CN111012903A

CN111012903A - Method for inducing formation and proliferation of mucosa colonizing memory T cells

Info

Publication number: CN111012903A
Application number: CN201910874907.3A
Authority: CN
Inventors: 徐建青; 张晓燕; 贺倩
Original assignee: SHANGHAI PUBLIC HEALTH CLINICAL CENTER
Current assignee: SHANGHAI PUBLIC HEALTH CLINICAL CENTER
Priority date: 2019-09-17
Filing date: 2019-09-17
Publication date: 2020-04-17

Abstract

The invention relates to the field of mucosal immune cells, in particular to a method for inducing the formation and proliferation of mucosal resident memory T cells. Wherein, the primary immunization mode of the part outside the intestinal tract at least comprises but is not limited to muscle inoculation, intradermal inoculation, subcutaneous inoculation, nasal drip inoculation, aerosol inhalation inoculation and sublingual inoculation. The intestinal site boosting mode includes but is not limited to rectal cavity inoculation, small intestine cavity inoculation through oral enteric-coated vaccine.

Description

Method for inducing formation and proliferation of mucosa colonizing memory T cells

Technical Field

The invention relates to the field of mucosal immune cells, in particular to a method for inducing the formation and proliferation of mucosal resident memory T cells.

Background

Human Immunodeficiency Virus (HIV) is a type of virus that seriously threatens public health and safety. The HIV-1 epidemiological data published by the united states aids program (uneds) show that about 3690 million people infected with HIV-1 globally in 2017, of which 180 million are new cases, and the rate of infection and transmission is rapidly increasing mainly through sexual contact including genital tract and rectal mucosal pathways, especially male contacts (Man who have sex with Man, MSM). Epidemiological investigations have shown that men are at a risk of HIV transmission of 1/10-1/600, which is much higher than the risk of heterosexual transmission of 1/200-1/1000, and therefore blocking the transmission of sexual pathways, including the rectal mucosa, is the key to the current HIV preventive vaccine study.

In the past decade, numerous large-scale clinical trials of HIV-1 vaccines have been conducted. For example, in the AIDS vaccine STEP II phase clinical test carried out in 2004, the vaccine takes replication-defective adenovirus Ad5 as a vaccine vector to express immunogen Gag-Nef-Pol, so as to induce the organism to generate system T cell response. But since the infection rate of HIV in individuals with high levels of pre-existing immunity in the vaccine group was rather increased, the test was terminated early. In the subsequent clinical test of the HVTN505 vaccine initiated in 2009, a strategy of primary immunization of a DNA vaccine and boosting immunization of an Ad5 vector vaccine (the immunogen is Gag-Pol-Env) is adopted, a systemic T cell response is mainly induced, a humoral immune response is assisted, and the protective effect of the vaccine is not observed. The clinical trial achievement of the HIV vaccine RV144 implemented in thailand was published in the new england journal of medicine, 10 months 2009. The study started in 2003 with participation of over 16000 adult volunteers, and a strategy of boosting with a recombinant canarypox virus vector vaccine ALVAC-HIV (immunogen Gag-Pol-Env) primary immunization in combination with gp120 protein (AIDSVAX B/E) was observed to have 31.2% protection in the vaccine group, the protection mechanism mainly consisting in the induction of neutralizing antibodies.

The results of the above vaccine clinical trials prompted us to argue what immune responses should be induced to provide effective protection? At the same time, where should the effector sites of these protective immune responses be effective in preventing pathogen infection?

The research results of the infection mechanism of the mucosal virus suggest that the virus in the early stage of infection (within the first week) has a small number and a weak histocompatibility in the local part of the mucosa, and additionally, the virus is easy to be eliminated by the specific immune response in the local part of the mucosal Tissue.

