Исследование антигенной специфичности Т-клеточных иммунных реакций в ответ на иммунизацию лабораторных мышей рекомбинантным аденовирусным вектором, кодирующим Spike-белок SARS-CoV-2

• Атауллаханов Равшан Иноятович
• Ушакова Екатерина Игоревна
• Пичугин Алексей Васильевич
• Лебедева Екатерина Семеновна
• Иванов Сергей Валерьевич
• Ожаровская Татьяна Андреевна
• Попова Ольга
• Щербинин Дмитрий Николаевич
• Банделюк Алина Сергеевна
• Зубкова Ольга Вадимовна
• Шмаров Максим Михайлович

Abstract

Введение. Рекомбинантные аденовирусные векторы являются одной из ведущих технологических платформ при разработке и производстве современных вакцин. В России и в других странах мира на базе рекомбинантных аденовирусов созданы и зарегистрированы вакцинные препараты против вируса лихорадки Эбола и коронавирусной инфекции SARS-CoV-2. Проводятся клинические испытания вакцин против гриппа, вируса Марбург, вирусов папилломы человека.

Закодированный в ДНК аденовирусного вектора целевой антиген экспрессируется в организме вакцинированного и против этого целевого белка-антигена развиваются адаптивные иммунные реакции. Векторная частица является вирусной и содержит в себе десятки вирусных антигенов. Следовательно, наряду с иммунными реакциями на целевой антиген в организме вакцинированного могут развиваться иммунные реакции на антигены самого вектора.

Цель данной работы – исследовать, как соотносятся между собой по интенсивности и качеству две разнонаправленные по антигенной специфичности иммунные реакции: против целевого антигена коронавируса и антигенов аденовирусного вектора.

Материал и методы. У мышей C57BL/6 исследовали интенсивность иммунных реакций CD4- и CD8-Т-клеток в ответ на иммунизацию рекомбинантным аденовирусным вектором, кодирующим S-белок SARS-CoV-2 (Ad5-S). Через 2 и 3 мес после иммунизации в селезенке мышей определяли количество и антигенную специфичность CD4- и CD8-T-клеток памяти. Реакцию Т-клеток индуцировали in vitro в совместной культуре с антиген-презентирующими дендритными клетками. Очищенные популяции CD4- и CD8-T-клеток получали сортировкой на лазерном проточном сортировщике BD FACS Aria II. Антиген-презентирующие клетки трансдуцировали аденовирусным вектором Ad5-S, кодирующим S-белок коронавируса, или контрольным рекомбинантным аденовирусным вектором без целевой вставки (Ad5-0). Ответ Т-клеток определяли методом ELISPOT по числу клеток, секретирующих ИФН-γ. В отдельных экспериментах для реактивации CD4-Т-клеток антиген-презентирующие дендритные клетки нагружали рекомбинантным RBD-фрагментом S-белка SARS-CoV-2. Для усиления ответа Т-клеток антиген-презентирующие дендритные клетки стимулировали агонистом TLR4.

Результаты. Установлено, что однократная интраназальная иммунизация вектором Ad5-S в дозе 10⁸ БОЕ индуцирует сильные системные Т-клеточные иммунные реакции у мышей C57BL/6. Через 2 мес после иммунизации в селезенке мышей обнаруживается около 100–200 тыс. антиген-реактивных Т-клеток памяти, секретирующих ИФН-γ при реактивации in vitro дендритными клетками, презентирующими целевой S-антиген SARS-CoV-2. Большинство антиген-реактивных CD8-T-клеток памяти специфично к S-антигену SARS-CoV-2. Содержание таких клеток после иммунизации вектором Ad5-S превышает 1 % от всех CD8-T-клеток. Количество антиген-реактивных CD8-T-клеток памяти, специфичных к аденовирусным антигенам вектора, было приблизительно в 3 раза ниже, чем количество антиген-реактивных CD8-T-клеток памяти, специфичных к целевому S-антигену. Интенсивность иммунной реакции CD4-T-клеток на иммунизацию вектором Ad5-S была сравнима с интенсивностью иммунной реакции CD8-T-клеток. Подавляющее большинство антиген-реактивных CD4-T-клеток памяти было специфично к антигенам аденовирусного вектора. Эти CD4-Т-клетки секретировали ИФН-γ в ответ на рестимуляцию in vitro дендритными клетками, трансдуцированными рекомбинантным аденовирусным вектором Ad5-0 без целевой вставки. Число CD4-T-клеток, реагирующих на рестимуляцию дендритными клетками, нагруженными рекомбинантным RBD, было исчезающе малым.

Показана возможность повышения интенсивности ответа CD8-T-клеток, специфичных к целевому S-антигену, путем увеличения дозы вектора Ad5-S при трансдукции антиген-презентирующих дендритных клеток, а также путем использования TLR4-агониста для стимуляции антиген-презентирующих дендритных клеток.

Предложены возможные механизмы кооперативного взаимодействия CD8-T-клеток, специфичных к целевому S-антигену SARS-CoV-2, и CD4-T-клеток, специфичных к антигенам аденовирусного вектора. Рассматриваются возможные пути усиления ответа CD4-T-клеток на целевой S-антиген.

