Vaccines are considered one of the most effective ways to end the new pandemic and help revive the global economy. A joint research team from Shanghai Jiaotong University, Fudan University and other institutions recently designed a new mRNA crown vaccine. The vaccine combines the functions of inactivated vaccines and mRNA vaccines by simulating coronavirus surface proteins and internal nucleic acids, providing a new vaccine platform for global anti-epidemic strains. The vaccine, called ShaCoVacc, induces a strong proto-protrusion-specific body fluid immune response with a single injection and has effective hydration and activity.
值得注意的是，目前mRNA新冠疫苗还未有动物实验的详细数据披露，该团队提供的数据也是全球首个。以上研究来自当地时间5月15日，《单剂量SARS-CoV-2模拟颗粒疫苗诱导有效的中和活性》（A single dose SARS-CoV-2 simulating particle vaccine induces potent neutralizing activities），研究人员来自上海交通大学、复旦大学、上海本导基因技术有限公司、国家北京药物安全评价研究中心、贵州医科大学。该项研究的通讯作者为复旦大学基础医学院应天雷研究员、复旦大学附属上海眼耳鼻喉科医院副主任医师洪佳旭，以及上海交通大学系统生物医学研究院蔡宇伽研究员。
To produce a natural-like immunogen that does not cause infection, the researchers modified the protrusion protein on the surface of the virus simulation particle by injecting the codemed mRNA protoprotein (Spike) into the viral simulation particles (VSPs), which are derived from slow-virus particles. The study characterized the number of mRNA copies, glycosylation status, transduction efficiency and innate immune properties of the new vaccine platform.
Importantly, studies have shown that ShaCoVacc induces a strong protrusion-specific body fluid immune response with a single injection and has effective hydration and activity. In addition, the researchers used peptide microarrays to disclose the epitope of proto-specific antibodies and to reveal epitopes that are sensitive to specific neutralizing antibodies. These results support ShaCoVacc as a candidate vaccine for COVID-19, which can be further developed, while viral simulation particles can serve as a new vaccine platform for emerging infectious diseases.
Over the past few decades, many vaccine platforms have been approved for market or clinical trials. Detox-reducing live vaccines are weakened pathogens that can cause strong body fluids and cellular immune responses, but there is also a risk of infection, especially in people with low immune function. Inactivated vaccines kill pathogens with complete structures and destroy their genetic material, so the risk is low, but the efficacy is also low.
Protein subunit vaccines, DNA vaccines, and mRNA vaccines are generally safe, but it is difficult to essentially reflect the conformation of viral immunogens. Viral-like particles (VLPs) are mutilated particles that have the ability to present viral protrusions with their natural conformations and trigger structural dependence and neutralizing antibodies. VlP, which is more similar to the pathogen, has a protrusion structure on its surface and encoded antigen nucleic acid spouts inside. What kind of vaccine platform is actually available for SARS-CoV-2 remains unknown, which makes it important to develop new vaccine platforms. Since neutralizing antibodies has been detected during the rehabilitation of new corona patients, the vaccine that simulates SARS-CoV-2 can transmit antigens to the immune system in almost the same way as a real virus, triggering a similar effective immune response.
The researchers designed a candidate vaccine by envelogling the protrusion protein in viral simulation particles (VSPs) and modifying its surface. The virus simulation particles simulate wild-type SARS-CoV-2 in the form of mRNA and protein, respectively, in the form of slow virus particles. It is assumed that the expression of full-length protrusions can be achieved by the production of slow-virus particles carrying mRNA.
To wrap the full-length protrusion mRNA into the virus simulation particles, the researchers also designed a protrusion structure that expresses a protoprotein with 6X MS2 stem loop on its transcript, which allows the prototyping mRNA to be packaged into the virus simulation particles by interacting with The GagPol in the MS2 coat.
At the same time, as a encapsulation protein, the protrusion protein is automatically assembled into the membrane of the virus simulation particles.
To check whether the protoprotein mRNAs were packaged into slow virus particles as designed, the researchers conducted RT-qPCR and found that each virus simulation particle had an average of three or four copies of the protoprotein mRNA. To verify whether the protoprotein had been assembled into viral analog particles and their glycolysitied state, the researchers performed a protein imprint analysis of the cleavage of the virus simulation particles by integrating the defective slow virus (IDLV) against the virus simulation particles. The analysis found that it successfully modified the protoprotein with or without the virus simulation particle mutation, while at the same time can load more mutations of this protoprotein.