Thus, large quantities of TRM obtained from the intestinal mucosa are useful for combating infection by pathogens via the intestinal tract. The classical vaccination mode (such as intramuscular injection, intradermal injection, subcutaneous injection and the like) can effectively amplify the effector memory T cells and the central memory T cells in vivo, but the systemic immunity and the mucosal immunity are relatively independent, and the classical vaccination mode cannot effectively induce the formation and proliferation of the mucosal TRM on an animal model. Due to the division of systemic mucosal sites into different immune compartments, vaccination of one mucosal site alone cannot efficiently induce T cell expansion of other mucosal sites, e.g. it is difficult to induce efficient T cell proliferation at intestinal sites by nasal mucosal vaccination. Vaccination via the gut alone faces the challenge of gut tolerance and digestive enzymes to enzymatically cleave immunogens. Therefore, a method for inducing the formation and proliferation of mucosal TRM has been developed, which can be used for preventing and treating mucosal invasion of pathogens by effectively inducing the proliferation of mucosal T cells, thereby obtaining a large amount of mucosal TRM.

Disclosure of Invention

The invention provides a method for effectively inducing the formation and proliferation of memory T cells at a mucosal part, so that a large number of mucosa-colonizing memory T cells are obtained to prevent the invasion of intestinal pathogens.

In a specific embodiment of the present invention, in the combined vaccination process, the vaccination mode of the part outside the intestinal tract at least includes but is not limited to muscle vaccination, intradermal vaccination, subcutaneous vaccination, nasal drip vaccination, aerosol inhalation vaccination, sublingual vaccination and the like; modes of vaccination at intestinal sites include, but are not limited to, intra-rectal vaccination, intra-intestinal vaccination by oral enteric vaccine, etc.

In a specific embodiment of the invention, in the combined vaccination procedure described above, subcutaneous vaccination, nasal drop vaccination or aerosol inhalation vaccination is used as the primary immunization, and intrarectal vaccination is used as the boost, to effectively activate the combination of intrarectal TRMs.

In a specific embodiment of the present invention, in the combined vaccination process, the parenteral vaccination forms include, but are not limited to, recombinant plasmid vaccines, recombinant protein subunit vaccines, recombinant bacterial vector vaccines, recombinant viral vector vaccines, virus-like particle vaccines, nanoparticle vaccines, attenuated live vaccines, inactivated virus and bacterial vaccines, and combinations of different vaccine forms; the vaccine forms for the intestinal tract vaccination include but are not limited to recombinant plasmid vaccine, recombinant protein subunit vaccine, recombinant bacterial vector vaccine, recombinant viral vector vaccine, virus-like particle vaccine, nanoparticle vaccine, attenuated live vaccine, inactivated virus and bacterial vaccine, and different vaccine forms combination.

In a specific embodiment of the present invention, in the combined vaccination process, the source of the vaccine immunogen includes, but is not limited to, hiv, herpesvirus, poliovirus, coxsackievirus, echovirus, enterovirus, rotavirus, calicivirus (norovirus and sapovirus), astrovirus, enteroadenovirus, hepatitis virus, human papillomavirus, listeria, helicobacter pylori, vibrio cholerae, vibrio parahaemolyticus, escherichia, shigella, salmonella, klebsiella, proteus, enterobacter, serratia, citrobacter, rhizogenes, and the like.

In a specific embodiment of the present invention, during the combined vaccination of the vaccine, adjuvants include, but are not limited to, aluminum adjuvant, cholera toxin and its subunit, oligodeoxynucleotide, manganese ion adjuvant, freund's adjuvant, MF59 adjuvant, QS-21 adjuvant, and the like.

The vaccine and immunization technology of the invention can be used for vaccinating people to prevent the interpersonal spread of pathogens including but not limited to AIDS virus, herpes virus, poliovirus, coxsackievirus, echovirus, novel enterovirus, rotavirus, calicivirus (norovirus and sapovirus), astrovirus, enteroadenovirus, hepatitis virus, human papilloma virus, listeria, helicobacter pylori, vibrio cholerae, vibrio parahaemolyticus, escherichia coli, shigella, salmonella, klebsiella, proteus, enterobacter, serratia, citrobacter, rhizogenes and the like, or reduce the pathogenicity of the pathogens.