Заключение. Иммунизация рекомбинантным аденовирусным вектором, кодирующим S-антиген SARS-CoV-2, индуцирует сильные CD8- и CD4-T-клеточные иммунные реакции с формированием массивного пула антиген-реактивных Т-клеток памяти. CD8- и CD4-T-клетки, реагирующие на иммунизацию вектором Ad5-S, различаются своей специфичностью к антигенам. CD8-T-клетки памяти в основном специфичны к целевому S-антигену SARS-CoV-2, CD4-T-клетки памяти специфичны к антигенам аденовирусного вектора.

Keywords:рекомбинантный аденовирусный вектор; S-антиген SARS-CoV-2; Т-клетки памяти; антигенная специфичность

Introduction

Recombinant adenoviral vectors have become one of the leading technological platforms in the development and production of modern vaccines. In Russia, vaccine preparations against the Ebola virus based on recombinant human adenovirus serotype 5 [1], as well as vaccine preparations against SARS-CoV-2 infection/COVID-19 based on recombinant human adenoviruses serotypes 5 and 26, have been registered [2, 3]. In other countries of the world, vaccines against COVID-19 based on recombinant human and chimpanzee adenoviruses have been registered - Ad5-nCov (CanSino Biologics, China) [4], COVID-19 Ad26COVs1 (Johnson & Johnson, Netherlands/USA) [5] and ChAdOx1 nCoV-19 (Oxford University/AstraZeneca, UK) [6]. In addition to the above, vaccines based on adenoviral vectors against influenza [7], Marburg virus [8], and human papillomaviruses [9] have been developed and are at various stages of clinical trials.

The target antigen encoded in the DNA of the adenoviral vector is expressed in the body of the vaccinated person, and adaptive immune reactions develop against this target antigen protein. Specific antibodies are produced, T-cells multiply and differentiate, capable of protecting against infection and regulating various types of anti-infective defense, long-lived T- and B-memory cells are formed, which provide the body with long-term protection.

The vector particle is viral and contains dozens of viral antigens. Consequently, along with immune reactions to the encoded target antigen, immune reactions to the antigens of the vector itself can develop in the body of the vaccinated person. In the case of an adenoviral vector encoding a coronavirus target antigen, these two antigenic entities are antigens from two completely different viruses - the adenovirus and the coronavirus. Both groups of antigens are immunogenic. The body of the vaccinated person can develop adaptive immune reactions against the target coronavirus antigen and against adenovirus antigens.

How do two immune reactions, different according to their antigenic specificity i. e. directed against the target coronavirus antigen and adenoviral vector antigens, relate to each other in intensity and quality? This article is exploring the answers to this question.

We studied the intensity and antigen specificity of the adaptive responses of CD4- and CD8-T-cells after immunization of laboratory mice with a coronavirus vaccine, which is an adenoviral vector encoding the S-antigen of SARS-CoV-2. We determined the intensity and quality of T-cell reactions directed against the protein components of the vector and the target S-antigen encoded in this vector according to the number of splenic CD4- and CD8-T-cells specific to the S-antigen or antigens of the adenoviral vector. The data obtained are very interesting, since they not only characterize the intensity and nature of T-cells that differ in specificity to adenovirus and S-antigens, but also indicate a previously unknown synergy between CD4- and CD8-T-cells with different antigen specificities. Practically useful aspects of obtained data for improvement of immunogens based on adenoviral vectors encoding the target antigen are discussed.

Material and methods

Experimental animals. Inbred C57BL/6 mice, females, 2 months of age were obtained from the laboratory animal nursery of the Institute of Bioorganic Chemistry, Russian Academy of Sciences (Pushchino, Moscow region). The animals were kept under standard conditions in the vivarium of the N.F. Gamaleya FRC of Epidemiology and Microbiology, MOH of Russia. All experimental procedures with animals were performed according to the Rules regulating research work with laboratory animals at the N.F. Gamaleya FRC of Epidemiology and Microbiology, MOH of Russia and NRC Institute of Immunology, FMBA of Russia.

Recombinant adenoviral vectors. A recombinant human adenovirus, serotype 5 (Ad5-S), expressing the S-protein gene of the SARS-CoV-2 coronavirus strain Wuhan (uniprot id: P0DTC2) was used in this work. Artificial gene synthesis was carried out by the Evrogen (Russia), the nucleotide sequence of the gene was optimized for expression in human cells and the Kozak sequence was added.

The Ad5-S∆N recombinant adenoviral vector containing SARS-CoV-2 S-protein gene with the deleted genetic sequence, encoding the 13 N-terminal amino acid residues that make up the leader peptide, was also used in this study. The recombinant adenoviral vector Ad5-0 without the target insert was obtained by a team of authors previously [10].

Immunization. Mice were immunized with the Ad5-S vector at a dose of 10⁸ PFU by a single intranasal administration in a volume of 50 μl. Control mice received a single intranasal dose of 50 μl buffer in which a suspension of vector particles was administered.

In vitro cell cultures. Suspensions of mouse spleen and bone marrow cells were obtained under aseptic conditions using standard methods. All cell cultures were incubated in complete medium (CM) composed of DMEM (Thermo Fisher Scientific, USA) with 25 mM HEPES, supplemented with a mixture of non-essential amino acids, 10 % fetal calf serum (Cytiva, GE Healthcare Life Sciences HyClone, USA), 2 mM L-glutamine, 1 mM sodium pyruvate, 50 μM β-mercaptoethanol and 10 μg/ml gentamicin (all PanEco reagents, Russia), at 37 °C, in a humidified atmosphere containing 5 % CO₂.