Because glycosylation affects the immunogenicity and immune advantages of the vaccine, the experiment examined the glycolytying state of the surface protrusion of the virus simulation particles. It is worth noting that after PNGase F treatment, the S2 band moved downwards, indicating that the protogenic protein on the virus simulation particles was modified by N-connected glycosylation, which is consistent with recent findings of SARS-CoV-2 revealed by mass spectrometry.
Next, the researchers transferred the virus simulation particles to 293T cells and evaluated the expression of the protoprotein. Cells were harvested 36 hours after infection for Western imprinting. Here, the researchers still observed the protrusion protein expression of virus-simulated particles in 293T cells, indicating that VSV-G was co-assembled into viral simulation particles, thus expanding their directionality. The researchers found two main protrusion zones, possibly the full-length single protrusion of glycosyl and its dipoly/dipolymer form.
In addition, the researchers identified 293T cells that transfected or transducted 293T using confocal analysis, which expressed protrusions. In order to check the innate immune properties of viral analog particles, using THP-1-derived macrophages as nucleic acid sensing models, it was found that the genes isG-15 and osteotic acid-induced genes stimulated by type I interferon (IFN) and IFN did not significantly increase I (RIG-I). Because the protrusion-driven virus-simulated particles are more effectively mixed with the protrusion mRNA and protein than their wild counterparts, the researchers chose it as a candidate vaccine for inviva evaluation (designated ShaCoVacc).
To obtain The Immunogenicity of ShaCoVacc, the researchers injected the candidate vaccine into C57BL / 6J mice. Two weeks after vaccination, enzyme-linked immunosorption (ELISA) was performed on the serum of mice to obtain protogen-specific IgG. The researchers observed significant induction of proto-specific IgG.
In order to evaluate neutralizing the production of antibodies, neutralizing hiv, a mature pseudoviral, was also used to neutralise the protrusion pseudo-HIV that encodes fireflies. Interestingly, in the study, a single injection of ShaCoVacc induced an immediate and effective immune response to SARS-CoV-2, whereas an inactivated vaccine required at least two or three doses.
The researchers also used the protrusion pseudo-slow virus, which encodes GFP to transducte huh-7 cells. The experiment found that precultured with 1:40 diluted serum from vaccinated mice almost completely eliminated fluorescence, which was evident in the placebo group and positive controls. Interestingly, serum from vaccinated mice did not inhibit transduction of VSV-G pseudo-slow virus in Huh-7 cells, indicating that neutralizing antibodies had protrusion specificity.
T-cell immune response is usually important for the vaccine’s ability to control viral infections. However, in COVID-19, overproduction of cytokines is associated with the severity of symptoms. Therefore, the team believes that for any SARS5 CoV-2 vaccine, cellular immunity must be cautious.
In this study, t-cell immune response was evaluated by simulating spleen cells with a thyme-derived peptide pool. The researchers did not find an increase in expression of IFN-gamma and IL-2, suggesting that for ShaCoVacc, the protogenitive-specific cell immune response was not significant. This is consistent with recent inactivated SARS-CoV-2 vaccine studies, which have a protective effect but have not been found to have significant changes in the percentage of lymphocytes and key cytokines vaccinated against macaques. In addition, no weight loss caused by ShaCoVacc was found during the vaccination process, indicating no apparent toxicity.
By dissecting the vaccinated mice to gain further understanding of the linear epitope characteristics of proto-specific antibodies, the researchers used a newly developed peptide microarray that contains short peptides that cover the entire length of the protrusion. The researchers found that the signal strength corresponding to some of the protopeptides in the vaccination group varied, while the mice treated with a placebo did not observe the signal.
The team also quantified the signal strength of antibodies for the S1 domain and the receptor binding domain (RBD) respectively. The serum of the inoculated mice produced significantly higher signals in both domains, which was consistent with ELISA analysis and neutralizing of previous proto-specific antibodies.
To get a panoramic view of the epitope, they mapped the heats of all the vaccinated mice and found that the antigen-determined cluster characteristics of each vaccinated mouse were different. However, the study also found that 66.7 percent of vaccinated mice had three common epitopes (S2-22, S2-76, and S2-83). Antibodies to this epitope extracted from the recovering serum have shown strong neutrality activity. It is worth noting that the S2-76 and S2-83 table is conservative and is shared by SARS-CoV and SARS-CoV-2.
Overall, the study provided a new vaccine platform by simulating coronavirus surface proteins and internal nucleic acids, which combined the functions of inactivated vaccines and mRNA vaccines. Because of the limited resources of SARS-CoV-2, researchers are currently unable to re-infect vaccinated animals with real viruses. In the future, researchers will further reveal the epitope profile of vaccinated mice and epitopes that are susceptible to specific neutralizing antibodies, which may contribute to the development of drugs and antibodies.