In yet another aspect of the present invention, there is provided a combined vaccination method for simultaneously and effectively activating memory T cells in the respiratory, reproductive and rectal regions, the combined vaccination method comprising at least one primary immunization in a region other than the rectum and one booster vaccination in the rectal region to prevent infectious diseases transmitted in the respiratory and/or reproductive and/or rectal region.

In a specific embodiment of the present invention, in the combined vaccination process, the vaccination mode of the parts other than rectum at least includes but is not limited to nasal drop vaccination, aerosol inhalation vaccination, muscle, subcutaneous, etc., and especially, the nasal drop vaccination or aerosol inhalation vaccination and vaccination for 2 times and more are preferred; rectal vaccination is preferably 2 times or more.

In a specific embodiment of the present invention, in the combined vaccination process, the form of the parenteral vaccination includes, but is not limited to, recombinant plasmid vaccine, recombinant protein subunit vaccine, recombinant bacterial vector vaccine, recombinant viral vector vaccine, virus-like particle vaccine, nanoparticle vaccine, attenuated live vaccine, inactivated virus and bacterial vaccine, and combinations of different vaccine forms; the form of the vaccine for rectal vaccination includes, but is not limited to, recombinant plasmid vaccine, recombinant protein subunit vaccine, recombinant bacterial vector vaccine, recombinant viral vector vaccine, virus-like particle vaccine, nanoparticle vaccine, attenuated live vaccine, inactivated virus and bacterial vaccine, and different vaccine forms combination.

In a specific embodiment of the present invention, in the combined vaccination process of the vaccine, the source of the vaccine immunogen includes, but is not limited to, hiv, herpesvirus, human papilloma virus, syphilis, poliovirus, coxsackie virus, echovirus, enteroadenovirus, novel enterovirus, and the like. The vaccines and immunization techniques of the present invention can be used to vaccinate humans to prevent the interpersonal spread of or reduce the pathogenicity of pathogens including, but not limited to, HIV, herpes virus, human papilloma virus, syphilis, poliovirus, Coxsackie virus, echovirus, enteroadenovirus, new enterovirus, and the like.

The vaccination mode of the part outside the intestinal tract comprises but is not limited to muscle vaccination, intradermal vaccination, subcutaneous vaccination, nasal drip vaccination, aerosol inhalation vaccination, sublingual vaccination and the like; modes of vaccination at intestinal sites include, but are not limited to, intra-rectal vaccination, intra-intestinal vaccination by oral enteric vaccine, etc.

The immunization method can induce high-level antigen-specific memory T cell response locally on the pulmonary mucosa, the genital tract mucosa and the rectal mucosa at the same time, so that the immunization method has application prospects in prevention and reduction of pathogen mucosal infection.

The method has the advantages that in the process of the method for inducing the proliferation of the mucosa-colonizing memory T cells, an immunization mode of primary immunization of parts outside the intestinal tract and intestinal tract part boosting immunization, namely a mode of combining system immunization and mucosa immunization, is adopted, and meanwhile, the response of mucosa systems and intestinal tract T cells is induced, so that compared with the independent system immunization or mucosa immunization, the method can effectively stimulate the proliferation of the mucosa-colonizing memory T cells, induce high-level antigen-specific mucosa-colonizing memory T cells, and have application prospects in preventing and reducing pathogen mucosa infection.

Drawings

FIG. 1 shows the effect of different immunization programs on the induction of mouse rectal mucosa specific memory T cells.