Mice were euthanized using cervical dislocation. The spleen was isolated under aseptic conditions. Cell suspension was prepared in phosphate buffer containing 0.5 % bovine albumin (PBS-BSA). The fraction of mononuclear cells was obtained by centrifugation in a Ficoll gradient (PanEko, Russia). Cells were washed with PBS-BSA. To sort CD4- and CD8-T-cells, the suspension was stained with CD4-PE and CD8-APC antibodies (BD Biosciences, USA). Leukocytes were counted using CD45-BV510 staining (BioLegend, USA). The viability of suspensions was determined by staining with DAPI. Cell sorting was performed using a BD FACS Aria II flow cytometer-sorter (Becton-Dickinson, USA). The purity of the resulting CD4- and CD8-T-cell populations was 98 %.

Preparation of antigen-presenting dendritic cells. Dendritic cells and macrophages from mouse bone marrow were in vitro differentiated in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF, Sigma, USA). The femurs and tibias of mice were isolated under aseptic conditions. The bone marrow was washed out with a syringe (25G needle) into a Petri dish with a PBS-BSA solution and thoroughly suspended. Cells were washed with PBS-BSA. Erythrocytes were removed by osmotic lysis. Nucleated cells were cultured in CM with the addition of 10 ng/ml GM-CSF at 37 °C and 5 % CO₂. After 3-4 days, an equal volume of CM with GM-CSF was added to the cultures. On day 7, dendritic cells were activated with lipopolysaccharide (LPS) from E. coli (Sigma, USA) at a final concentration of 0.1 μg/ml and loaded with antigen. Recombinant adenoviral vectors rAd5-S, rAd5-S∆N, rAd5-0, as well as a recombinant RBD-fragment of the SARS-CoV-2 S-protein were used as antigens.

Analysis of T-cell responses specific to target antigens. The antigen-specific T-cell response was assessed by IFN-γ secretion 2 and 3 months after immunization. A specific T-cell response was induced in a co-culture of T-cells with antigen-presenting dendritic cells preloaded with the target antigen. To analyze T-cells recognizing epitopes of the SARS-CoV-2 S-antigen, dendritic cells were transduced with Ad5-S or Ad5-SΔN vectors, or loaded with a recombinant RBD-fragment of the SARS-CoV-2 S-protein. To analyze T-cells recognizing adenoviral vector antigens, dendritic cells were transduced with the adenoviral Ad5-0 vector without any target insert. When transducing dendritic cells with one of the three Ad-vectors used, a concentration of 100 PFU of Ad-vector per 1 dendritic cell was used. In separate experiments, two concentrations of Ad5-S were compared: 30 and 100 PFU per 1 dendritic cell.

70 000 mononuclear cells, or 30 000 CD4-T-cells, or 30 000 CD8-T-cells isolated from mouse spleen using the BD FACS Aria II sorter were placed into the wells of the ELISPOT plate (BD TM ELISPOT Mouse IFN-γ, cat. 551083, BD Bioscience, USA). 50 000 dendritic cells were added to the same wells of the culture plate as antigen-presenting cells. Co-cultures of T- and dendritic cells were incubated for 36 h at 37°C and 5 % CO₂, thereafter IFN-γ-secreting cells were detected according to the ELISPOT manufacturer’s instructions. Photographs of each well were taken using a Levenhuk DTX 700 LCD microscope (Levenhuk, USA), the number of spots was determined using the ImageJ computer program (NIH, USA). The number of IFN-γ-secreting antigen-reactive T cells was presented per 1 mln CD4- or CD8-T-cells.

The number of antigen-reactive memory T-cells was determined by the secretion of IFN-γ after in vitro reactivation with the corresponding antigen. The number of effector T-cells was determined by the secretion of IFN-γ in cultures without antigen restimulation.

Statistical analysis. Statistical data analysis was performed using Graph-Pad Prism 9.0 software (GraphPad Software Inc., USA). To assess the significance of differences between two independent groups, the Mann-Whitney U-test was used. The Wilcoxon test was used to analyze the difference between two related groups. The Friedman method was used to analyze the three related groups. Differences between groups with p-values below 0.05 were considered significant. The graphs show medians and the 25^th and 75^th percentiles. In Tab. 1 and Fig. 9 the mean values and standard deviations are shown.

Results

Immunization of C57BL/6 mice with the Ad5-S vector induces a potent adaptive T-cell immune response

The schematic diagram of the experiments is shown in Figure 1. C57BL/6 mice were immunized once with the recombinant Ad5-S vector. After 2 and 3 months, the number of T-cells specifically responding by IFN-γ secretion to adenovirus antigens or the SARS-CoV-2 S-antigen, encoded in the vector, was determined in the spleens of immunized mice. Individual IFN-γ-secreting cells were identified by ELISPOT. IFN-γ-secreting cells in populations of splenic mononuclear cells or sorted CD4- and CD8-T-cells were studied using various variants of this method. The levels of T-cell response to the Ad-vaccine were compared with the background level of IFN-γ-secreting cells in the spleen of control mice that received a sham immunization with an appropriate volume of buffer solution.

