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The outbreak of Coronavirus Disease 2019 (COVID-19) has posed a serious threat to global public health, calling for the development of safe and effective prophylactics and therapeutics against infection of its causative agent, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), also known as 2019 novel coronavirus (2019-nCoV). The CoV spike (S) protein plays the most important roles in viral attachment, fusion and entry, and serves as a target for development of antibodies, entry inhibitors and vaccines. Here, we identified the receptor-binding domain (RBD) in SARS-CoV-2 S protein and found that the RBD protein bound strongly to human and bat angiotensin-converting enzyme 2 (ACE2) receptors. SARS-CoV-2 RBD exhibited significantly higher binding affinity to ACE2 receptor than SARS-CoV RBD and could block the binding and, hence, attachment of SARS-CoV-2 RBD and SARS-CoV RBD to ACE2-expressing cells, thus inhibiting their infection to host cells. SARS-CoV RBD-specific antibodies could cross-react with SARS-CoV-2 RBD protein, and SARS-CoV RBD-induced antisera could cross-neutralize SARS-CoV-2, suggesting the potential to develop SARS-CoV RBD-based vaccines for prevention of SARS-CoV-2 and SARS-CoV infection.

The current COVID-19 pandemic has led to a devastating impact across the world. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) (the virus causing COVID-19) is known to use the receptor-binding domain (RBD) at viral surface spike (S) protein to interact with the angiotensin-converting enzyme 2 (ACE2) receptor expressed on many human cell types. The RBD-ACE2 interaction is a crucial step to mediate the host cell entry of SARS-CoV-2. Recent studies indicate that the ACE2 interaction with the SARS-CoV-2 S protein has a higher affinity than its binding with the structurally identical S protein of SARS-CoV-1, the virus causing the 2002-2004 SARS outbreak. However, the biophysical mechanism behind such binding affinity difference is unclear. This study utilizes combined single-molecule force spectroscopy and steered molecular dynamics (SMD) simulation approaches to quantify the specific interactions between SARS-CoV-2 or SARS-CoV-1 RBD and ACE2. Depending on the loading rates, the unbinding forces between SARS-CoV-2 RBD and ACE2 range from 70 to 105 pN and are 30-40% higher than those of SARS-CoV-1 RBD and ACE2 under similar loading rates. SMD results indicate that SARS-CoV-2 RBD interacts with the N-linked glycan on Asn90 of ACE2. This interaction is mostly absent in the SARS-CoV-1 RBD-ACE2 complex. During the SMD simulations, the extra RBD-N-glycan interaction contributes to a greater force and prolonged interaction lifetime. The observation is confirmed by our experimental force spectroscopy study. After removing N-linked glycans on ACE2, its mechanical binding strength with SARS-CoV-2 RBD decreases to a similar level of the SARS-CoV-1 RBD-ACE2 interaction. Together, the study uncovers the mechanism behind the difference in ACE2 binding between SARS-CoV-2 and SARS-CoV-1 and could help develop new strategies to block SARS-CoV-2 entry.

SARS-CoV-2-caused COVID-19 cases are growing globally, calling for developing effective therapeutics to control the current pandemic. SARS-CoV-2 and SARS-CoV recognize angiotensin-converting enzyme 2 (ACE2) receptor via the receptor-binding domain (RBD). Here, we identified six SARS-CoV RBD-specific neutralizing monoclonal antibodies (nAbs) that cross-reacted with SARS-CoV-2 RBD, two of which, 18F3 and 7B11, neutralized SARS-CoV-2 infection. 18F3 recognized conserved epitopes on SARS-CoV and SARS-CoV-2 RBDs, whereas 7B11 recognized epitopes on SARS-CoV RBD not fully conserved in SARS-CoV-2 RBD. The 18F3-recognizing epitopes on RBD did not overlap with the ACE2-binding sites, whereas those recognized by 7B11 were close to the ACE2-binding sites, explaining why 7B11 could, but 18F3 could not, block SARS-CoV or SARS-CoV-2 RBD binding to ACE2 receptor. Our study provides an alternative approach to prevent SARS-CoV-2 infection using anti-SARS-CoV nAbs., Highlights • Identified six anti-SARS-CoV-RBD mAbs with cross-reactivity against SARS-CoV-2 RBD. • Two SARS-CoV RBD-specific mAbs had cross-neutralizing activity against SARS-CoV-2. • 18F3 mAb recognized conserved epitopes on SARS-CoV and SARS-CoV-2 RBDs. • 7B11 mAb blocked binding of SARS-CoV and SARS-CoV-2 RBDs to ACE2 receptor.

