Why has the spread of new crown mutants increased?

Since December 2019, the new crown virus has spread to 223 countries around the world, with more than 210 million infections and more than 4.
44 million deaths.
It has become the world's largest health crisis since the 1918 influenza pandemic
.

In particular, the delta variant, which has received widespread attention recently, can infect 8-9 people on average, and the transmission capacity has been greatly improved.
Moreover, the infection of the delta variant is often accompanied by more severe clinical symptoms of new coronary pneumonia, which has brought great effects to the prevention and control of the epidemic.
Challenge
.

Figure 1: Identification of new coronavirus-specific CD8+ T cell epitopes.
Why can the variants of the new coronavirus have such a strong infectious ability? To explain this reason, the Tan Xu laboratory of the Beijing Advanced Innovation Center for Structural Biology and the Jin Tengchuan Laboratory of the School of Life Sciences of the University of Science and Technology of China identified the specificity of the new coronavirus through a high-throughput screening method from the perspective of cellular immunity.
CD8+ T cell antigen recognition epitope, and found evidence that the new coronavirus variant escapes cellular immunity, providing new ideas for subsequent vaccine design and optimization
.

Related papers were recently published in Cell Reports, a subsidiary of Cell
.

About the new coronavirus variants 1.
The naming of the new coronavirus variants The naming of the new coronavirus delta variants comes from the classification system of the CDC and WHO.
As the new coronavirus is constantly mutating during its transmission, in order to better define the new coronavirus variants, CDC A classification system has been established independently with the WHO to divide the variants of the new coronavirus into variants of concern (Variants of Concern) and variants of interest (Variants of interest)
.

The characteristics of the variants concerned are: 1) it is more infectious, that is, it can infect more people; 2) it induces more serious diseases (that is, the number of hospitalizations or the number of deaths increases); 3) the effectiveness of treatment and vaccines is reduced; 4 ) May evade diagnostic testing; the concerned variants include a total of four variants: Alpha (B.
1.
1.
7), Beta (B.
1.
351), Gamma (P.
1), Delta (B.
1.
617.
2)
.

The variants to be watched are considered to be less dangerous variants of concern.
The WHO updated the list of variants to be watched on June 22, 2021, including 7 variants: Epsilon (B.
1.
427 and B.
1.
429), Zeta (P .
2), Eta (B.
1.
525), Xita (P.
3), Iota (B.
1.
526), ​​Kappa (B.
1.
617.
1)) and Lambda (C.
37), which has received wide attention recently
.

2.
The spread of new coronavirus variants The Global Influenza Shared Database (GISAID) summarizes the proportion of all new coronavirus sequences submitted on the official website in 2021 (Figure 2), and you can see the dynamic changes of new coronavirus variants in the global infection range
.

Figure 2: Statistics on the proportion of new coronavirus sequences included in the official website of the Global Influenza Shared Database (GISAID).
As can be seen from Figure 2, from January 2021, the alpha variants will first take the advantage of spreading
.

However, starting in May, the Delta variant gradually replaced the Alpha variant, becoming an absolute dominant variant spreading around the world
.

The delta variant was first discovered in the Indian state of Maharashtra in October 2020
.

It then spread to the whole of India in only 2 months at the end of 2020, and surpassed the original 73 pedigrees spread in India, which shows the strong communication ability
.

So far, the delta variant has spread to more than 200 countries and is currently the most noteworthy variant
.

3.
Research on the establishment of advantages of new coronavirus variants and the importance of cellular immune response.
The establishment of advantages of new coronavirus variants is related to the infection ability, immune escape ability, and transmission ability of the virus
.

The infection ability takes the delta variant as an example.
It has been reported that compared with the alpha variant, the S protein of the delta variant can be cleaved by the host cell protease more efficiently to form a functional protein, which is greatly improved at the organoid level.
The efficiency of delta variants invading cells also speeds up the replication speed of delta variants in the early stage of infection, providing ideas for the establishment of the advantages of delta variants (Mlcochova et al.
, 2021)
.