The results of a representative experiment in Tab. 1 and Fig. 2 demonstrate the immune response of C57BL/6 mice to vaccination with the Ad5-S vector. More than 5000 IFN-γ-secreting cells per 1 mln mononuclear cells accumulated in the spleens of immunized mice. The number of IFN-γ-secreting cells per spleen reached 150 000-200 000, which proves a high level of immune response to immunization.

With two vectors, Ad5-S and Ad5-S∆N, which encode two different structures of the SARS-CoV-2 S-protein, we could transduce antigen-presenting dendritic cells with each of these vectors to compare the two versions of the S-antigen. The fundamental difference between them was that the full-length S-protein, synthesized during Ad5-S transduction, penetrates the endoplasmic reticulum using the leading N-terminal peptide. In contrast, the S-protein encoded in Ad5-S∆N lacks the N-terminal peptide. It is unable to penetrate the endoplasmic reticulum and remains in the cytosol, which is optimal for subsequent proteasome processing and presentation of peptide protein fragments in the context of MHC class I. A comparison of the IFN-γ secreting cell numbers upon reactivation by dendritic cells transduced with Ad5-S or Ad5-S∆N showed a slight advantage of Ad5-S∆N, but it was not statistically significant (Tab. 1, Fig. 2).

When transducing antigen-presenting dendritic cells with the Ad5-0 adenoviral vector without a target insert, we are able to present peptide fragments of adenovirus protein antigens on the surface of the dendritic cell. In this case, the most probable is the presentation of adenovirus antigenic peptides in the context of MHC class II, since the Ad5-0 vector is a non-replicating recombinant virus and, therefore, adenovirus proteins cannot be de novo synthesized in a dendritic cell. They must be processed in a manner typical for the processing and presentation of endocytosed antigens, which are characterized by expression on the surface of the antigen-presenting cell in complex with MHC class II.

The results presented in Fig. 2 and in Tab. 1 indicate that after immunization of C57BL/6 mice with the Ad5-S adenoviral vector encoding the S-antigen of SARS-CoV-2, in the spleen of mice accumulated not only IFN-γ secreting cells responsive to in vitro reactivation by the S-antigen (in form Ad5-S or Ad5-S∆N), but also cells secreting IFN-γ in response to in vitro reactivation by adenovirus antigens (in the form Ad5-0). Thus, reactivation with dendritic cells transduced with the Ad5-0 adenoviral vector without any target insert revealed more than 6000 IFN-γ-secreting cells per 1 million splenic mononuclear cells, which was quite comparable to the level of the immune response in response to the target S-antigen. In other words, immunization with the adenoviral Ad5-S vector, encoding the S-antigen of the coronavirus, induces immune reactions against the target S-antigen, as well as antigens of the adenoviral vector itself. These two differently directed immune reactions are equal in intensity.

By examining unsorted spleen cells or a fraction of mononuclear cells using the ELISPOT method, it is impossible to determine the nature of the cells that secrete IFN-γ in response to in vitro reactivation by dendritic cells presenting on their surface adenoviral or coronaviral antigenic peptides, or both. To identify the nature of the responding cells, we examined the responses of CD4- and CD8-T-cells purified by cell sorting from the spleens of immunized or control mice.

CD8 T cells that specifically respond to the target S-antigen and adenoviral vector antigens

CD8 T cells recognize foreign antigenic peptides in the context of MHC class I. Antigenic peptides of proteins de novo synthesized in the dendritic cell associate with MHC class I, and such a complex is suitable for recognition by CD8-T-cell receptors [9]. With this classical knowledge in mind, we loaded dendritic cells with vectors encoding the S antigen (Ad5-S or Ad5-SΔN) or the Ad5-0 vector without an expression cassette with a target insert. In the latter case, there should not have been any presentation of any foreign antigenic peptides in complex with MHC class I, since the Ad5-0 vector does not induce de novo synthesis of any viral proteins. There are reports in the literature of so-called cross-presentation, when the classical pathway of antigen presentation is disrupted and peptide fragments of endocytosed proteins are presented in the context of MHC class I. For this reason, we checked whether cross-presentation of recombinant adenovirus proteins occurs, which could be seen by the reactivation of CD8-T-cells in their contact with dendritic cells pre-loaded with Ad5-0.

In our experiments, by culturing CD8-T-cells isolated by sorting from the spleen of Ad5-S immunized mice, together with antigen-presenting dendritic cells transduced with Ad5-S, or Ad5-SΔN, or Ad5-0, we assessed the number of CD8-T-cells reacting to the SARS-CoV-2 S-antigen encoded in these vectors or to the own antigens of the adenoviral vector.

The results of the study are presented in Fig. 3 and 4. They accurately determine the antigenic specificity of memory CD8-T-cells formed in the mouse body 2 or 3 months after immunization with the recombinant Ad5-S adenoviral vector encoding the SARS-CoV-2 S-protein. On average, more than 10 000 CD8-T-cells that specifically recognize the target coronavirus S-antigen (per 1 million CD8-T-cells) accumulated in the spleen of mice immunized with Ad5-S. Consequently, the immune response of C57BL/6 mice to immunization with the Ad5-S vector was so strong that the number of memory CD8-T-cells specifically recognizing the target S-antigen exceeded 1 % of all CD8-T-cells.

It is known that only the largest clonotypes of memory T-cells, specific for any antigen, reach sizes bigger than 1 % of the entire T-cell population.