As vaccine-induced non-neutralizing antibodies may cause antibody-dependent enhancement of Zika virus (ZIKV) infection, we test a vaccine that induces only specific cytotoxic T lymphocytes (CTLs) without specific antibodies. We construct a DNA vaccine expressing a ubiquitinated and rearranged ZIKV non-structural protein 3 (NS3). The protein is immediately degraded and processed in the proteasome for presentation via major histocompatibility complex (MHC) class I for CTL generation. We immunize Ifnar1-/- adult mice with the ubiquitin/NS3 vaccine, impregnate them, and challenge them with ZIKV. Our data show that the vaccine greatly reduces viral titers in reproductive organs and other tissues of adult mice. All mice immunized with the vaccine survived after ZIKV challenge. The vaccine remarkably reduces placenta damage and levels of pro-inflammatory cytokines, and it fully protects fetuses from damage. CD8+ CTLs are essential in protection, as demonstrated via depletion experiments. Our study provides a strategy to develop safe and effective vaccines against viral infections.

Vaccination is one of the most successful strategies to prevent human infectious diseases. Combinatorial adjuvants have gained increasing interest as they can stimulate multiple immune pathways and enhance the vaccine efficacy of subunit vaccines. We investigated the adjuvanticity of Aluminum (alum) in combination with rASP-1, a protein adjuvant, using the Middle East respiratory syndrome coronavirus MERS-CoV receptor-binding-domain (RBD) vaccine antigen. A highly enhanced anti-MERS-CoV neutralizing antibody response was induced when mice were immunized with rASP-1 and the alum-adjuvanted RBD vaccine in two separate injection sites as compared to mice immunized with RBD + rASP-1 + alum formulated into a single inoculum. The antibodies produced also significantly inhibited the binding of RBD to its cell-associated receptor. Moreover, immunization with rASP-1 co-administered with the alum-adjuvanted RBD vaccine in separate sites resulted in an enhanced frequency of TfH and GC B cells within the draining lymph nodes, both of which were positively associated with the titers of the neutralizing antibody response related to anti-MERS-CoV protective immunity. Our findings not only indicate that this unique combinatorial adjuvanted RBD vaccine regimen improved the immunogenicity of RBD, but also point to the importance of utilizing combinatorial adjuvants for the induction of synergistic protective immune responses.

Middle East respiratory syndrome coronavirus (MERS-CoV) has infected at least 2040 patients and caused 712 deaths since its first appearance in 2012, yet neither pathogen-specific therapeutics nor approved vaccines are available. To address this need, we are developing a subunit recombinant protein vaccine comprising residues 377–588 of the MERS-CoV spike protein receptor-binding domain (RBD), which, when formulated with the AddaVax adjuvant, it induces a significant neutralizing antibody response and protection against MERS-CoV challenge in vaccinated animals. To prepare for the manufacture and first-in-human testing of the vaccine, we have developed a process to stably produce the recombinant MERS S377-588 protein in Chinese hamster ovary (CHO) cells. To accomplish this, we transfected an adherent dihydrofolate reductase-deficient CHO cell line (adCHO) with a plasmid encoding S377-588 fused with the human IgG Fc fragment (S377-588-Fc). We then demonstrated the interleukin-2 signal peptide-directed secretion of the recombinant protein into extracellular milieu. Using a gradually increasing methotrexate (MTX) concentration to 5 μM, we increased protein yield by a factor of 40. The adCHO-expressed S377-588-Fc recombinant protein demonstrated functionality and binding specificity identical to those of the protein from transiently transfected HEK293T cells. In addition, hCD26/dipeptidyl peptidase-4 (DPP4) transgenic mice vaccinated with AddaVax-adjuvanted S377-588-Fc could produce neutralizing antibodies against MERS-CoV and survived for at least 21 days after challenge with live MERS-CoV with no evidence of immunological toxicity or eosinophilic immune enhancement. To prepare for large scale-manufacture of the vaccine antigen, we have further developed a high-yield monoclonal suspension CHO cell line.