Immune escape ability In addition to the improvement of infection ability, attention to variants also enhances immune escape ability.
Take the delta variant as an example: compared with wild-type new coronavirus, the neutralizing ability of the serum neutralizing antibody of the new crown virus recoverer against the delta variant is average Decreased by 6 times; the protective effect of vaccines (ChAdOx-1 and BNT162b2) on delta variants decreased by an average of 8 times; the clinically approved monoclonal antibodies bamlavinimab and imdevimab for the treatment of new coronavirus-related diseases have the ability to neutralize delta variants, respectively Decrease by 1000 times and 50 times (Mlcochova et al.
, 2021)
.

Although the concerned variants are less sensitive to antibody-mediated humoral immunity, most people are infected for the first time during the spread of the concerned variants, and there are no neutralizing antibodies in the body, so the weak response to known antibodies can indicate The protection of our existing vaccines against these variants will decrease, but it cannot fully explain the large-scale spread of these variants
.

In addition, according to experimental data, the degree of reduction in the neutralizing antibody sensitivity of the delta variant to the rehabilitated serum or post-vaccine serum is similar to that of the beta variant (B.
1.
315), but the degree of transmission is much higher than that of the beta variant (Plante et al.
, 2021).
This suggests that in addition to humoral immunity, the escape of CD8+ T cell-mediated cellular immune responses may also promote the continuous spread of dominant variants of the new coronavirus
.

Previous studies have shown that the cellular immune response is not only related to the severity and development of the new crown disease (Sette and Crotty, 2021).
In particular, the new crown virus-specific CD8+ T cells were detected earlier than neutralizing antibodies, indicating cellular immunity The response begins to play a role in the early stage of viral infection, and this early immunosuppression may be very important for the body to defend against the new coronavirus and inhibit the spread of the new coronavirus
.

In addition, a long-term study of patients who recovered from SARS in 2003 showed that cellular immunity was maintained for a long time, some even up to 17 years (Le Bert et al.
, 2020).
Relatively speaking, antibody immunity of recovered patients Memory is much shorter
.

A systematic understanding of the cellular immune response of patients infected with the new coronavirus, especially recovered patients, is of great significance for us to understand the immune determinants of the patient's disease development, help new vaccine design and even predict the patient's response to the vaccine
.

T-Scan technology screens new coronavirus-specific CD8+ T cell epitopes.
An important step in exploring the cellular immune response is to identify the antigen recognition epitopes of new coronavirus-specific CD8+ T cells
.

The research team of Xu Tan of Tsinghua University used T-Scan technology to study the distribution of epitopes of the new coronavirus that can cause human CD8 T cells to respond, and found that mutations in 4 amino acid positions in the new coronavirus genome may give attention to the variants escape The ability of cellular immunity to promote the spread of viruses provides the basis for the direction of cellular immunity for the prevention of variants and variants (Figure 1)
.

T-Scan technology is a high-throughput screening platform for identifying T cell antigens invented by Professor Stephen Elledge of Harvard Medical School in 2019 (Kula et al.
, 2019)
.

The principle of using T-Scan technology to construct a target cell library presented by the new coronavirus antigen is shown in Figure 1
.

First, use DNA chip large-scale synthesis technology to synthesize polypeptide gene sequences covering all proteins encoded by the new coronavirus
.
Each polypeptide has 56 amino acids and a total of 9179 sequences .

Then use the lentiviral vector to transfer the gene library of the new coronavirus polypeptide antigen to the target cell.
The expression of the polypeptide antigen gene and the endogenous processing of the polypeptide antigen are finally performed by the main histocompatibility complex of the target cell ( MHC) molecules present the new coronavirus antigen to the cell surface, which better mimics the process of presenting the viral antigen to the cell surface after the cell is infected by the virus
.

In addition, the target cells also carry the IFP fluorescent reporter gene that responds to the activity of Granzyme B (Granzyme B, GzB) (Figure 3), which is used to screen target cells recognized by CD8+ T cells
.

