From a single cell
The method of Siberian scientists will bring the development of a vaccine against HIV closer
The staff of the Institute of Molecular and Cellular Biology SB RAS proposed a simple and effective way to obtain antibodies from single B-lymphocytes. This will significantly reduce the time and cost of creating vaccines against most viral and bacterial agents, including HIV. An article about this was published in the journal BioTechniques (Guselnikov et al., A simple way to increase recovery of the expressed VH and VL genes in single-sorted human B cells).
The human immune system is able to recognize and destroy various pathogens that enter the body: viruses, bacteria, fungi, parasites, as well as its own defective cells, such as tumor cells. Such a specific reaction to a foreign agent (antigen) is carried out by cells of the immune system – lymphocytes.
B-lymphocytes play an important role in the acquired immune response: they synthesize protein molecules (immunoglobulins, or antibodies) against specific foreign structures and secrete them into the blood. These molecules attach to the antigens, so that the latter are identified as foreign and subsequently destroyed.
"We can't know in advance what diseases we will encounter, but we don't need to: our B-lymphocytes are able to produce antibodies against a lot of pathogens. If a person once met with any of them, so-called memory cells from the disease remain in his body. By isolating the DNA of immunoglobulin genes from such cells, we can obtain antibodies against any foreign substance, be it a virus, a bacterium, a fungus or a tumor cell. In the future, they can be used to treat other people," says Sergey Vladimirovich Guselnikov, a senior researcher at the Laboratory of Immunogenetics of the IMKB SB RAS, Candidate of Biological Sciences.
According to the researcher, more than 40 years ago, for the diagnosis and treatment of viral and bacterial infections, a technology was invented that made it possible to isolate immunoglobulin genes from individual B cells of animals - mice, rabbits, monkeys – and on their basis to produce antibodies against certain diseases: HIV, hepatitis, malaria and even Ebola. Of course, it is impossible to treat a person simply with antibodies, for example, mice, constantly – a secondary immune response can develop against them. "Therefore, the efforts of physicians and biologists were aimed at obtaining chimeric antibodies – "stitched" from mouse and human – as well as humanized ones, in which some fragments originally taken from the mouse, necessary to fight the disease, were replaced with human ones. Over the past 10-15 years, the focus of scientists' attention has shifted to obtaining completely human antibodies: they are the best suited for creating therapeutic drugs and vaccines," comments Andrey Alexandrovich Gorchakov, PhD, senior researcher at the Laboratory of Immunogenetics of the IMKB SB RAS.
One of the ways to obtain such antibodies in a laboratory is to isolate B cells from the donor's body that produce antibodies against the desired pathogen, identify their genetic passport and multiply. "Our colleagues from the Moscow Institute of Immunology, led by Professor, Doctor of Biological Sciences Alexander Vasilyevich Filatov, are developing a technology that allows millions of copies of it to be obtained from a single B-cell isolated from the body and thus create a ready–made cell culture that produces antibodies," says Sergey Guselnikov. – We are developing another version of this technology: we isolate single B cells, viable and not, and directly determine the DNA sequence in their immunoglobulin genes, separately for heavy, separately for light antibody chains. It would seem that it is not difficult, but in practice it is possible to establish the sequences of both antibody chains only for 30-70% of single B cells, even in very reputable foreign molecular genetic laboratories."
Novosibirsk biologists have found a way to increase this amount to 90-100%. Sergey Guselnikov clarifies that finding memory B-lymphocytes with a given specificity is a difficult task, because there are very few of them in the body. "Our approach allows us to extract the necessary immune cells from just 1-2 ml of blood with a high degree of probability," says the researcher. To do this, scientists mark them with the target protein of interest. "Rare memory B cells can form antibodies to this agent, and some of them are on the surface. If we are looking for antibodies against the conditional “protein x”, then the B-cell will firmly bind to this protein. We mark the protein, for example, with green fluorescent paint. He sticks to the right B-cell, and it also turns green. The principle is about the same as that of fishing: we catch fish with a very exquisite bait, which is to the taste of only one of their species. The stained cells – and only them – we then sort by individual test tubes, after which the magic of PCR begins," explains Andrey Gorchakov.
Polymerase chain reaction, or PCR, is a method of molecular biology that allows you to easily and multiply increase the number of DNA fragments of interest in a sample. To find out which DNA sequence in a given individual cell encodes an antibody that specifically recognizes the target, scientists must first turn the RNA into so-called complementary DNA (cDNA), and only then conduct PCR. PCR can also be performed directly with DNA, but this is less effective, since there is only one tiny B cell in each test tube, and only one DNA molecule with rearranged immunoglobulin genes in each cell, while there are several orders of magnitude more matrix RNA molecules (and therefore complementary DNA).
PCR from individual cells is not obtained every time. "It can be compared to going to a casino: I got a PCR product - I won, no – I lost. If you received a PCR product immediately for both heavy and light chains - bingo! – you have all the information for antibody synthesis. Sergey suggested not to use the entire volume of complementary DNA entirely for one reaction, as is traditionally done, but to divide it into three parts – roughly speaking, to make not two (one for the light and one for the heavy chain), but six casino bets. First for the heavy chain: it didn't work out – it didn't work out - it worked out. Then for an easy one: it didn't work out – it worked out – it didn't work out. In both cases, you won at least once. This is already enough to leave the casino as a winner," notes Andrey Gorchakov.
"Thanks to this simple procedure, in the experiments given as an example in our article, we received complete information about the structure of antibodies for 21 of the 24 isolated B cells, that is, for almost all," says Sergey Guselnikov.
Scientists say that they initially developed the technique for themselves so that it would be more convenient for them to work and minimize valuable cellular material. Now they are successfully using it for research under a grant from the Russian Foundation for Basic Research (No. 18-29-08051 "Design and validation of artificial non–viral immunogens for induction of germ precursors of broad-spectrum antibodies against HIV", head A.A. Gorchakov), whose goal is to find such components of a future HIV vaccine that will trigger the formation of an antiviral response without adverse reactions.
"We work with B-cells obtained exclusively from the blood of healthy donors," emphasizes Andrey Gorchakov. – Yes, antibodies against HIV that can neutralize most variants of HIV-1 have been found, but in sick people, and in order for them to appear in a healthy person, you need to try hard. And we are trying. The more information we get about the number of B cells capable of forming antibodies against HIV-1, the better."
The ultimate goal of scientists is to reach clinical trials. "It is important for us to see if there are B cells in the blood of an uninfected person that interact with the antigen of interest to us. Specifically for this, we have developed a bait with which we hope to catch the precursor of broadly neutralizing antibodies against HIV-1. If in the future we find in a healthy person the necessary number of B-lymphocytes that have the right properties (they carry exactly the immunoglobulin genes that we are counting on), then we have reason to proceed to tests at the next stage – first on mice and monkeys, and in the future on humans," he emphasizes Sergey Guselnikov.
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