Highly pathogenic influenza viruses continue to cause serious threat to public health due to their pandemic potential, calling for an urgent need to develop effective, safe, convenient, and universal vaccines against influenza virus infection. In this study, we constructed two recombinant protein vaccines, 2H5M2e-2H7M2e-H5FP-H7FP (hereinafter M2e-FP-1) and 2H5M2e-H5FP-2H7M2e-H7FP (hereinafter M2e-FP-2), by respectively linking highly conserved sequences of two molecules of ectodomain of M2 (M2e) and one molecule of fusion peptide (FP) epitope of hemagglutinin (HA) of H5N1 and H7N9 influenza viruses in different orders. The Escherichia coli-expressed M2e-FP-1 and M2e-FP-2 proteins induced similarly high-titer M2e-FP-specific antibodies in the immunized mice. Importantly, both proteins were able to prevent lethal challenge of heterologous H1N1 influenza virus, with significantly reduced viral titers and alleviated pathological changes in the lungs, as well as increased body weight and complete survivals, in the challenge mice. Taken together, our study demonstrates that highly conserved M2e and FP epitope of HA of H5N1 and H7N9 influenza viruses can be used as important targets for development of safe and economical universal influenza vaccines, and that the position of H7N9 M2e and H5N1 HA epitope sequences in the vaccine components has no significant effects on the immunogenicity and efficacy of M2e-FP-based subunit vaccines.

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.

Zika virus (ZIKV) infection during pregnancy leads to severe congenital Zika syndrome, which includes microcephaly and other neurological malformations. No therapeutic agents have, so far, been approved for the treatment of ZIKV infection in humans, as such, there is a need for a continuous effort to develop effective and safe antiviral drugs to treat ZIKV-caused diseases. After screening a natural product library, we have herein identified four natural products with anti-ZIKV activity in Vero E6 cells, including gossypol, curcumin, digitonin, and conessine. Except for curcumin, the other three natural products have not been reported before to have anti-ZIKV activity. Among them, gossypol exhibited the strongest inhibitory activity against almost all 10 ZIKV strains tested, including six recent epidemic human strains. The mechanistic study indicated that gossypol could neutralize ZIKV infection by targeting the envelope protein domain III (EDIII) of ZIKV. In contrast, the other natural products inhibited ZIKV infection by targeting the host cell or cell-associated entry and replication stages of ZIKV. A combination of gossypol with any of the three natural products identified in this study, as well as with bortezomib, a previously reported anti-ZIKV compound, exhibited significant combinatorial inhibitory effects against three ZIKV human strains tested. Importantly, gossypol also demonstrated marked potency against all four serotypes of dengue virus (DENV) human strains in vitro. Taken together, this study indicates the potential for further development of these natural products, particularly gossypol, as the lead compound or broad-spectrum inhibitors against ZIKV and other flaviviruses, such as DENV.