GzB is a serine protease, which is usually stored in the cytotoxic particles in CD8+ T cells.
When CD8+ T cells recognize the viral antigen presented by the target cell and are activated, CD8+ T starts to perform the killing function and forms holes in the cell membrane through perforin.
GzB-loaded cytotoxic particles are specifically secreted into target cells
.

GzB activates the IFP fluorescent reporter gene after entering the target cell
.

Next, the identified target cells can be enriched by flow screening, and the antigen sequences presented by the target cells can be identified by next-generation sequencing
.

Figure 3: Recognition of CD8+ T cells in response to Granzyme B (GzB) fluorescent reporter system is restricted to MHC class I molecules, and can only recognize antigens presented by MHC molecules that match their own MHC genotype
.

MHC-I molecules include three genotypes of A, B, and C, called HLA-A, HLA-B and HLA-C.
Each genotype includes multiple alleles, so the diversity of MHC molecules Very rich
.

The author selected four HLA-A genotypes with the highest frequency in the Chinese population for research, including HLA-A*02:01, HLA-A*02:07, HLA-A*11:01 and HLA-A*24: 02 (respectively accounting for 10.
9%, 9.
2%, 21.
4% and 14.
4% of the Chinese population, http:// 
.

The author constructed a library of target cells overexpressing HLA-A*02:01, HLA-A*02:07, HLA-A*11:01 or HLA-A*24:02 to present epitopes of the new coronavirus
.

After testing the HLA-A genotypes of the new coronavirus survivors, incubate the genotype-matched target cell library with the CD8 + T cells of the new coronavirus survivors, from 10 new coronaviruses covering the above four HLA genotypes Memory CD8+ T cells were isolated from the PBMC of the recovered patients for T-Scan screening, and a total of 70 potential new coronavirus-specific CD8+ T cell antigen recognition epitopes were screened, which significantly enriched the new coronavirus ORF1a, ORF1b and S protein.
Polypeptide fragments indicate that these three genes may be important antigen sources for CD8+ T cell-mediated cellular immunity
.

Identification of CD8+ T cell epitopes 1.
Identification of conservative CD8+ T cell epitopes Figure 4: Conservative CD8+ T cell epitopes The author first reported the new coronavirus and other coronaviruses (including SARS, and other 4 common types) The coronavirus that causes the common cold) has a highly conserved epitope in the amino acid sequence (Figure 4), and the intracellular INF-y staining experiment verified that 7 of the peptides can induce CD8 + T cell growth in healthy people.
Immune response, which also validates the previous research on the pre-existing cellular immune memory of people who are not infected with the new coronavirus, showing that if there is a cellular immune response against other coronaviruses in the human body, there will also be a certain cross-immune response against the new coronavirus, and this The level of pre-existing cellular immunity may also partly determine the differences in the population's condition (Le Bert et al.
, 2020)
.

2.
Identification of CD8+ T cell epitopes carrying mutations of interest , And therefore received the most attention
.

Figure 5 lists the origins of the four variants of interest and their mutation sites on the S protein of the new coronavirus
.

Figure 5: Four global attention variants of the new coronavirus (Focus variants) and the amino acid mutation sites they carry (from the website https://covariants.
org) In order to explore whether the point mutations of the attention variants help the virus escape CD8+ T cells For immune recognition, the author focused on the four epitopes covering the mutation sites of the concerned variant: GVY, KIA, NYN, and IIW; these four epitopes include the Y144-deletion mutation, K417N/T mutation, and K417N/T mutation on the S protein, respectively.
L452R mutation and I2230T mutation on ORF1a protein
.

Through intracellular IFN-γ detection and MHC-tetramer staining methods, the authors verified that these four epitopes can effectively cause CD8+ T cell immune responses in patients who have recovered from the new crown or volunteers after vaccine immunization
.