In the spleen of immunized mice, 2 and 3 months after immunization with Ad5-S, the content of memory CD8-T-cells that react to adenovirus antigens (reactivation with the Ad5-0 vector) was several times lower than the content of S-antigen antigen-reactive CD8-T-cells (reactivation with Ad5-S or Ad5-S∆N vectors, Fig. 3 and 4). Consequently, cross-presentation of the adenoviral vector’s own proteins occurred to some extent, ensuring the presentation of adenoviral antigenic peptides in the context of MHC class I and allowing CD8-T-cells to recognize and respond to recombinant adenoviral vector antigens, despite the lack of de novo adenoviral protein synthesis in antigen-presenting dendritic cells.

A comparison of the number of memory CD8-T-cells, secreting IFN-γ in response to the S-antigen or adenovirus antigens, 2 and 3 months after immunization did not reveal significant differences between these two time points of the C57BL/6 mice immune response to Ad5-S immunization (Fig. 5). This means that in 2 months after immunization the exponential (proliferative) phase of the immune reaction ended, and contraction of the T-cell expanded population occurred with the formation of a pool of long-lived memory CD8-T-cells. The number of effector CD8-T-cells in the spleen of mice 2 and 3 months after Ad5-S immunization was an order of magnitude lower than the number of memory CD8-T-cells, which also indicated the completion of the effector phase of the immune reaction and the onset of the long-term immune memory phase (Fig. 6).

CD4-T-cells that specifically respond to the target S-antigen and adenoviral vector antigens

A study of the antigen specificity of a purified population of CD4-T-cells isolated by sorting from the spleen of mice immunized with Ad5-S showed that almost all memory CD4-T-cells respond to adenoviral vector antigens. CD4-T-cells specific to the coronavirus S-protein were either very few or absent in the spleen of mice immunized with Ad5-S (Fig. 7, 8). When reactivated by dendritic cells loaded with Ad5-0, the number of the IFN-γ-secreting CD4-T-cells was the same as when reactivated by dendritic cells loaded with Ad5-S or Ad5-SΔN. These data indicate that virtually all antigen-reactive memory CD4-T-cells recognize adenoviral vector antigens rather than the target coronavirus S-antigen. This conclusion is also consistent with the lack of response of CD4-T-cells from the spleen of mice immunized with Ad5-S to in vitro reactivation by dendritic cells loaded with the recombinant RBD-fragment of the coronavirus S-protein (Fig. 8, bottom panel).

Possibility of enhancing the T-cell response by increasing the dose of Ad5-S vector and/or using a molecular adjuvant

In previous sections of the article, we presented evidence that Ad5-S immunization induces an intense CD8-T-cell response specific to the target coronavirus S-protein. At the same time, the vaccine induces an intensive CD4-T-cell response specific to adenoviral vector antigens, but does not induce the desired CD4-T-cell response to the target S-antigen.

The intensity of weak immune reactions can be increased. There are various methods for this. One simple way is to increase the dose of the immunogen. Another method is to use an immune response enhancer, a molecular adjuvant that acts at the level of antigen-presenting cells. In this work, we tested both of these methods to enhance the response of T-cells specific to the S-antigen of SARS-CoV-2. The results are presented in Fig. 9-11.

Increasing the dose of immunogen can indeed significantly increase the response of T-cells recognizing a given antigen on the surface of antigen-presenting dendritic cells. When the Ad5-S dose was increased from 30 to 100 PFU (per dendritic cell), the number of CD8-T-cells responding to the S antigen increased approximately 2-fold (Fig. 9A). Even greater enhancement was achieved by activating antigen-presenting cells using a molecular adjuvant. We used a TLR4 agonist, an acidic peptidoglycan with a molecular weight bigger than 2 million Dalton (Avexima, Russia, Fig. 9B and 10). At a dose of 30 PFU of Ad5-S, stimulation of dendritic cells with a TLR4 agonist increased the number of responding CD8-T-cells by approximately 6-fold, and at a dose of 100 PFU of Ad5-S, the T-cell response was enhanced by 4-fold (Fig. 9 and 10).

Notably, the use of the same TLR4-agonistic molecular adjuvant failed to significantly enhance the vanishingly weak CD4-T-cell response to RBD (Fig. 11). Under the influence of the TLR4 agonist, the ELISPOT reaction revealed a few IFN-γ-secreting CD4-T-cells, but their number remained too small to consider this a reliable immune response of T-cells to the RBD-antigen.

Discussion

In this work, we conducted a detailed study of the intensity and antigen specificity of the CD8- and CD4-T-cell immune responses induced in mice by the recombinant adenoviral Ad5-S vector encoding S-protein of SARS-CoV-2. Immunization with such an adenoviral vector, in principle, has the potential to induce immune responses directed against antigens of two completely different viruses, coronavirus and adenovirus. Fig. 12 shows the major adenovirus antigens and their number per each adenovirus particle. When a person is immunized with a dose of 10¹¹ particles of the adenoviral vector, the total mass of only major viral proteins is ~ 75 μg. This dose is sufficient to induce immune reactions against adenovirus antigens. It is well known that upon immunization with such recombinant vectors containing a target insert, quite intensive immune reactions develop in the body of the vaccinated person not only against the target antigen, but also against the antigens of the vector itself. To minimize the impact of antibodies specific to the adenoviral vector on the effectiveness of immunization, the Sputnik V vaccine against coronavirus infection COVID-19 is composed of two components based on recombinant human adenoviruses serotypes 5 and 26 [2, 3].

