Vaccines are considered to be one of the most effective ways to end the new crown pandemic and help restore the global economy. A joint scientific research team from Shanghai Jiaotong University, Fudan University and other institutions recently designed a new mRNA crown vaccine. By simulating the surface protein and internal nucleic acid of coronavirus, the vaccine combines the functions of inactivated vaccine and mRNA vaccine, which provides a brand new vaccine platform for global anti-epidemic. This vaccine is named ShaCoVacc, and it can induce a strong spike-specific humoral immune response with a single injection, and has effective neutralizing activity.
It is worth noting that at present, no detailed data of animal experiments on the new mRNA crown vaccine has been disclosed, and the data provided by the team is also the first in the world. The above research is from May 15, local time, "Single dose SARS-CoV-2 simulating particle vaccine induces potent neutralizing activities", the researchers come from Shanghai Jiaotong University, Fudan University, Shanghai Bendao Gene Technology Co., Ltd., National Beijing Drug Safety Evaluation and Research Center, Guizhou Medical University. Corresponding authors of this study are Researcher Ying Tianlei of the School of Basic Medicine of Fudan University, Hong Jiaxu, deputy chief physician of Shanghai Eye, Ear, Nose and Throat Hospital affiliated to Fudan University, and Researcher Cai Yujia of the Institute of Systems Biomedicine, Shanghai Jiaotong University.
In order to produce a similar natural immunogen without causing infection, the researchers injected the mRNA spike protein (Spike) into the interior of virus mimic particles (VSPs), which are derived from lentiviral particles; the researchers also The spike protein on the surface of the virus mimic particles was modified. This study characterized the mRNA copy number, glycosylation status, transduction efficiency and innate immunity characteristics of the new vaccine platform.
Importantly, studies have shown that ShaCoVacc can induce a strong spike-specific humoral immune response with a single injection and has effective neutralizing activity. In addition, the researchers used peptide microarrays to disclose the epitopes of spike-specific antibodies and revealed epitopes sensitive to specific neutralizing antibodies. These results support ShaCoVacc as a candidate vaccine for COVID-19, which can be further developed, and virus simulation particles can be used 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. Live attenuated vaccines are weakened pathogens that can cause a strong humoral and cellular immune response, but there is also a risk of infection, especially for people with low immune function. Inactivated vaccines can kill pathogens with complete structure and destroy their genetic material, so the risk is lower, but the efficacy is also lower.
Protein subunit vaccines, DNA vaccines and mRNA vaccines are generally safe, but it is difficult to essentially reflect the conformational structure of viral immunogens. Virus-like particles (VLPs) are defective particles that have the ability to present viral spikes in their natural conformation and elicit conformation-dependent neutralizing antibodies. The VLP, which is more similar to the pathogen, has a spiked structure on the surface and a nucleic acid encoding the antigen inside. Which vaccine platform is really suitable for SARS-CoV-2 is still unknown, which makes the development of new vaccine platforms of great significance. Since the neutralizing antibody has been detected during the rehabilitation period of the new crown patient, the SARS-CoV-2 vaccine can deliver the antigen to the immune system, which is almost the same as the real virus, thus stimulating a similar effective immune response.
The researchers designed a candidate vaccine by encapsulating spike protein in virus-like particles (VSP) and modifying its surface. The virus mimic particles in the form of lentiviral particles mimic wild-type SARS-CoV-2 with mRNA and protein, respectively. It is assumed that the expression of full-length spikes can be achieved during the production of lentiviral particles carrying mRNA.
To package the full-length spike mRNA into the virus mimic particles, the researchers also designed a spike construct that expresses a spike protein with a 6X MS2 stem loop on its transcript, which makes the spike Spike mRNA is packaged into virus mimic particles by interacting with GagPol fused to the MS2 coat.
At the same time, as an envelope protein, spike protein will automatically assemble into the membrane of the virus mimic particles.
In order to check whether the spike protein mRNA has been packaged into lentiviral particles as designed, the researchers conducted RT-qPCR and found that each virus simulated particle had an average of 3 or 4 copies of spike protein mRNA. In order to verify whether the spike protein has been assembled into the virus mimic particles and their glycosylation status, the researchers performed western blot analysis on the lysates of the virus mimic particles with the integration-deficient lentivirus (IDLV) as a control. The analysis found that it successfully modified the spike protein with or without the virus simulation particle mutation, and at the same time can load more mutant spike protein.
Since glycosylation affects the immunogenicity and immunological advantages of the vaccine, the experiment examined the glycosylation status of the spikes on the surface of the virus-simulated particles. It is worth noting that after PNGase F treatment, the S2 band moved down, indicating that the spike protein on the virus simulated particles was modified by N-linked glycosylation, which is consistent with the recent findings of SARS-CoV-2 revealed by mass spectrometry .