In order to further explore the impact of mutations carried by the variants on CD8+ T cell immunity, the authors synthesized antigenic polypeptides carrying mutation sites and compared the antigenic effectiveness of wild-type antigenic polypeptides and mutant polypeptides
.

The comparison found that compared with the wild-type peptide, the CD8+ T cells of the volunteers after immunization with the new crown rehabilitation patients/vaccines have a reduced ability to respond to the antigenic peptides carrying the mutation sites (Figure 6)
.

This shows that the amino acids at these sites are very important for antigen recognition and the production of cellular immunity, and the mutation of these key amino acids by the variants concerned may reduce the virus's stimulation of cellular immunity, thereby achieving the escape of cellular immunity
.

Figure 6: Epitope mutations reduce the efficiency of CD8+ T cell activation in vaccinators and COVID-19 patients.
Focus on the structural mechanism of mutations destroying HLA molecules to present CD8+ T cell epitopes.
Professor Jin Tengchuan’s research group Through the method of crystallography, the crystal structure of the complex of two epitopes and corresponding MHC molecules was analyzed (Figure 7)
.

It can be clearly seen that in the KIA-HLA-A*02:01 complex (Figure 7A), the positively charged side chain of the lysine at position 417 interacts with the W167 of the HLA-A*02:01 protein through the Pi cation.
Firmly binding, the K417N/T mutation carried by the beta variant and the gamma variant will break this interaction, causing the HLA-A*02:01 protein to fail to recognize this epitope.
The author also proved this through biochemical methods.
Conclusion
.

In the NYN-HLA-A*24:02 complex (Figure 7B), the hydrophobic side chain of Leucine at position 452 is buried in the hydrophobic pocket on the surface of the HLA-A*24:02 protein, which is tightly interacted by hydrophobic interactions.
In combination, the mutation of the 452 amino acid at the key position may help the virus escape the cellular immunity mediated by the epitope NYN
.

Figure 7: The crystal structure of KIA&HLA-A*02:01 complex (A) and NYN&HLA-A*24:02 complex (B).
Taking the delta variant as an example, the L452R mutation carried by the delta variant will introduce a positive The charged arginine R completely destroys the original hydrophobic interaction, thereby destroying the presentation of the NYN epitope
.

In July 2021, Motozono et al.
reported in Cell Host & Microbe that L452R mediates the cellular immune escape of the new coronavirus (Motozono et al.
, 2021).
This study also provides a further structural basis for this
.

Summary The escape of the new coronavirus to the immune system by the variants concerned has greatly increased the complexity of the global vaccination plan.
Understanding the immune escape mechanism of the variants concerned is of great significance to the design of future vaccines
.

Previous studies have confirmed that one of the evolutionary dynamics of HIV is to evade cellular immunity by mutating the epitope recognized by CD8+ T cells (Allen et al.
, 2005; Kawashima et al.
, 2009; Moore et al.
, 2002) )
.

In this article, the author used T-Scan technology to screen and identify four key epitopes recognized by the new coronavirus-specific CD8+ T cells, and the new coronavirus focuses on variants (alpha, beta, gamma and delta) at least One of the key epitopes was mutated, revealing that escaping human cellular immunity is a common feature of the new coronavirus epidemic
.

In addition, the conservative epitopes of coronaviruses screened by the author also provide a theoretical basis for the subsequent design of universal vaccines for coronaviruses
.

Zhang Hang, a doctoral student in the School of Pharmacy of Tsinghua University, and Shasha Deng, a doctoral student in the Department of Life Sciences and Medicine of the University of Science and Technology of China, are co-authors of this article
.
Professor Tan Xu and Professor Jin Tengchuan are the corresponding authors of this article .

The research was funded by the National Natural Science Foundation of China, the Chunfeng Fund of Tsinghua University and the pilot project of the Chinese Academy of Sciences
.

Tsinghua University’s Dong Chen laboratory, Lin Xin laboratory and Shi Yan laboratory provided great help to this project
.

Link to the paper: https:// Open for reprint