The mice used in the experiment are 6-8 weeks old C57/BL6, and the immunogens are model antigens chicken Ovalbumin (OVA) (A) and HIV-1 an extracellular domain gp120(B) of a membrane protein. (A) Shown is the ratio of rectal antigen specific TRM measured by flow cytometry at week 4 (immune memory) after OVA immunization. The abscissa represents different immunization modes and the ordinate represents OVA induced by OVA_257-264Proportion of specific TRM (CD45.1+ cells). (B) Shown is the rectal mucosa specific memory T cell response at week 4 after detection of immunization by an enzyme-linked immunospot assay. The control group and the experimental group (gp120 group) on the abscissa both adopt a strategy of rectal boosting after one week of nasal drop priming, except that the gp120 group is immunized with an immunogen gp120 and an adjuvant Cholera Toxin (CT), while the control group is free of immunogen and is immunized with the adjuvant CT only. Ordinate is per 10⁶Among the individual lymphocytes, the T cells that secrete IFN-. gamma.under the stimulation of specific antigenic peptides are Spot Forming Cells (SFCs). Denotes p<0.05, represents p<0.001。

FIG. 2 is a graph showing the effect of infiltration of recipient mouse rectal mucosa-specific T cells by allo-adoptive administration of mouse activated antigen-specific T cells to wild-type mice.

The proportion of specific T cells in the rectal tissue of recipient mice was determined by flow cytometry at day 7 after rectal immunization. The abscissa (upper) represents the tissue source of adoptive activated specific T cells, spleen, peripheral blood, respectively, the abscissa (lower) represents the rectal immunization with immunogenic OVA, (+) represents the OVA and CT immunization, and (-) represents the adjuvant CT immunization. Ordinate is OVA_257-264Proportion of specific T cells (CD45.1+ cells). Denotes p<0.05, represents p<0.001。

FIG. 3 shows the effect of different priming strategies on the induction of mouse rectal antigen specific memory T cells.

Detection of OVA in rectal tissue of mice at week 4 after completion of immunization by flow cytometry_257-264Proportion of specific T cells. The abscissa is different priming strategies, with control groups immunized with adjuvant CT and experimental groups grouped according to different priming strategies (including different immunogen types, adjuvants and immunization sites). OVA induced by different immunization strategies in ordinate_257-264Proportion of specific TRM (CD45.1+ cells). ChartShows p<0.05, represents p<0.01, represents p<0.001。

FIG. 4 shows the induction and toxicity protection effects of different rectal boosting strategies on mouse rectal antigen specific memory T cells.

(A) Shown is flow cytometry to detect rectal OVA of mice at week 4 (immune memory period) after immunization_257-264Ratio of specific TRMs. The abscissa is different rectal boosting strategies, where the control group was immunized with adjuvant CT and the experimental groups were grouped according to the rectal boosting strategy (including different immunotypes and adjuvants). OVA induced by different immunization strategies in ordinate_257-264Proportion of specific TRM (CD45.1+ cells). (B) Shows the bacterial load of the rectal tissue of mice on day 3 after rectal challenge with recombinant listeria (LM-OVA) expressing OVA. The abscissa is the different rectal boosting strategies (same as fig. 4A) and the ordinate is the number of LM-OVA contained per rectal tissue, i.e. Colony Forming Units (CFU). Denotes p<0.01, represents p<0.001 denotes p<0.0001。

FIG. 5 shows the effect of different immunization protocols on the induction of mucosal-specific memory T cells in various tissue sites in mice.

(A) The ratio of antigen-specific memory T cells in the rectum, reproductive tract and pulmonary mucosa of mice at week 4 after OVA immunization is detected by flow cytometry. The abscissa represents different immunization modes and the ordinate represents OVA induced by OVA_257-264Proportion of specific TRM (CD45.1+ cells). (B) Shown is the specific memory T cell response in the rectum, the lung mucosa and the spleen of the mice at the 4 th week after the OVA immunization is detected by an enzyme-linked immunosorbent assay. The abscissa is different immunization regimens and the ordinate is every 10⁶Among the individual lymphocytes, the T cells that secrete IFN-. gamma.under the stimulation of antigen-specific peptides, i.e., spot-forming cells (SFCs). Denotes p<0.05。

Detailed description of the preferred embodiments

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention. These and other features and advantages of the present invention will become apparent upon reading the following detailed description of the disclosed embodiments and the appended claims.