Viral subunit vaccines generally show low efficacy. In this study, we revealed an intrinsic limitation of subunit vaccine designs: artificially exposed surfaces of subunit vaccines contain epitopes unfavorable for vaccine efficacy. More specifically, we identified an epitope on Zika virus (ZIKV) envelope protein domain III (EDIII) that is buried in the full-length envelope protein but becomes exposed in recombinant EDIII. We further shielded this epitope with a glycan, and the resulting mutant EDIII vaccine demonstrated significantly enhanced efficacy over the wild-type EDIII vaccine in protecting animal models from ZIKV infections. Therefore, the intrinsic limitation of subunit vaccines can be overcome through shielding these artificially exposed unfavorable epitopes. The engineered EDIII vaccine generated in this study is a promising vaccine candidate that can be further developed to battle ZIKV infections., Zika virus (ZIKV) infection in pregnant women can lead to fetal deaths and malformations. We have previously reported that ZIKV envelope protein domain III (EDIII) is a subunit vaccine candidate with cross-neutralization activity; however, like many other subunit vaccines, its efficacy is limited. To improve the efficacy of this subunit vaccine, we identified a nonneutralizing epitope on ZIKV EDIII surrounding residue 375, which is buried in the full-length envelope protein but becomes exposed in recombinant EDIII. We then shielded this epitope with an engineered glycan probe. Compared to the wild-type EDIII, the mutant EDIII induced significantly stronger neutralizing antibodies in three mouse strains and also demonstrated significantly improved efficacy by fully protecting mice, particularly pregnant mice and their fetuses, against high-dose lethal ZIKV challenge. Moreover, the mutant EDIII immune sera significantly enhanced the passive protective efficacy by fully protecting mice against lethal ZIKV challenge; this passive protection was positively associated with neutralizing antibody titers. We further showed that the enhanced efficacy of the mutant EDIII was due to the shielding of the immunodominant nonneutralizing epitope surrounding residue 375, which led to immune refocusing on the neutralizing epitopes. Taken together, the results of this study reveal that an intrinsic limitation of subunit vaccines is their artificially exposed immunodominant nonneutralizing epitopes, which can be overcome through glycan shielding. Additionally, the mutant ZIKV protein generated in this study is a promising subunit vaccine candidate with high efficacy in preventing ZIKV infections in mice. IMPORTANCE Viral subunit vaccines generally show low efficacy. In this study, we revealed an intrinsic limitation of subunit vaccine designs: artificially exposed surfaces of subunit vaccines contain epitopes unfavorable for vaccine efficacy. More specifically, we identified an epitope on Zika virus (ZIKV) envelope protein domain III (EDIII) that is buried in the full-length envelope protein but becomes exposed in recombinant EDIII. We further shielded this epitope with a glycan, and the resulting mutant EDIII vaccine demonstrated significantly enhanced efficacy over the wild-type EDIII vaccine in protecting animal models from ZIKV infections. Therefore, the intrinsic limitation of subunit vaccines can be overcome through shielding these artificially exposed unfavorable epitopes. The engineered EDIII vaccine generated in this study is a promising vaccine candidate that can be further developed to battle ZIKV infections.

Middle East respiratory syndrome (MERS) coronavirus (MERS-CoV), an infectious coronavirus first reported in 2012, has a mortality rate greater than 35%. Therapeutic antibodies are key tools for preventing and treating MERS-CoV infection, but to date no such agents have been approved for treatment of this virus. Nanobodies (Nbs) are camelid heavy chain variable domains with properties distinct from those of conventional antibodies and antibody fragments. We generated two oligomeric Nbs by linking two or three monomeric Nbs (Mono-Nbs) targeting the MERS-CoV receptor-binding domain (RBD), and compared their RBD-binding affinity, RBD–receptor binding inhibition, stability, and neutralizing and cross-neutralizing activity against MERS-CoV. Relative to Mono-Nb, dimeric Nb (Di-Nb) and trimeric Nb (Tri-Nb) had significantly greater ability to bind MERS-CoV RBD proteins with or without mutations in the RBD, thereby potently blocking RBD–MERS-CoV receptor binding. The engineered oligomeric Nbs were very stable under extreme conditions, including low or high pH, protease (pepsin), chaotropic denaturant (urea), and high temperature. Importantly, Di-Nb and Tri-Nb exerted significantly elevated broad-spectrum neutralizing activity against at least 19 human and camel MERS-CoV strains isolated in different countries and years. Overall, the engineered Nbs could be developed into effective therapeutic agents for prevention and treatment of MERS-CoV infection.

Middle East respiratory syndrome coronavirus (MERS-CoV) is continuously spreading and causing severe and fatal acute respiratory disease in humans. Prophylactic and therapeutic strategies are therefore urgently needed to control MERS-CoV infection. Here, we generated a humanized monoclonal antibody (mAb), designated hMS-1, which targeted the MERS-CoV receptor-binding domain (RBD) with high affinity. hMS-1 significantly blocked MERS-CoV RBD binding to its viral receptor, human dipeptidyl peptidase 4 (hDPP4), potently neutralized infection by a prototype MERS-CoV, and effectively cross-neutralized evolved MERS-CoV isolates through recognizing highly conserved RBD epitopes. Notably, single-dose treatment with hMS-1 completely protected hDPP4 transgenic (hDPP4-Tg) mice from lethal infection with MERS-CoV. Taken together, our data suggest that hMS-1 might be developed as an effective immunotherapeutic agent to treat patients infected with MERS-CoV, particularly in emergent cases.

