Evaluation of New Coronavirus Vaccine Potency

As of November 26, a total of 60,425,242 people have been diagnosed in the global COVID-19 epidemic, and a total of 1,421,494 people have died.

The COVID-19 epidemic has become normal in most parts of the world, and the arrival of winter in the northern hemisphere will bring a new wave of epidemic peaks, which will bring further threats to human health and the global economy. In the fight against the epidemic, countries around the world have focused on “prevention” and have high hopes for the COVID-19 vaccine.

Why vaccines can protect us?

Vaccination can activate the immune system, protect the body from virus invasion, and produce memory immune cells so that they can “come forward” when needed.

Vaccine intramuscular injection activates immune response

After the vaccine enters the human body as a foreign antigen (or antigen expressed by a nucleic acid vaccine), it first activates the “innate immune system”/”innate immunity”: antigen presenting cells (including macrophages and dendritic cells) Phagocytosis of foreign antigens.

After that, the antigen presenting cells transmit the antigen information to T cells and B cells and activate the “acquired immune system”/”adaptive immunity” (adaptive immunity): 1. Activate T cells, CD4+ T cells secrete a variety of cytokines, To further activate the immune response, CD8+ T cells eliminate virus-infected cells; 2. Activate B cells to produce specific antibodies, and the neutralizing antibodies can protect cells from virus infection.

Finally, the immune system will produce targeted memory B cells and T cells, which are stored in bone marrow and other places for a long time. After that, since the human body has memory immune cells, if it is infected again by the virus, the human body can produce an obvious immune response in a short time, effectively protecting the body.

Vaccine protection performance evaluation

Due to the limitation of the vaccine itself, the vaccinators cannot be protected 100% (clinical protection data: Moderna mRNA vaccine 94.5%, BioNtech mRNA vaccine 95%, AstraZeneca adenovirus vaccine 62-90%), the potency of the vaccine itself, and the vaccine It is necessary to evaluate the protective effect after vaccination.

Neutralizing antibody evaluation

As a neutralizing antibody with antiviral activity, it only accounts for a small part of the antibodies secreted by B cells. The blood titer of the neutralizing antibody is very important. The most important indicator for evaluating the effectiveness of the new crown vaccine is the subject’s body The content of neutralizing antibodies. The principle of neutralizing antibodies to protect the body: binding to the surface protein of the new coronavirus and blocking the binding of the virus protein to the cell surface specific receptor ACE-2, thereby preventing the virus from invading cells (non-neutralizing antibodies have no such function). When the neutralizing antibody titer in the antibody is insufficient, it may lead to the deterioration of the antibody-dependent enhancement (ADE) disease: the virus binds to the Fc receptor on the cell surface through the antibody, and then infects the macrophages. More types of cells lead to disordered immune response.

There are virus infection verification methods and immune blocking verification methods for evaluating the detection of neutralizing antibodies induced by the new crown vaccine.

Virus infection verification

The principle of using neutralizing antibodies to prevent viruses from infecting cells, observe the decrease in the number of virus-infected cells after adding neutralizing antibodies, and calculate the blocking effect.

Due to the high infectivity and pathogenicity of the new coronavirus, the use of live new coronavirus for infection verification must be carried out in a biosafety level 3 or above laboratory, which is limited by laboratory conditions and the source of the virus. In addition, due to the different strains, culture conditions, and evaluation criteria of the results, there are often some differences in the results of live virus detection in different laboratories. In terms of safety and operability, it is more convenient to use pseudoviruses, and pseudoviruses are easy to achieve standardization, which is helpful for evaluating the antiviral effects of vaccines in preclinical and clinical stages.

Neutralizing antibodies inhibit new coronavirus infection of cells

Pseudovirus detection system: New coronavirus pseudovirus (NovoproteinCat.XCV03) has no self-replication ability. It expresses full-length S protein on its surface and contains green fluorescent protein (GFP) and luciferase (Luciferase) reporter genes, which can be specifically infected and expressed ACE-2 cells (Novoprotein Cat. XCC14). The cells infected by the virus express GFP and Luciferase and emit fluorescence, so that the results can be conveniently observed and detected by a chemiluminescence microplate reader to realize quantitative analysis. Pseudovirus assays can be further optimized to achieve high-throughput detection and standardized detection. By replacing the S protein expression plasmid (protein coding gene), the cross-neutralization of anti-S protein antibodies against different mutant strains can be studied.

The neutralizing antibody inhibits the pseudovirus from infecting 293 cells expressing ACE-2, the positive rate of fluorescence and MFI value are detected by flow cytometry, and the fluorescence photos of cells after infection are taken by the inverted fluorescence microscope; 293 cells not expressing ACE-2 are not infected

Neutralizing antibody inhibits pseudovirus from infecting 293 cells expressing ACE-2, chemiluminescence microplate reader quantitatively detects the expression of luciferase; 293 cells not expressing ACE-2 are not infected

Summary: 1. The system has high virus titer, good detection accuracy and repeatability; 2. Pseudovirus contains fluorescent reporter gene, which is conducive to quantification and standardization; 3. Pseudovirus has no self-replication ability and only infects cells expressing ACE-2 ,Safety;

Immune blocking verification

The virus infection verification method is to verify the effectiveness of neutralizing antibodies at the cellular level, and is suitable for the vaccine development stage. For the population after mass vaccination, to quickly assess the protective effect of neutralizing antibodies for each person, a simpler and faster immune blocking verification method can be used.