Example 1: evaluation of Effect of different vaccination programs on inducing mucosal rectal memory T cells

Immunizing C57/BL6 mice by different immunization programs, wherein the immunogen is a model antigen of chicken Ovalbumin (OVA), the adjuvant is Cholera Toxin (Cholera Toxin, CT), and after four weeks of immunization (immune memory period), evaluating the different immunization programs to induce OVA_257-264Effect of specific rectal memory T cells. Both egg white protein and cholera toxin were purchased from Sigma. The experimental procedure was as follows:

(1) c57BL/6 mice 6-8 weeks old are randomly divided into 5 groups, and named as rectum + rectum group, nose drop + nose drop group, nose drop + genital tract group, nose drop + rectum group according to different subsequent immunization programs.

(2) OT-I cell adoptivity was performed 24 hours prior to immunization. Magnetic beads (brand: Stemcell, cat # 19853) were used to sort CD8+ T cells from OT-I mice, which were positive for CD45.1 and were adoptively 2X 10 per C57BL/6 mouse⁵The OT-I cells are adoptive in the orbital venous plexus. CD8+ T cells of OT-I mice have the function of specifically recognizing chicken ovalbumin epitope peptide OVA_257-264The TCR can generate specific T cell response after chicken ovalbumin immunization, and express a marker molecule CD45.1, thereby being convenient for detection.

(3) Mice were immunized with the immunogen OVA and adjuvant CT, and the specific immunization procedure is shown in table 1. Before the intranasal instillation immunization operation, 100 microliters of 1% pentobarbital were intraperitoneally injected into each mouse for anesthesia. The dose of OVA inoculation is 10 micrograms/piece, the dose of CT inoculation is 1 microgram/piece, and the two are mixed and inoculated. The inoculation volume is adjusted by using normal saline according to different inoculation positions, wherein the dropping volume in a rectal cavity is 10 microliters, the dropping volume in a nasal cavity is 50 microliters, and the dropping volume in a genital tract cavity is 10 microliters. The vaccine immunization interval was 1 week.

TABLE 1 mouse experiments with different vaccination programs to induce tissue memory T cells

(4) At week 4 after the end of the last immunization, rectal area OVA induction by different vaccination procedures was detected by flow cytometry_257-264Proportion of specific memory T cells (i.e., CD45.1+ cells). A single cell suspension of isolated rectal tissue is prepared and rectal tissue lymphocytes are sorted using a lymphocyte separation medium. The lymphocytes obtained by the isolation were surface-stained, and the flow antibodies used were an anti-mouse CD3 antibody labeled with PerCP-CY5.5 (brand: BD, cat # 560527), an anti-mouse CD8a antibody labeled with APC (brand: BD, cat # 561093), and an anti-mouse CD45.1 antibody labeled with FITC (brand: BD, cat # 561871), respectively. After dyeing is finished, detecting OVA in a flow mode_257-264The proportion of specific T cells (i.e., CD45.1+ cells) in CD8+ T cells.

Different immunization programs for mice OVA_257-264The induction effect of specific rectal memory T cells is shown in figure 1A: the average value of the ratio of the rectal cells to the rectal cells, the nasal drops to the nasal drops and the nasal drops to the genital tract cells of CD45.1+ T cells is close to or lower than 1%, and the OVA is shown_257-264Weak specific T cell immune response; compared with the three groups, the nasal drop and rectum group has OVA at the rectal part_257-264The immune response of specific T cells is improved, the difference is significant, and the proportion of the specific T cells in CD8+ T cells is close to 5%; the mean of the rectal CD45.1+ T cell ratios of nasal drops, rectum and rectum groups was close to 12%, showing OVA_257-264The specific T cell immune response is further enhanced.

The experiment proves that rectal resident memory T cells can be effectively induced by respiratory tract vaccination priming and intestinal tract vaccination boosting.

