key: cord-0895262-2qljx9cq authors: He, Chunting; Qin, Ming; Sun, Xun title: Highly pathogenic coronaviruses: thrusting vaccine development in the spotlight date: 2020-05-30 journal: Acta Pharm Sin B DOI: 10.1016/j.apsb.2020.05.009 sha: 2a5adcf030d5dcb0642890cbe396cec2b5afdb5d doc_id: 895262 cord_uid: 2qljx9cq Coronaviruses (CoVs) are a large family of viruses that cause illness ranging from the common cold to more severe diseases such as Severe Acute Respiratory Syndrome (SARS) and Middle East Respiratory Syndrome (MERS). Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2) disease (COVID-19) has caused major public health crises. There have been more than 4,400,000 reported cases of COVID-2019 and more than 300,000 reported deaths to date (16/05/2020). SARS-CoV, MERS-CoV and SARS-CoV-2 have attracted widespread global attention due to their high infectivity and pathogenicity. To date, there is no specific treatment proven effective against these viral infectious diseases. Vaccination is considered one of the most effective strategies to prevent viral infections. Therefore, the development of effective vaccines against highly pathogenic coronaviruses is essential. In this review, we will briefly describe coronavirus vaccine design targets, summarize recent advances in the development of coronavirus vaccines, and highlight current adjuvants for improving the efficacy of coronavirus vaccines. Vaccination is the most effective and economical way to prevent viral infections. This review describes coronavirus vaccine design targets, summarizes recent advances and potential strategies for coronavirus vaccine development, and highlights promising technological routes and adjuvants for improving the effectiveness of coronavirus vaccines. In 2019, a novel strain of coronavirus was found in humans 1 . On February 11, 2020, WHO announced a new name for the epidemic disease: Corona Virus Disease . Meanwhile, the International Committee on Taxonomy of Viruses named the novel coronavirus as Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). As of May 16th, 2020, the epidemic of COVID-19 has caused more than 4,400,000 laboratory-confirmed cases and more than 300,000 reported deaths 2 . COVID-19 is the third known zoonotic coronavirus disease 3 SARS is a zoonosis caused by SARS-CoV, which has infected 8096 humans, including 774 deaths (mortality rate 9.6%), in at least 29 countries 4 . Another highly pathogenic coronavirus, MERS-CoV, has been reported in 27 countries with reported viral infection and 858 associated deaths (mortality rate 34.4%). 5 Research indicates that SARS-CoV was transmitted from civet cats to humans and MERS-CoV was transmitted from dromedary camels to humans. However, the intermediate host of SARS-CoV-2 has not been identified 6, 7, 8 . SARS-CoV-2, together with SARS-CoV and MERS-CoV, has posed significant threats to international health due to theirs high pathogenicity and infectivity. Vaccination is an important strategy to provide protection from infectious diseases. However, to date, no vaccine has been approved to prevent coronavirus infection, indicating the need for further development of novel and effective vaccines against coronavirus infection. In this review, we will illustrate vaccine design targets, review current advances and potential strategies for vaccine development based on the spike (S) protein of SARS-CoV and MERS-CoV, and focus on how to improve the efficacy of vaccines through adjuvant formulations. Overall, these strategies may provide useful guidance for vaccine development of SARS-CoV-2. infections in animals and humans. According to the phylogenetic relationships, coronavirus can be divided into four genera: Alpha, Beta, Gamma and Delta. Alpha and beta genera can infect mammals, while gamma and delta genera are mostly avian coronaviruses 9 . There are seven known coronavirus that can infect humans: 229E, OC43, NL63, HKU1, SARS-CoV, MERS-CoV and SARS-CoV-2. The first four viruses cause only mild minor respiratory illness. The other three strains-SARS-CoV, MERS-CoV and SARS-CoV-2-are zoonotic and lead to severe respiratory syndrome 10-12 . Coronaviruses are the largest single positive-strand RNA viruses