Zika virus (ZIKV) infection during pregnancy can cause significant problems, particularly congenital Zika syndrome. Nevertheless, the potential deleterious consequences and associated mechanisms of transfusion-transmitted ZIKV infection on pregnant individuals and their fetuses and babies have not been investigated. Here we examined transmissibility of ZIKV through blood transfusion in ZIKV-susceptible pregnant A129 mice. Our data showed that transfused-transmitted ZIKV at the early infection stage led to significant viremia and broad tissue tropism in the pregnant recipient mice, which were not seen in those transfused with ZIKV-positive (ZIKV+) plasma at later infection stages. Importantly, pregnant mice transfused with early-stage, but not later stages, ZIKV+ plasma also exhibited severe placental infection with vascular damage and apoptosis, fetal infection and fetal damage, accompanied by fetal and pup death. Overall, this study suggests that transfusion-related transmission of ZIKV during initial stage of infection, which harbors high plasma viral titers, can cause serious adverse complications in the pregnant recipients and their fetuses and babies.

The newly emerged Middle East respiratory syndrome coronavirus (MERS-CoV) continues to infect humans and camels, calling for efficient, cost-effective, and broad-spectrum strategies to control its spread. Nanobodies (Nbs) are single-domain antibodies derived from camelids and sharks and are potentially cost-effective antivirals with small size and great expression yield. In this study, we developed a novel neutralizing Nb (NbMS10) and its human-Fc-fused version (NbMS10-Fc), both of which target the MERS-CoV spike protein receptor-binding domain (RBD). We further tested their receptor-binding affinity, recognizing epitopes, cross-neutralizing activity, half-life, and efficacy against MERS-CoV infection. Both Nbs can be expressed in yeasts with high yield, bind to MERS-CoV RBD with high affinity, and block the binding of MERS-CoV RBD to the MERS-CoV receptor. The binding site of the Nbs on the RBD was mapped to be around residue Asp539, which is part of a conserved conformational epitope at the receptor-binding interface. NbMS10 and NbMS10-Fc maintained strong cross-neutralizing activity against divergent MERS-CoV strains isolated from humans and camels. Particularly, NbMS10-Fc had significantly extended half-life in vivo; a single-dose treatment of NbMS10-Fc exhibited high prophylactic and therapeutic efficacy by completely protecting humanized mice from lethal MERS-CoV challenge. Overall, this study proves the feasibility of producing cost-effective, potent, and broad-spectrum Nbs against MERS-CoV and has produced Nbs with great potentials as anti-MERS-CoV therapeutics.IMPORTANCE Therapeutic development is critical for preventing and treating continual MERS-CoV infections in humans and camels. Because of their small size, nanobodies (Nbs) have advantages as antiviral therapeutics (e.g., high expression yield and robustness for storage and transportation) and also potential limitations (e.g., low antigen-binding affinity and fast renal clearance). Here, we have developed novel Nbs that specifically target the receptor-binding domain (RBD) of MERS-CoV spike protein. They bind to a conserved site on MERS-CoV RBD with high affinity, blocking RBD's binding to MERS-CoV receptor. Through engineering a C-terminal human Fc tag, the in vivo half-life of the Nbs is significantly extended. Moreover, the Nbs can potently cross-neutralize the infections of diverse MERS-CoV strains isolated from humans and camels. The Fc-tagged Nb also completely protects humanized mice from lethal MERS-CoV challenge. Taken together, our study has discovered novel Nbs that hold promise as potent, cost-effective, and broad-spectrum anti-MERS-CoV therapeutic agents.

