4, and that mutations of the key residues in the receptor-binding motif of the RBD reduce the production of antibody responses, including neutralizing antibodies, and significantly diminish the RBD-DPP4 binding.^18,26 Since the RBD with an optimal dosage and immunization interval identified in this study induced in the immunized mice a higher antibody titer with stronger neutralizing activity, demonstrating a great potential of these antibodies in inhibiting RBD-receptor binding, thus protecting against MERS-CoV infection.

To summarize, this study emphasizes the importance of immunogen doses and vaccination intervals in determining an appropriate immunization schedule, identifies an ideal interval and suitable dose for MERS-CoV RBD vaccines to induce effective neutralizing antibody responses in protecting against lethal MERS-CoV infection, and elucidates the potential for developing MERS vaccines targeting viral RBD as a promising subunit vaccine candidate. Taken together, the data in this report provide a solid scientific foundation and technical guidance for the design, optimization, and application of a safe and effective MERS subunit vaccine for clinical trials. The identified optimal immunization schedule (2-dose-4-week interval) may also be applied to vaccination against other enveloped viruses with class I glycoproteins.

Materials and methods

Ethics statement

Female BALB/c mice at 6–8 weeks and male and female hDPP4-Tg mice at 4 months were used. Animal studies were carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. Animal protocols were approved by the Committee on the Ethics of Animal Experiments of the New York Blood Center (Approval number: 194.17) and Beijing Institute of Microbiology and Epidemiology (Permit Number: PMB15-0012).

Construction, expression and purification of recombinant proteins

Construction, expression and purification of recombinant MERS-CoV RBD and S1 proteins were described previously.^18,22,24,25 Briefly, S377-588-Fc was constructed by fusion of MERS-CoV RBD (residues 377–588) with human IgG-Fc, expressed in 293T cell culture supernatant, and purified by Protein A affinity chromatography (GE Healthcare). MERS-CoV S1 (residues 18–725) containing a C-terminal His₆ (S1-His) was expressed in the 293T cell culture supernatant and purified by Ni-NTA Superflow (Qiagen).

Mouse immunization and sample collection

This was performed following our previously described protocols with some modifications.^18,22,24,25 Briefly, BALB/c mice were s.c. immunized with S377-588-Fc (10 μg/100 μl/mouse) in the presence of MF59 adjuvant and boosted either once at 1-, 2-, 3-, or 4-week intervals, respectively, or not boosted. Sera were collected at 10 and 40 days post-last vaccination and measured for MERS-CoV S1-specific IgG, subtypes, and neutralizing antibodies.

ELISA

ELISA was performed to evaluate MERS-CoV S1-specific antibody responses in mouse sera as previously described.^18,22,24,25 Briefly, ELISA plates were coated with MERS-CoV S1-His protein overnight at 4°C and blocked with 2% non-fat milk for 2 h at 37°C. Serially diluted mouse sera were added to the plates and incubated for 1 h at 37°C, followed by four washes with PBST. Horseradish peroxidase (HRP)-conjugated anti-mouse IgG (1:3,000, GE Healthcare), IgG1 (1:2,000) or IgG2a (1:5,000) (Invitrogen) antibodies were added to the plates and incubated for 1 h at 37°C. The substrate 3,3′,5,5′-tetramethylbenzidine (TMB, Invitrogen) was added to the plates, and the reaction was stopped by H₂SO₄ (1 N). The absorbance at 450 nm (A450) was measured by ELISA plate reader (Tecan).

Pseudovirus neutralization assay

Neutralizing antibodies were detected in mouse sera using a pseudovirus neutralization assay as previously described.^38,52,53 Briefly, 293T cells were co-transfected with a plasmid encoding Env-defective, luciferase-expressing HIV-1 genome (pNL4-3.luc.RE) and a plasmid encoding MERS-CoV (EMC-2012 strain) S protein using the Calcium Phosphate Method. Supernatants containing MERS pseudovirus were harvested 72 h post-transfection and used for single-cycle infection. MERS pseudovirus was incubated with serially diluted mouse sera at 37°C for 1 h before adding to Huh-7 cells, followed by addition of fresh medium 24 h later, and culture of the cells for an additional 48 h. Cells were then lysed using cell lysis buffer (Promega) and transferred to 96-well luminometer plates. Luciferase substrate (Promega) was added to the cell lysates, and relative luciferase activity was measured in an Infinite® 200 PRO Luminator (Tecan). MERS pseudovirus neutralization was calculated⁵⁴ and expressed as NT_50.

Live MERS-CoV neutralization assay

Neutralizing antibodies were confirmed using a standard microneutralization assay as previously described.^22,24,25,38 Briefly, serially diluted mouse sera were incubated for 1 h at room temperature with ∼100 infectious MERS-CoV (EMC-2012 strain) before addition to Vero E6 cells and culture for 72 h at 37°C. Neutralizing antibodies were assessed in mouse sera by determining the presence or absence of CPE. Neutralizing antibody titers were calculated as the reciprocal of the highest dilution of sera that completely inhibited virus-induced CPE in at least 50% of the wells (NT₅₀).

Animal challenge experiments

Using our established MERS-CoV-susceptible small animal model, hDPP4-Tg mice,²⁸ the optimal immunogen dosage and immunization interval identified above were applied for the challenge studies as previously described.²⁷ Briefly, mice were immunized with S377-588-Fc protein (5 μg/mouse) and alum adjuvant, boosted once at 4 weeks, challenged with the lethal dose of MERS-CoV (EMC-2012 strain, 10⁴ TCID50) 12 weeks post-boost, and investigated for 3 weeks to detect pathological changes and survival. The correlation between neutralizing antibodies and mouse survival was compared and recorded.

Statistical analysis

Values are presented as mean with SD. Statistical significance among different immunization groups was calculated by Student's t-test using GraphPad Prism statistical software. P values less than 0.05 were considered statistically significant.

Disclosure of potential conflicts of interest

No potential conflicts of interest were disclosed.

Funding

This study was supported by NIH grants (R01AI098775, U01AI124260, R21AI109094, and R21 AI128311) to LD and SJ, the National Program of Infectious Disease Fund of China (2014ZX10004001004) to YZ, and the National Key Research and Development Program of China (2016YFC1201000) to SJ.