with a genome of 27-32 kb 13 . It is named for its corona-like appearance ( SARS-CoV, MERS-CoV, and SARS-CoV-2 have strong human-to-human characteristics, which is attributed to the interaction between S protein and host cell surface receptors. SARS-CoV and SARS-CoV-2 use the angiotensin-converting enzyme 2 (ACE2) as a receptor 15, 16 , whereas MERS-CoV uses dipeptidyl peptidase 4 (DPP4; also known as CD26) as a receptor 17 Table 1 2-8,12,15-17 . S protein is a large type I transmembrane glycoprotein whose trimers constitute the spike structure on the surface of the virus. The S protein (Fig. 1B) can be divided into two functional subunits: an N-terminal S1 domain contains signal peptide and receptor binding domain (RBD), and a C-terminal S2 domain contains fusion peptide and two heptapeptide repeats (HR1 and HR2) to facilitate viral fusion. RBD mediates the binding of virus and cell receptor, which then triggers a conformational change of the S protein, exposing HR1 and HR2 to form a 6-helix bundle fusion core structure, further leading to membrane fusion and viral RNA release [18] [19] [20] [21] . Furthermore, S protein carries B-cell epitopes, which induces the body to produce neutralizing antibody and provides immune protection 22 . Because the S protein is involved in viral infection and is responsible for inducing host immune response and virus-neutralizing antibodies, it has been considered a key target for vaccine design. Antigen-specific targets of S protein include full-length S protein, S1 subunits, RBD and S2 subunits. Viral vector vaccines encoding full-length S protein or S1 subunits have been demonstrated to induce high levels of neutralizing antibodies in various animal models 23, 24 . However, some non-neutralizing epitopes on full-length S protein or S1 subunits may compete with neutralizing epitopes, leading to several safety concerns, including inflammatory and immunopathological effects such as pulmonary eosinophilic infiltration and antibody-dependent enhancement (ADE) following subsequent viral challenge of vaccinated animals 22, 25, 26 . ADE is a phenomenon in which non-neutralizing antibodies are produced following an infection or a vaccination leads to enhanced infection 27 . One approach to mitigate the adverse effects of ADE is to narrow the immune response to target only critical or beneficial epitopes 28 . Vaccines based on RBD elicited a robust protective immune response and neutralizing antibodies. At the same time, RBD does not contain non-neutralizing epitopes that may cause harmful immune responses, which is a hot spot for CoV vaccine development. It is worth mentioning that RBD has relatively low immunogenicity and often requires repeated doses and adjuvants [29] [30] [31] . Because the S2 subunit is highly conserved and not prone to mutation, S2 region has become an important target for the development of protective vaccines. However, reports regarding the presence of neutralizing epitopes in S2 and a protective role for antibodies to S2 have been inconsistent. Several studies demonstrated that S2 domain could induce specific cellular immune response and a high level of total IgG but little neutralizing antibodies against coronavirus infection 32, 33 . On the contrary, there are also reports that showed that S2 domain contains neutralizing epitopes and could induce neutralizing antibodies 34, 35 . N protein serves multiple functions in viral replication, transcription, and assembly of the viral genome complex, which is more conservative than other proteins, such as S and M. Therefore, N protein has been also widely reported as a target antigen. N proteins have been shown to be highly immunogenic and capable of triggering T cell responses 36 . Remarkably, many studies indicated that the serum containing anti-N protein does not contain neutralizing antibodies against coronavirus infection 37, 38 . In addition, vaccines based on N protein not only failed to protect from homologous or heterologous challenge, but resulted in enhanced immunopathology with eosinophilic infiltrates within the lungs of SARS-CoV-challenged mice 39 . It is critical for CoV vaccines to induce robust humoral and cellular