Next, the researchers transduced the virus mimic particles into 293T cells and evaluated the expression of spike protein. The cells were harvested 36 hours after infection and subjected to Western blot analysis. Here, the researchers still observed the spike protein expression of the virus mimic particles in 293T cells, indicating that VSV-G was co-assembled into virus mimic particles, thereby expanding their tropism. The researchers discovered two main spikes, which may be glycosylated full-length single spikes and their dimer / trimer forms.
In addition, the researchers confirmed the transfected or transduced 293T cells using confocal analysis, which expressed spikes. In order to examine the innate immune characteristics of the virus mimic particles, THP-1 derived macrophages were used as a nucleic acid sensing model, and it was found that the type I interferon (IFN) and IFN stimulated genes ISG-15 and retinoic acid induced genes were not obvious Increase I (RIG-I). Because spiked virus mimic particles incorporated spiked mRNA and protein more efficiently than their wild-type counterparts, the researchers chose it as a candidate vaccine for in vivo evaluation (designated ShaCoVacc).
To obtain the immunogenicity of ShaCoVacc, the researchers injected candidate vaccines into C57BL / 6J mice. Two weeks after vaccination, mouse serum was subjected to enzyme-linked immunosorbent assay (ELISA) to obtain spike-specific IgG. The researchers observed a significant induction of spike-specific IgG.
To assess the production of neutralizing antibodies, a spiked pseudotype HIV encoding firefly luciferase was also used for the neutralization assay—a mature pseudovirus neutralization assay. Interestingly, in the study, a single injection of ShaCoVacc can induce an immediate and effective immune response against SARS-CoV-2, while an inactivated vaccine requires at least two or three doses.
The researchers also used the spike pseudo-type lentivirus, which encodes GFP to transduce Huh-7 cells. The experiment found that pre-incubation with 1:40 diluted serum from vaccinated mice almost completely eliminated fluorescence, which was obvious for the placebo group and the positive control. Interestingly, the sera from the inoculated mice did not inhibit the transduction of VSV-G pseudotyped lentivirus in Huh-7 cells, suggesting that the neutralizing antibody is spike-specific.
T cell immune responses are often important for the function of vaccines to control viral infections. However, in COVID-19, the excessive production of cytokines is related to the severity of symptoms. Therefore, the research team believes that for any SARS5 CoV-2 vaccine, cellular immunity must be cautious.
In this study, T cell immune responses were evaluated by simulating spleen cells with spike-derived peptide pools. The researchers did not find an increase in the expression of IFN-γ and IL-2, indicating that for ShaCoVacc, the spike-specific cellular immune response was not significant. This is consistent with recent studies on the inactivated SARS-CoV-2 vaccine, which has a protective effect, but no significant changes were found in the percentage of lymphocytes and key cytokines inoculated with rhesus monkeys. In addition, no weight loss due to ShaCoVacc was found during vaccination, indicating no significant toxicity.
By dissecting the vaccinated mice, we can further understand the linear epitope characteristics of spike-specific antibodies. The researchers used a newly developed peptide microarray containing short peptides covering the entire length of the spike. The researchers found that the signal intensity corresponding to some spike peptides in the vaccinated group varied, but no signal was observed in the placebo-treated mice.
The research team also quantified the signal intensity of antibodies directed against the S1 domain and the receptor binding domain (RBD), respectively. The serum of the vaccinated mice elicited a significantly higher signal in both domains, which is consistent with the previous ELISA analysis and neutralization assay of spike-specific antibodies.
In order to obtain a panoramic view of the epitope, they drew a heat map of all vaccinated mice and found that the characteristics of the epitope of each vaccinated mouse were different. However, the study also found that 66.7% of the vaccinated mice had three common epitopes (S2-22, S2-76, and S2-83). The antibody against this epitope extracted from the serum of the recovered person has shown strong neutralizing activity. It is worth noting that the S2-76 and S2-83 epitopes are conserved epitopes and are shared by SARS-CoV and SARS-CoV-2.
In summary, this study combined the functions of inactivated vaccines and mRNA vaccines by simulating coronavirus surface proteins and internal nucleic acids, thus providing a new vaccine platform. Due to the limited resources of SARS-CoV-2, researchers cannot currently infect vaccinated animals with real viruses. In the future, researchers will further reveal the epitope profile of vaccinated mice and the epitopes susceptible to specific neutralizing antibodies, which may help the development of drugs and antibodies.
Reporter He Liping