Zika virus (ZIKV) infection remains a serious health threat due to its close association with congenital Zika syndrome (CZS), which includes microcephaly and other severe birth defects. As no vaccines are available for human use, continuous effort is needed to develop effective and safe vaccines to prevent ZIKV infection. In this study, we constructed three recombinant proteins comprising, respectively, residues 296–406 (E296-406), 298–409 (E298-409), and 301–404 (E301-404) of ZIKV envelope (E) protein domain III (EDIII) fused with a C-terminal Fc of human IgG. Our results demonstrated that E298-409 induced the highest titer of neutralizing antibodies against infection with nine ZIKV strains isolated from different hosts, countries, and time periods, and it maintained long-term anti-ZIKV immunogenicity to induce neutralizing antibodies. Pups born to mice immunized with E298-409 were fully protected against lethal challenge with two epidemic human ZIKV strains, 2015/Honduras (R103451) and 2015/Colombia (FLR). Passive transfer of anti-E298-409 mouse sera protected pups born to naive mice, as well as type I interferon receptor-deficient adult A129 mice, from lethal challenge with human ZIKV strains R103451 and FLR, and this protection was positively correlated with neutralizing antibodies. These data suggest that the critical neutralizing fragment (i.e., a fragment that can induce highly potent neutralizing antibodies against divergent ZIKV strains) of ZIKV EDIII is a good candidate for development as an effective and safe ZIKV subunit vaccine to protect pregnant mothers and their fetuses against ZIKV infection. The E298-409-specific antibodies can be used for passive immunization to prevent ZIKV infection in newborns or immunocompromised adults.

The Zika virus (ZIKV), a flavivirus transmitted by Aedes mosquitoes, has emerged as a global public health concern. Pre-existing cross-reactive antibodies against other flaviviruses could modulate immune responses to ZIKV infection by antibody-dependent enhancement, highlighting the importance of understanding the immunogenicity of the ZIKV envelope protein. In this study, we identified a panel of human monoclonal antibodies (mAbs) that target domain III (DIII) of the ZIKV envelope protein from a very large phage-display naive antibody library. These germline-like antibodies, sharing 98%-100% hoLogy with their corresponding germline IGHV genes, bound ZIKV DIII specifically with high affinities. One mAb, m301, broadly neutralized the currently circulating ZIKV strains and showed a synergistic effect with another mAb, m302, in neutralizing ZIKV in vitro and in a mouse model of ZIKV infection. Interestingly, epitope mapping and competitive binding studies suggest that m301 and m302 bind adjacent regions of the DIII C-C' loop, which represents a recently identified cryptic epitope that is intermittently exposed in an uncharacterized virus conformation. This study extended our understanding of antigenic epitopes of ZIKV antibodies and has direct implications for the design of ZIKV vaccines.

Zika virus (ZIKV), a mosquito-borne flavivirus, has attracted global attention due to its close association with congenital Zika syndrome and neurological diseases, and transmission through additional routes, such as sexual contact. Currently there are no vaccines approved for ZIKV, and thus, there is an urgent need to develop an effective and safe ZIKV vaccine. Domain III (DIII) of the ZIKV envelope (E) protein is an important vaccine target, and a vaccine developed using a mutant DIII of E (EDIII) protein protects adult and pregnant mice, and unborn offspring, against ZIKV infection. Here, we have used immunocompetent BALB/c mice treated with anti-interferon-&alpha, /&beta, receptor 1 (Ifnar1) antibodies to investigate whether three adjuvants (aluminum (Alum), monophosphoryl lipid A (MPL), and MF59), either alone or in combination, could improve the efficacy of this EDIII subunit vaccine. Our data show that, although vaccine formulated with a single adjuvant induced a specific antibody and cellular immune response, and reduced viral load in mice challenged with ZIKV, the combination of Alum and MPL adjuvants led to a more robust and balanced immune response, stronger neutralizing activity against three recent ZIKV human strains, and greater protection against a high-dose ZIKV challenge. Particularly, the combination of Alum with MPL significantly reduced viral titers and viral RNA copy numbers in sera and tissues, including the male reproductive organs. Overall, this study has identified the combination of Alum and MPL as the most effective adjuvant for ZIKV EDIII subunit vaccines, and it has important implications for subunit vaccines against other enveloped viruses, including non-ZIKV flaviviruses.