immunities. Nucleic acid vaccines, including DNA and RNA vaccines, are based on plasmids or messenger RNA that encode vaccine antigens, and they are introduced into the host to produce immunological response to protect organisms against diseases 59 induced durable immune responses, as most participants maintained detectable S1 binding antibodies and had cellular immune responses at almost 1 year after the last vaccination 63 . Nevertheless, CoV DNA vaccines based on full-length S protein may cause a Th2-related harmful immune response, leading to liver damage in vaccinated animals. One study comparing the immunogenicity of MERS-CoV DNA vaccines expressing S or S1 in mice showed that plasmids expressing the S1 (pS1) subunit triggered a balanced Th1/Th2 response, thereby avoiding the risk of immunopathological risk associated with Th2 response 64 . Moreover, immunization of mice with pS1 vaccine induced significantly higher levels of IFN-γ compared to pS vaccine 64 . Messenger RNA (mRNA) vaccines carry transcripts encoding antigens, and use the host cell translational machinery to produce the antigens, which then stimulates an immune response 65 . Because of high yields of in vitro transcription reactions, mRNA has the potential for rapid, inexpensive and scalable manufacturing, which greatly shortens the development time and can respond quickly to epidemics. Compared to DNA vaccines, mRNA vaccines do not need to pass an additional membrane barrier (nuclear membrane), so it does not have safety concerns about integration into the host genome 66 . Due to the above advantages, mRNA vaccines are becoming a powerful tool against coronavirus infection. However, their application has been restricted by the instability and inefficient in vivo delivery of nucleic acid (DNA or mRNA) 67, 68 . To provide protection from degradation and facilitate their entry into targeted cells, efficient delivery systems for nucleic acid vaccines, particularly the nanocarriers, have been explored extensively. Viral vectors have a molecular mechanism that assists the target gene to enter cells and infect them, which is an important vector platform for CoV candidate vaccines. 71, 72 . Furthermore, compared with MERS-CoV S-encoding Ad5 vaccines, MERS-CoV S1-encoding Ad5 vaccines might induce higher levels of neutralizing antibodies 73 . In a recent study, rAd5 constructs expressing CD40-targeted S1 fusion protein (rAd5-S1/F/CD40L) exhibited full protection against lethal MERS-CoV challenge, and prevented severe perivascular hemorrhage within the lungs as compared to non CD40-targeted vaccine (rAd5-S1) 74 Virus-like particles (VLPs) are multiprotein structures that mimic the organization and Subunit vaccines are composed of highly purified antigens which require only a part of the pathogen to generate a protective immune response. Subunit vaccines are characterized by high security, controllable performance and easy production on a large scale, thereby gradually becoming the focus of more and more researchers. Table 2 Highly purified proteins in subunit vaccines are usually not inherently immunogenic, as they generally do not directly stimulate the innate immune system. However, the development of effective CoV vaccines requires the activation of powerful humoral and cellular immunity to induce protective immunity and virus clearance in the body. Therefore, adjuvants are needed to be incorporated in subunit vaccines to enhance the immunogenicity of these weaker antigens and evoke the required antigen-specific immune response phenotype, thus improving the overall potency of poorly immunogenic subunit vaccines. The following review will discuss adjuvants commonly used in subunit vaccines against coronavirus infection. Aluminum (Alum) adjuvant is the longest and most frequently used adjuvant in licensed vaccines, with an extensive safety record. Alum is a Th2-type adjuvant that induces strong humoral immune response, including the production of neutralizing antibodies 86 . Therefore, Alum is incorporated into a range of vaccines against viral infection where neutralizing antibodies to viral antigens are required for protection, including human papillomavirus, rabies and hepatitis B 87 . Aluminum adjuvant has been widely used in the development of CoV vaccine due to a variety of advantages noted above. Several studies have indicated that RBD-based subunit vaccines in the presence of alum induce powerful serum-specific and neutralizing antibodies, providing a degree of protection against viral challenges 85, 88 . It is noteworthy to mention that eliciting powerful cellular and humoral immunity is critical for a potential CoV vaccine. Virus-specific T cells can secrete IFN-γ and promote virus clearance. Meanwhile, effector T cells can further differentiate into memory T cells, which is expected to respond quickly and effectively to subsequent CoV infection 89, 90 . Although alum successfully induces antibody-mediated protective immunity, its ability to induce cellular immune responses is limited. One approach to overcome the limitations of alum is to use it in combination with other adjuvants to enhance cellular immune responses. Another approach that has an extensive history of use as CoV vaccine adjuvants are emulsions. Freund's adjuvant is a water-in-oil emulsion, divided into complete The innate immune system recognizes pathogen-associated molecular patterns Table 3 83, 85, 87, 88, [91] [92] [93] [94] [98] [99] [100] [101] [102] [103] [104] [105] [106] 108, 109, 112, 113, 118, 122, 123, 125, 126, 128 . In addition to safety considerations, the design of adjuvants must also pay attention to the ability to selectively induce and regulate the types of immune responses in the body, so as to effectively promote the humoral and cellular immunity to combat coronavirus infection. It is also noteworthy to mention that an existing well-established adjuvant could be combined with new immunostimulants (e.g., TLRs agonist) to improve the breadth and intensity of the immune responses, which has become a potential strategy for exploring efficient adjuvant systems. SARS-CoV-2 has spread rapidly since its outbreak and has now posed a risk to There are no conflicts of interest to declare. 2. Induces the production of type-I IFN, elicits T cell responses 113 . TLR4 agonist LPS/MPLA Induces SARS-CoV S-specific antibody and virus-specific antibody (>1:10 4 ) 118 . Induces the production of Th1 cytokines, elicits T cell responses 118 . TLR9 agonist CpG DNA No-report 1. Induces the production of IFN-a and IFN-γ, enhance NK cell cytotoxicity 122 . Stimulator of interferon genes (STING ) agonists STING agonist cdGMP 1. Activates the production of host defense molecules and cytokines 2. 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Induces neutralizing antibodies in mice (1:160) immunized with RV-SARS-S vector vaccine Induces neutralizing antibodies in mice (1: 64-128) immunized with MVA-MERS-S vector vaccine, neutralizes MERS-CoV; elict T-cell responses and reduces virus titers in the lung 71 Induces S-specific antibody responses and neutralizing antibodies in mice (>1:10 3 ) immunized with Ad5/Ad41-MERS-S vector vaccine, neutralizes pseudotyped MERS-CoV; elicts T-cell responses 72 Induces S-specific IgG subtype antibody (IgG1 and IgG2a) and neutralizing antibodies in mice (>1:10 3 ) immunized with Ad5-MERS-S1 vector vaccine, neutralizes MERS-CoV strain 3 /1:10 3 -10 4 ) immunized with rAd5-S1/F/CD40 vaccine, neutralizes pseudotyped and live MERS-CoV 74 . neutralizing antibodies in mice (1: 200± 97.7), neutralizes SARS-CoV; reduces virus titers in the lung 78 . 2. Induce neutralizing antibodies in mice reduces virus titers in the lung and protects all mice against challenge with virulent virus 79 Induced RBD-specific antibody responses and neutralizing antibodies in mice (1: 320), neutralizes pseudotyped MERS-CoV; elict T-cell responses 80 Induce S-specific antibody responses and neutralizing antibodies in mice (1:4.0×10 3 ±3.5×10 2 ), neutralizes SARS-CoV BJ01 strain; protects 80% of the mice from the virus challenge 81 4×10 2 ), neutralizes pseudotyped and live SARS-CoV; elict T-cell responses and protects all mice against challenge with virulent virus 82 Induced RBD-specific antibody responses and neutralizing antibodies in in rhesus monkey (1:1600), neutralizes pseudotyped MERS-CoV; elicts T-cell responses and reduces virus titers 85