Immune blocking verification uses the principle that neutralizing antibodies block the binding of S-RBD protein to ACE-2. After adding neutralizing antibodies, detecting the reduced amount of S-RBD protein bound to ACE-2 is used to calculate the drop of neutralizing antibodies. degree.

TRFILF, Time-resolved fluorescence immunochromatography: The vaccinated serum is dropped on the sample area on the left, and the serum is chromatographed to the right under capillary action, and drives the fluorescent nano-micrometer on the binding pad. The ball marked S-RBD (Novoprotein Cat.DRA72) moves together. After the serum reaches the detection area, S-RBD binds to the ACE-2 (Novoprotein Cat.C05Y) immobilized on the detection line, and is fixed on the detection line, and the neutralizing antibody in the serum competitively inhibits S-RBD and ACE -2 combined to reduce the S-RBD fixed on the detection line. After the reaction, the detection area is scanned with ultraviolet light, and the fluorescent nanospheres on the detection line and the quality control line emit fluorescence. Through the intensity and ratio of the fluorescence intensity of the detection line and the quality control line, the fluorescence change can be accurately and quantitatively detected, thereby standardizing the calculation of the neutralizing antibody inhibitory effect.

Summary: 1. The immunochromatographic operation is simple and the detection time is short (<30min), which can be used for on-site screening and large-scale detection; 2. Low cost, high cost performance, and conducive to large-scale detection; 3. Quantitative detection.

T cell immune response assessment

Some current studies have shown that induction of neutralizing antibody responses alone cannot fully protect the body, and the immune response of T cells also plays a very important role. Studies have shown that neutralizing antibodies, helper T cells and killer T cells can be detected in all fully recovered individuals. Generally speaking, people with extensive and well-coordinated immune responses have milder, special, and strong illnesses. The new coronavirus-specific T cell response is a precursor to milder symptoms. Some mild patients will trigger a strong memory T cell response even if no virus-specific antibody response is detected. People over 65 years of age are more likely to have poor T cell responses and uncoordinated immune responses, so they will have a more serious or fatal COVID-19. The large part of the susceptibility of the elderly to COVID-19 seems to be a weaker adaptive immune response, which may be because the elderly have fewer initial T cells. In addition, memory T cells after vaccination will continue to protect the body. Therefore, it is important to evaluate the T cell immune response induced by the vaccine.

Studies have found that new coronavirus N protein-specific T cells have also been detected in people who have not been infected with the new crown, and that the specific T cells in SARS recovered patients have strong cross-reactivity with SARS-CoV-2. This indicates that the common β-coronavirus infection can induce the human body to produce multi-specific and durable T cell immunity to the structural protein NP of the coronavirus. Therefore, to verify the specific T cells induced by the vaccine should use the new crown-specific S-trimer Protein as the main.

To evaluate the T cell immune response triggered by the vaccine, the gold standard is IFN-γ ELISpot to detect the immune response of PBMC (peripheral blood mononuclear cells), that is, use S-trimer Protein to stimulate the PBMC in the same volunteer before and after vaccination. The degree of IFN-γ secreted by the cells should be several times before inoculation.

Enzyme-Linked Immunospot Assay (ELISpot) can detect antibody-secreting cells or cytokine-secreting cells at the single-cell level. IFN-γ, as a cytokine secreted by immunocompetent cells, plays an important role in inducing antiviral immunity, including activation of cytotoxic T lymphocytes (CTL), natural killer cells (NK) cells And phagocytes, etc. The level of IFN-γ produced by the body after vaccine immunization actually reflects the activity of helper T cells. Therefore, using ELISpot to detect the level of IFN-γ is an indirect detection of helper T cell activity.

IFN-γ ELISpot operation process:

1. Coat the bottom of the detection hole with anti-IFN-γ monoclonal antibody;

2. Separate the PBMC of the sample to be tested;

3. Put the PBMC to be tested into the detection hole, and add the stimulus S-trimer Protein (Novoprotein Cat.DRA49) at the same time. During the incubation period, T lymphocytes that respond to S protein will be activated and begin to secrete specific cytokine IFN-γ. These cytokines are simultaneously captured by the monoclonal antibody at the bottom of the plate; cells that do not respond to S protein are not. It is stimulated and does not secrete specific cytokine IFN-γ;

4. Remove the cells, leaving potential “images” of cytokines at the bottom of the plate;

5. Add a biotin-labeled detection antibody, which combines with the cytokines on the “image” to form a sandwich structure of “antibody-antigen-antibody”;

6. Add streptavidin-labeled enzyme solution and combine with the labeled biotin on the detection antibody through streptavidin to form a complex;

7. Adding the chromogenic substrate, under the catalytic decomposition of enzymes, insoluble pigments are generated, which are deposited on the local membrane to form spots and counted.

Each spot represents a specific T lymphocyte that responds to a specific antigen. The number of spots reflects the recognition status of the sample’s cellular immunity: more spots indicate a good immune recognition status, and fewer spots indicate a poor immune recognition status or immune tolerance.

Summary: 1. ELISpot has high sensitivity. Only one positive cell secreting cytokine can be detected in one million negative cells. 2. ELISpot detects the secretion of a single cell, not the average secretion of a cell population. 3. The cytokines detected by ELISpot do not exist in the normal state of cells, but are secreted by living cells after being stimulated by stimuli during the detection process. The strength and number of spots directly reflect the ability of cells to respond to stimuli and to secrete cytokines. 4. The result is intuitive, one positive cell corresponds to one spot.

With the frequent occurrence of good news in the progress of vaccine experiments, dawn has already appeared at the end of the tunnel. In the future, there will be different types of new crown vaccines for the world to choose.