The results of the study described in this article demonstrate that the Ad5-S vaccine vector induces strong CD4- and CD8-T-cell immune responses in C57BL/6 mice. Two months after immunization, the number of T-cells recognizing the target coronavirus S-antigen exceeded 1 % of all splenic T-cells (Fig. 3 and 4). This large size of the T-cells population responding to a single antigen is comparable to the largest (hyper-expanded) T-cell clonotypes produced in mice or humans as a result of intensive immune reactions to infections or vaccinations.

The intensive T-cell response was systemic in nature, since the vaccine was administered intranasally, and a large number of S-antigen-specific CD8-T-cells was recorded in the spleen of immunized mice. The numbers of S-antigen-specific memory CD8-T-cells compared in 2 and 3 months after immunization proves that the proliferative and effector phases of the immune response were completed during 2 months. By this time, a stable pool of long-lived memory T-cells had formed. Two months after immunization with the Ad5-S vector, 9 out of 10 CD8-T-cells specific to S-antigen were represented by long-lived memory T-cells, and only 1 out of 10 cells had the properties of effector T-cells (Fig. 5 and 6).

Memory CD8-T-cells, which specifically respond to antigens of the adenoviral vector, were also found in fairly large numbers in the spleen of mice 2-3 months after immunization with Ad5-S. Although the number of these cells was several times less than the number of memory CD8-T-cells that specifically react to the target S-antigen of the coronavirus, the results obtained indicate cross-presentation of antigens belonging to the recombinant adenovirus capsid.

The intensity of the CD4-T-cell response to vaccination with the Ad5-S vector was as high as the intensity of the CD8-T-cell response. However, the vast majority of memory CD4-T-cells in the spleen of mice 2-3 months after immunization with Ad5-S were specific to the antigens of the adenoviral vector, and not to the target S-antigen encoded in this vector (Fig. 7 and 8).

We have shown that the robust S-antigen-specific CD8-T-cell response can be further enhanced by increasing the dose of adenoviral vector (Fig. 9) and/or by using a TLR4 agonist (Fig. 10) that stimulates antigen-presenting cells [11]. The vanishingly weak response of CD4-T-cells to S-antigen could not be increased using a TLR4 agonist (Fig. 11).

The deficient response of S-antigen-specific CD4-T-cells can be significantly improved. To do this, it is enough to slightly modify the target S-protein gene in the Ad5-S adenoviral vector. A secretory version of the coronavirus S-protein to be encoded in the vector, together with or instead of the membrane version of the same S-protein. The secretory form of the S-protein to be released in the environment around the dendritic cell in which the Ad5-S vector encoding the secretory S-protein is expressed. Such a release will allow endocytosis of the secretory S-protein, processing of this antigen along the endocytic pathway and presentation of its peptide fragments in the context of MHC class II. This type of presentation of S-protein fragments on the surface of an antigen-presenting dendritic cell ensures a response of CD4-T-cells specific to S-antigen epitopes. The S-antigen specific T-helper cell shortage will be overcome.

It is well known that the help of CD4-T-cells is required at the initial stage of the primary CD8-T-cell response, in the effector phase and during the formation of long-term CD8-T-cell memory, as well as during the induction of a secondary CD8-T-cell response [12-16]. Our study showed that after immunization with Ad5-S, an intensive CD8-T-cell response to S-antigen with the formation of large numbers of memory CD8-T-cells occurred in the absence of an S-antigen-specific CD4-T-cell response. At the same time, a strong CD4-T-cell response to the adenoviral vector antigens occurred. These data suggest that in their response to S-antigen, CD8-T-cells received assistance from numerous CD4-T-cells specific for adenoviral vector antigens. This is quite possible because the adenoviral vector Ad5-S carries both collections of antigenic peptides - coronavirus S-protein and adenoviral capsid proteins. When Ad5-S vector particles transduce a dendritic cell, a mosaic presentation of antigens of different nature occurs on the surface of the same antigen-presenting cell. Antigenic peptides of the S-protein are displayed in complex with MHC class I, and peptides of adenoviral vector proteins are displayed on the surface of the same dendritic cell in complex with MHC class II (Fig. 13). CD8-T-cells recognize and respond to S-protein peptides in complex with MHC class I, while CD4-T-cells recognize and respond to antigenic peptides of adenoviral proteins in complex with MHC class II (Fig. 13A). Being in close proximity to each other on the surface of the same antigen-presenting cell, CD4- and CD8-T-cells may well interact. In particular, CD4-T-cells can promote the activation, proliferation, and differentiation of their neighboring CD8-T-cells with the help of IL-2, TNF-α, IFN-γ, and other secreted cytokines (Fig. 13B). This interaction of CD4- and CD8-T-cells will not depend in any way on the origin of the antigenic peptides (coronavirus or adenoviral) that these two T-cells recognize. The spatial proximity of CD4- and CD8-T-cells on one antigen-presenting cell is important, as well as the active state of both cells, i.e. the presence of signals necessary for the activation of these T-cells on the surface of the dendritic cell.

The mechanism of interaction between CD4- and CD8-T-cells on the surface of an antigen-presenting cell described above is a hypothesis that is subject to experimental testing. There are data in the literature about the cooperation of specific to the same antigen CD4- and CD8-T-cells upon their contact with an antigen-presenting dendritic cell [17]. The novelty of our hypothesis is that we assume the presence of interaction between CD4- and CD8-T-cells specific to two different antigens, under condition that the peptide fragments of these antigens are exposed on the surface of the same antigen-presenting cell.

