To ultimately combat the emerging COVID-19 pandemic, it is desired to develop an effective and safe vaccine against this highly contagious disease caused by the SARS-CoV-2 coronavirus. Our literature and clinical trial survey showed that the whole virus, as well as the spike (S) protein, nucleocapsid (N) protein, and membrane protein, have been tested for vaccine development against SARS and MERS. We further used the Vaxign reverse vaccinology tool and the newly developed Vaxign-ML machine learning tool to predict COVID-19 vaccine candidates. The N protein was found to be conserved in the more pathogenic strains (SARS/MERS/COVID-19), but not in the other human coronaviruses that mostly cause mild symptoms. By investigating the entire proteome of SARS-CoV-2, six proteins, including the S protein and five non-structural proteins (nsp3, 3CL-pro, and nsp8–10) were predicted to be adhesins, which are crucial to the viral adhering and host invasion. The S, nsp3, and nsp8 proteins were also predicted by Vaxign-ML to induce high protective antigenicity. Besides the commonly used S protein, the nsp3 protein has not been tested in any coronavirus vaccine studies and was selected for further investigation. The nsp3 was found to be more conserved among SARS-CoV-2, SARS-CoV, and MERS-CoV than among 15 coronaviruses infecting human and other animals. The protein was also predicted to contain promiscuous MHC-I and MHC-II T-cell epitopes, and linear B-cell epitopes localized in specific locations and functional domains of the protein. Our predicted vaccine targets provide new strategies for effective and safe COVID-19 vaccine development.
Our prediction of the potential SARS-CoV-2 antigens, which could induce protective immunity, provides a timely analysis for the vaccine development against COVID-19. Currently, most coronavirus vaccine studies use the whole inactivated or attenuated virus, or target the structural proteins such as the spike (S) protein, nucleocapsid (N) protein, and membrane (M) protein (Table 2). But the inactivated or attenuated whole virus vaccine might induce strong adverse events. On the other hand, vaccines targeting the structural proteins induce a strong immune response20,29,30. In some studies, these structural proteins, including the S and N proteins, were reported to associate with the pathogenesis of coronavirus21,31 and might raise safety concern. A study has shown increased liver pathology in the vaccinated ferrets immunized with modified vaccinia Ankara-S recombinant vaccine32.
Although there were no other adverse events reported in other animal studies, the safety and efficacy of these vaccination strategies has not been tested in human clinical trials. Our study applied the state-of-the-art Vaxign reserve vaccinology (RV) and Vaxign-ML machine learning strategies to the entire SARS-CoV-2 proteomes including both structural and non-structural proteins for vaccine candidate prediction. Our results indicate for the first time that many non-structural proteins could be used as potential vaccine candidates.
The SARS-CoV-2 S protein was identified by our Vaxign and Vaxign-ML analysis as the most favorable vaccine candidate. First, the Vaxign RV framework predicted the S protein as a likely adhesin, which is consistent with the role of S protein for the invasion of host cells. Second, our Vaxign-ML predicted that the S protein had a high protective antigenicity score. These results confirmed the role of S protein as the important target of COVID-19 vaccines. However, the S protein exists in many coronaviruses, and many non-pathogenic human coronaviruses also use S protein to cell invasion. For example, despite markedly weak pathogenicity, HCoV-NL63 also uses S protein and employs the angiotensin-converting enzyme 2 (ACE2) for cellular entry33. This suggests that the S protein is not the only factor determining the infection level of a human coronavirus. In addition, targeting only the S protein may induce high serum-neutralizing antibody titers but cannot induce sufficient protective efficacy34. Thus, alternative vaccine antigens may be considered.
The SARS-CoV-2 nsp3 protein was predicted to be a potential vaccine candidate, as shown by its predicted second-highest protective antigenicity score, adhesin property, promiscuous MHC-I & MHC-II T cell epitopes, and B cell epitopes. The nsp3 is the largest non-structural protein that includes multiple functional domains to support viral pathogenesis26. The multiple sequence alignment of nsp3 also showed higher sequence conservation in most of the functional domains in SARS-CoV-2, SARS-CoV, and MERS-CoV, than in all 15 coronavirus strains (Fig. 1B). The induction of nsp3-specific immunity would likely help the host to fight against the infection. Besides the S and nsp3 proteins, our study also suggested four additional vaccine candidates, including 3CL-pro, nsp8, nsp9, and nsp10. All these proteins were predicted as adhesins, and the nsp8 protein was also predicted to have a significant protective antigenicity score.
Our predicted non-structural proteins (nasp3, 3CL-pro, nsp8, nsp9, and nsp10) are not part of the viral structural particle, and none of the non-structural proteins have been evaluated as vaccine candidates.
The SARS/MERS/COVID-19 vaccine studies so far target the structural (S/M/N) proteins. Still, the non-structural proteins have been used effective vaccine antigens to stimulate protective immunity against many viruses. For example, the non-structural protein NS1 was found to induce protective immunity against the infections by flaviviruses35. The non-structural proteins of the hepatitis C virus were reported to induce HCV-specific vigorous and broad-spectrum T-cell responses36. The non-structural HIV-1 gene products were also shown to be valuable targets for prophylactic or therapeutic vaccines37. Therefore, it is reasonable to consider the SARS-CoV-2 non-structural proteins as possible vaccine targets, as suggested by the present study.
Instead of using a single protein as the vaccine antigen, we would like to propose the development of a “cocktail vaccine” as an effective strategy for COVID-19 vaccine development. A typical cocktail vaccine includes more than one antigen to cover different aspects of protection39,40. The licensed Group B meningococcus Bexsero vaccine, which was developed via reverse vaccinology, contains three protein antigens9. To develop an efficient and safe COVID-19 cocktail vaccine, it is possible to mix the structural (e.g., S protein) and non-structural (e.g., nsp3) viral proteins. The other proteins identified in our study may also be considered as possible vaccine targets. The benefit of a cocktail vaccine strategy could induce immunity that can protect the host against not only the S-ACE2 interaction and viral entry to the host cells, but also protect against the accessary non-structural adhesin proteins (e.g., nsp3), which might also be vital to the viral entry and replication.
The usage of more than one antigen allows us to reduce the volume of each antigen and thus reducing the induction of adverse events. Nonetheless, the potentials of these predicted non-structural protein targets in vaccine development need to be experimentally validated.
For rational COVID-19 vaccine development, it is critical to understand the fundamental host-coronavirus interaction and protective immune mechanism7. Such understanding may not only provide us guidance in terms of antigen selection but also facilitate our design of vaccine formulations. For example, an important foundation of our prediction in this study is based on our understanding of the critical role of adhesin as a virulence factor as well as protective antigen. The choice of DNA vaccine, recombinant vaccine vector, and another method of vaccine formulation is also deeply rooted in our understanding of pathogen-specific immune response induction. Different experimental conditions may also affect results41,42. Therefore, it is crucial to understand the underlying molecular and cellular mechanisms for rational vaccine development.
Resources & references: https://www.ncbi.nlm.nih.gov/
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