Example 2: evaluation of Effect of 'nasal drip priming and rectal enhancement' on inducing gp 120-specific rectal TRM

Mice were immunized with the extramembranous gp120 protein of the membrane protein of HIV-1 (strain AE 2F) and CT, the first needle was inoculated by nasal drip and the second needle was inoculated by rectal. 4 weeks after completion of immunization (immunological memory phase), the effect of induction of rectal-site gp 120-specific rectal-colonizing memory T cells was evaluated. The experimental procedure was as follows:

(1) c57BL/6 mice 6-8 weeks old were randomly divided into 2 groups, designated control and gp120 groups, respectively.

(2) Mice were immunized with the immunogen gp120 and the adjuvant CT, and the specific immunization procedure is shown in table 2. Before the intranasal instillation immunization operation, 100 microliters of 1% pentobarbital were intraperitoneally injected into each mouse for anesthesia. The inoculation dose of gp120 is 10 microgram/piece, the inoculation dose of CT is 1 microgram/piece, and the two are mixed and inoculated. The inoculation volume was adjusted with physiological saline according to the site of inoculation, with a 10. mu.l instillation volume in the rectal cavity and a 50. mu.l instillation volume in the nasal cavity. The vaccine immunization interval was 1 week.

TABLE 2 mouse experiments with gp120 protein induced rectal memory T cells

(3) At week 4 after the end of the last immunization, the rectal gp 120-specific memory T cell response was evaluated using Enzyme-linked immuno spot Assay (ELISpot). Rectal lymphocyte suspensions were obtained as described in example 1. An ELISPOT assay was performed using the mouse IFN-. gamma.ELISPOT assay kit (brand: BD, cat # 551083). After 20 hours of stimulation of rectal lymphocytes with the gp120 protein peptide library, dot visualization was performed and counted. Each positive spot represents antigen peptide specific T cells, and the positive spots generated by the stimulated cells are summed to obtain the number of gp120 specific T cells.

The results of the rectally colonized gp 120-specific memory T cell responses are shown in figure 1B: each 1 × 10⁶Of the lymphocytes, the gp120 group had more than 500 positive spots, while the control group had only about 70. Rectal colonization specific T cell responses were shown to be significantly stronger in gp120 group than in control group.

The experiment proves that the HIV-1 membrane protein vaccine is inoculated by adopting a combined inoculation method of nasal drip priming combined intestinal tract boosting immunization, and the HIV-1 specific rectum colonizing memory T cells can be effectively induced.

Example 3: evaluation of Effect of infiltration of circulating activated T cells into rectal tissue by rectal immunization

Spleen and peripheral blood antigen-specific T cells which are subjected to nasal drip immune activation are sorted out, and are successively given to a receptor mouse, and the infiltration effect of the antigen-specific T cells to rectal tissues is evaluated. The experimental procedure was as follows:

(1) OT-I cell adoptive was performed on C57BL/6 mice as described in example 1.

(2) The immune mice were injected intranasally with the same dose of immunogen OVA and adjuvant CT as in example 1.

(3) The spleen and peripheral blood of the mice are taken 7 days after immunization, and OVA is sorted by a magnetic bead sorting method (brand: American day, gentle, cat No. 130-_257-264Specific (i.e., CD45.1+ cells) T cells. The cells obtained were sorted as donor cells for subsequent experiments.

(4) Sorting the obtained donor cells (spleen-derived 5X 10)⁵ 2X 10 sources of peripheral blood⁴) Adoptively transferred to CD45.1 negative wild-type C57BL/6 mice via the orbital venous plexus, respectively.

(5) After 24 hours of adoptive transfer, recipient mice adoptively transfected with spleen-derived donor cells were randomly divided into two groups, group 1 for rectal immunization with adjuvant CT only and group 2 for rectal immunization with immunogen OVA and adjuvant CT. Recipient mice adoptively transfected with peripheral blood-derived donor cells were group 3 and subjected to rectal immunization using immunogen OVA and adjuvant CT.

(6) Rectal OVA detection by flow cytometry 7 days after the end of immunization_257-264The proportion of specific T cells (i.e., CD45.1+ cells) in CD8+ T cells. The flow detection method was as described in example 1.