This condition is fully met when the antigen-presenting cells are transduced with a recombinant virus encoding the target antigen. In this work, we considered a special case when this condition is met - this is an adenoviral vector encoding the coronavirus S-antigen.

Conclusions

1. Immunization of C57BL/6 mice with the Ad5-S recombinant adenoviral vector, encoding the full-length SARS-CoV-2 S-protein, induces intensive reactions of CD4- and CD8-T-cells, culminating after 2 months in the formation of a large population of long-lived antigen-reactive memory T cells.

2. Most of the antigen-reactive memory CD8-T-cells formed as a result of immunization with the recombinant Ad5-S vector specifically recognize and respond to antigenic epitopes of the coronavirus S-protein. CD4-T-cells induced by Ad5-S immunization recognize and respond to antigenic epitopes of the adenoviral vector.

Acknowlegements. The article is dedicated to the memory of Academician of the Russian Academy of Sciences Rakhim Musaevich Khaitov - mentor, colleague and friend.

References

1. Dolzhikova I.V., Zubkova O.V., Tukhvatulin A.I., Dzharullaeva A.S., Tukhvatulina N.M., Shcheblyakov D.V., Shmarov M.M., Tokarskaya E.A., Simakova Y.V., Egorova D.A., Scherbinin D.N., Tutykhina I.L., Lysenko A.A., Kostarnoy A.V., Gancheva P.G., Ozharovskaya T.A., Belugin B.V., Kolobukhina L.V., Pantyukhov V.B., Syromyatnikova S.I., Shatokhina I.V., Sizikova T.V., Rumyantseva I.G., Andrus A.F., Boyarskaya N.V., Voytyuk A.N., Babira V.F., Volchikhina S.V., Kutaev D.A., Bel’skih A.N., Zhdanov K.V., Zakharenko S.M., Borisevich S.V., Logunov D.Y., Naroditsky B.S., Gintsburg A.L. Safety and immunogenicity of GamEvac-Combi, a heterologous VSV- and Ad5-vectored Ebola vaccine: An open phase I/II trial in healthy adults in Russia. Hum Vaccin Immunother. 2017; 13 (3): 613–20. DOI: https://doi.org/10.1080/21645515.2016.1238535

2. Logunov D.Y., Dolzhikova I.V., Zubkova O.V., Tukhvatullin A.I., Shcheblyakov D.V., Dzharullaeva A.S., Grousova D.M., Erokhova A.S., Kovyrshina A.V., Botikov A.G., Izhaeva F.M., Popova O., Ozharovskaya T.A., Esmagambetov I.B., Favorskaya I.A., Zrelkin D.I., Voronina D.V., Shcherbinin D.N., Semikhin A.S., Simakova Y.V., Tokarskaya E.A., Lubenets N.L., Egorova D.A., Shmarov M.M., Nikitenko N.A., Morozova L.F., Smolyarchuk E.A., Kryukov E.V., Babira V.F., Borisevich S.V., Naroditsky B.S., Gintsburg A.L. Safety and immunogenicity of an rAd26 and rAd5 vector-based heterologous prime-boost COVID-19 vaccine in two formulations: two open, non-randomised phase 1/2 studies from Russia. Lancet. 2020; 396 (10255): 887–97. DOI: https://doi.org/10.1016/S0140-6736(20)31866-3

3. Logunov D.Y., Dolzhikova I.V., Shcheblyakov D.V., Tukhvatulin A.I., Zubkova O.V., Dzharullaeva A.S., Kovyrshina A.V., Lubenets N.L., Grousova D.M., Erokhova A.S., Botikov A.G., Izhaeva F.M., Popova O., Ozharovskaya T.A., Esmagambetov I.B., Favorskaya I.A., Zrelkin D.I., Voronina D.V., Shcherbinin D.N., Semikhin A.S., Simakova Y.V., Tokarskaya E.A., Egorova D.A., Shmarov M.M., Nikitenko N.A., Gushchin V.A., Smolyarchuk E.A., Zyryanov S.K., Borisevich S.V., Naroditsky B.S., Gintsburg A.L. Gam-COVID-Vac Vaccine Trial Group. Safety and efficacy of an rAd26 and rAd5 vector-based heterologous prime-boost COVID-19 vaccine: an interim analysis of a randomised controlled phase 3 trial in Russia. Lancet. 2021; 397 (10275): 671–81. DOI: https://doi.org/10.1016/S0140-6736(21)00234-8

4. Zhu F.C., Li Y.H., Guan X.H., Hou L.H., Wang W.J., Li J.X., Wu S.P., Wang B.S., Wang Z., Wang L., Jia S.Y., Jiang H.D., Wang L., Jiang T., Hu Y., Gou J.B., Xu S.B., Xu J.J., Wang X.W., Wang W., Chen W. Safety, tolerability, and immunogenicity of a recombinant adenovirus type-5 vectored COVID-19 vaccine: a dose-escalation, open-label, non-randomised, first-in-human trial. Lancet. 2020; 395 (10240): 1845–54. DOI: https://doi.org/10.1016/S0140-6736(20)31208-3