Middle East respiratory syndrome coronavirus (MERS-CoV) binds to cellular receptor dipeptidyl peptidase 4 (DPP4) via the spike (S) protein receptor-binding domain (RBD). The RBD contains critical neutralizing epitopes and serves as an important vaccine target. Since RBD mutations occur in different MERS-CoV isolates and antibody escape mutants, cross-neutralization of divergent MERS-CoV strains by RBD-induced antibodies remains unknown. Here, we constructed four recombinant RBD (rRBD) proteins with single or multiple mutations detected in representative human MERS-CoV strains from the 2012, 2013, 2014, and 2015 outbreaks, respectively, and one rRBD protein with multiple changes derived from camel MERS-CoV strains. Like the RBD of prototype EMC2012 (EMC-RBD), all five RBDs maintained good antigenicity and functionality, the ability to bind RBD-specific neutralizing monoclonal antibodies (MAbs) and the DPP4 receptor, and high immunogenicity, able to elicit S-specific antibodies. They induced potent neutralizing antibodies cross-neutralizing 17 MERS pseudoviruses expressing S proteins of representative human and camel MERS-CoV strains identified during the 2012-2015 outbreaks, 5 MAb escape MERS-CoV mutants, and 2 live human MERS-CoV strains. We then constructed two RBDs mutated in multiple key residues in the receptor-binding motif (RBM) of RBD and demonstrated their strong cross-reactivity with anti-EMC-RBD antibodies. These RBD mutants with diminished DPP4 binding also led to virus attenuation, suggesting that immunoevasion after RBD immunization is accompanied by loss of viral fitness. Therefore, this study demonstrates that MERS-CoV RBD is an important vaccine target able to induce highly potent and broad-spectrum neutralizing antibodies against infection by divergent circulating human and camel MERS-CoV strains. IMPORTANCE MERS-CoV was first identified in June 2012 and has since spread in humans and camels. Mutations in its spike (S) protein receptor-binding domain (RBD), a key vaccine target, have been identified, raising concerns over the efficacy of RBD-based MERS vaccines against circulating human and camel MERS-CoV strains. Here, we constructed five vaccine candidates, designated 2012-RBD, 2013-RBD, 2014-RBD, 2015-RBD, and Camel-RBD, containing single or multiple mutations in the RBD of representative human and camel MERS-CoV strains during the 2012-2015 outbreaks. These RBD-based vaccine candidates maintained good functionality, antigenicity, and immunogenicity, and they induced strong cross-neutralizing antibodies against infection by divergent pseudotyped and live MERS-CoV strains, as well as antibody escape MERS-CoV mutants. This study provides impetus for further development of a safe, highly effective, and broad-spectrum RBD-based subunit vaccine to prevent MERS-CoV infection.

Viral subunit vaccines often contain immunodominant non-neutralizing epitopes that divert host immune responses. These epitopes should be eliminated in vaccine design, but there is no reliable method for evaluating an epitope's capacity to elicit neutralizing immune responses. Here we introduce a new concept ‘neutralizing immunogenicity index' (NII) to evaluate an epitope's neutralizing immunogenicity. To determine the NII, we mask the epitope with a glycan probe and then assess the epitope's contribution to the vaccine's overall neutralizing immunogenicity. As proof-of-concept, we measure the NII for different epitopes on an immunogen comprised of the receptor-binding domain from MERS coronavirus (MERS-CoV). Further, we design a variant form of this vaccine by masking an epitope that has a negative NII score. This engineered vaccine demonstrates significantly enhanced efficacy in protecting transgenic mice from lethal MERS-CoV challenge. Our study may guide the rational design of highly effective subunit vaccines to combat MERS-CoV and other life-threatening viruses., Viral subunit vaccines contain epitopes that elicit non-neutralizing immune responses. Here, Du et al. mask an immunodominant non-neutralizing epitope on a MERS coronavirus subunit vaccine with a glycan probe, leading to significantly improved efficacy of the vaccine.

First identified in 2012, Middle East respiratory syndrome (MERS) coronavirus (MERS-CoV) is listed as a new Category C Priority Pathogen. While the high mortality of MERS-CoV infection is further intensified by potential human-to-human transmissibility, no MERS vaccines are available for human use. This review explains immune responses resulting from MERS-CoV infection, describes MERS vaccine criteria, and presents available small animal models to evaluate the efficacy of MERS vaccines. Current advances in vaccine development are summarized, focusing on specific applications and limitations of each vaccine category. Taken together, this review provides valuable guidelines toward the development of an effective and safe MERS vaccine. This article is written for a Special Focus Issue of Expert Review of Vaccines on ‘Vaccines for Biodefence’.