5. Solforosi L., Kuipers H., Jongeneelen M., Rosendahl Huber S.K., van der Lubbe J.E.M., Dekking L., Czapska-Casey D.N., Izquierdo Gil A., Baert M.R.M., Drijver J., Vaneman J., van Huizen E., Choi Y., Vreugdenhil J., Kroos S., de Wilde A.H., Kourkouta E., Custers J., van der Vlugt R., Veldman D., Huizingh J., Kaszas K., Dalebout T.J., Myeni S.K., Kikkert M., Snijder E.J., Barouch D.H., Böszörményi K.P., Stammes M.A., Kondova I., Verschoor E.J., Verstrepen B.E., Koopman G., Mooij P., Bogers W.M.J.M., van Heerden M., Muchene L., Tolboom J.T.B.M., Roozendaal R., Brandenburg B., Schuitemaker H., Wegmann F., Zahn R.C. Immunogenicity and efficacy of one and two doses of Ad26.COV2.S COVID vaccine in adult and aged NHP. J Exp Med. 2021; 218 (7): e20202756. DOI: https://doi.org/10.1084/jem.20202756

6. ChAdOx1-S vaccine for prevention of COVID-19. Aust Prescr. 2021; 44 (2): 59–61. DOI: https://doi.org/10.18773/austprescr.2021.012

7. Online database of Clinical trials. URL: https://www.clinicaltrials.gov/study/NCT01006798?cond=%20Influenza%20Vaccines&page=4&limit=100&rank=371

8. Online database of Clinical trials. URL: https://www.clinicaltrials.gov/study/NCT05817422?cond=adenovirus%20vectored%20vaccine%20&limit=100&rank=5

9. Kedzierska K., Koutsakos M. The ABC of major histocompatibility complexes and T cell receptors in health and disease. Viral Immunol. 2020; 33 (3): 160–78. DOI: https://doi.org/10.1089/vim.2019.0184

10. Shmarov M.M., Sedova E.S., Verkhovskaya L.V., Rudneva I.A., Bogacheva E.A., Barykova Y.A., Shcherbinin D.N., Lysenko A.A., Tutykhina I.L., Logunov D.Y., Smirnov Y.A., Naroditsky B.S., Gintsburg A.L. Induction of a protective heterosubtypic immune response against the influenza virus by using recombinant adenoviral vectors expressing hemagglutinin of the influenza H5 Virus. Acta Naturae. 2010; 2 (1): 111–18. (in Russian)

11. Lebedeva E., Bagaev A., Pichugin A., Chulkina M., Lysenko A., Tutykhina I., Shmarov M., Logunov D., Naroditsky B., Ataullakhanov R. The differences in immunoadjuvant mechanisms of TLR3 and TLR4 agonists on the level of antigen-presenting cells during immunization with recombinant adenovirus vector. BMC Immunol. 2018; 19 (1): 26. DOI: https://doi.org/10.1186/s12865-018-0264-x

12. Janssen E.M., Lemmens E.E., Wolfe T., Christen U., von Herrath M.G., Schoenberger S.P. CD4⁺ T cells are required for secondary expansion and memory in CD8⁺ T lymphocytes. Nature. 2003; 421 (6925): 852–56. DOI: https://doi.org/10.1038/nature01441

13. Sun J.C., Bevan M.J. Defective CD8 T cell memory following acute infection without CD4 T cell help. Science. 2003; 300 (5617): 339–42. DOI: https://doi.org/10.1126/science.1083317

14. Shedlock D.J., Shen H. Requirement for CD4 T cell help in generating functional CD8 T cell memory. Science. 2003; 300 (5617): 337–39. DOI: https://doi.org/10.1126/science.1082305

15. Novy P., Quigley M., Huang X., Yang Y. CD4 T cells are required for CD8 T cell survival during both primary and memory recall responses. J Immunol. 2007; 179 (12): 8243–51. DOI: https://doi.org/10.4049/jimmunol.179.12.8243

16. Phares T.W., Stohlman S.A., Hwang M., Min B., Hinton D.R., Bergmann C.C. CD4 T cells promote CD8 T cell immunity at the priming and effector site during viral encephalitis. J Virol. 2012; 86 (5): 2416–27. DOI: https://doi.org/10.1128/JVI.06797-11

17. Gressier E., Schulte-Schrepping J., Petrov L., Brumhard S., Stubbemann P., Hiller A., Obermayer B., Spitzer J., Kostevc T., Whitney P.G., Bachem A., Odainic A., van de Sandt C., Nguyen T.H.O., Ashhurst T., Wilson K., Oates C.V.L., Gearing L.J., Meischel T., Hochheiser K., Greyer M., Clarke M., Kreutzenbeck M., Gabriel S.S., Kastenmüller W., Kurts C., Londrigan S.L., Kallies A., Kedzierska K., Hertzog P.J., Latz E., Chen Y.E., Radford K.J., Chopin M., Schroeder J., Kurth F., Gebhardt T., Sander L.E., Sawitzki B., Schultze J.L., Schmidt S.V., Bedoui S. CD4⁺ T cell calibration of antigen-presenting cells optimizes antiviral CD8⁺ T cell immunity. Nat Immunol. 2023; 24 (6): 979–90. DOI: https://doi.org/10.1038/s41590-023-01517-x

All articles in our journal are distributed under the Creative Commons Attribution 4.0 International License (CC BY 4.0 license)