The infiltration effect of recipient mouse rectal specific T cells following allo-adoptive administration of mouse activated antigen specific T cells to wild mice is shown in figure 2: both the group 2 and the group 3 have more specific T cell infiltration, the group 1 has only a very small amount of specific T cell infiltration, the proportion is lower than 2 percent, the activated T cells in the circulation can infiltrate into rectal tissues under the action of rectal immunity, and the activated T cells in the circulation are difficult to colonize the rectum and further form TRM in the absence of rectal immunity.

This experiment demonstrates that any immune strategy that can induce activated T cells in the circulation, or the reinfusion of activated specific T cells followed by rectal boost, can induce a good rectal specific T cell response.

Example 4: evaluation of Effect of different priming strategies on Induction of intestinal memory T cells

Mice were immunized according to the nasal drop + rectal group immunization program as described in example 1. The boosting strategy for fixing the second needle and the third needle is to drop into the rectal cavity, change different priming strategies, and evaluate the effect of different priming strategies on inducing intestinal tract memory T cells after 4 weeks of the last needle (immunological memory period). The experimental procedure was as follows:

(1) randomly dividing 6-8 week-old C57BL/6 mice into 7 groups, and respectively naming the groups as control group and experimental group (including OVA + aluminum adjuvant subcutaneous group, OVA + CT muscle group, OVA + CT nose drop group, rTTV-OVA nose drop group, H9N2-OVA nose drop group) according to different follow-up priming strategies_257-264Nasal drip group).

(2) OT-I cell adoptivity was performed 24 hours prior to immunization. As described in example 1.

(3) Mice were immunized with different priming strategies (including different vaccination sites, adjuvants and immunogen types) and the specific immunization procedure is shown in table 3. Wherein rTTV-OVA is recombinant Tiantan strain vaccinia vector for expressing OVA, H9N2-OVA_257-264For expressing OVA epitope peptide OVA_257-264The recombinant H9N2 influenza virus vector of (1). The control group was primed with cholera toxin by nasal drip. Mice were anesthetized prior to nasal instillation, dose of inoculation, volume of inoculation, and immunization interval as described in example 1. Wherein the rTTV-OVA inoculation dose is 1 x 10 per mouse⁶PFU, PFU being a plaque forming unit; H9N2-OVA_257-264The inoculation dose is 1000TCID 50/mouse, and TCID50 is half of the tissue culture infection dose; inoculation with OVAThe dosage is 10 micrograms/mouse, the volume ratio of the aluminum adjuvant to the OVA inoculation is 1:1, and the mass ratio of the cholera toxin to the chicken ovalbumin inoculation is 1: 10.

(4) Mice in 6 experimental groups were immunized by rectal instillation with immunogen OVA and adjuvant CT at 1 week and 2 weeks after priming, and mice in control group were immunized by rectal instillation with adjuvant CT.

TABLE 3 mouse experiments with different priming strategies for induction of intestinal memory T cells

(5) Detecting different priming strategies to induce OVA by flow cytometry at 4 weeks after the last injection immunization_257-264Effect of specific rectal memory T cells. Flow assay OVA_257-264The method of specific T cells (i.e., CD45.1+ cells) is described in example 1.

Different priming strategies induce rectal area OVA_257-264The ratio of specific memory T cells is shown in figure 3: the control group did not detect CD45.1+ T cells in the rectum. OVA + aluminum adjuvant subcutaneous group, OVA + CT muscle group, OVA + CT nasal drop group, rTTV-OVA nasal drop group, H9N2-OVA_257-264The nasal drip group has more CD45.1+ T cells, which shows that 6 experimental groups have better OVA_257-264Specific T cell immune responses.

The experiment proves that the mice are primarily immunized by adopting different parenteral inoculation parts, different adjuvants and different vaccine carriers, and can effectively induce the rectum colonizing memory T cells by combining intestinal tract boosting immunization.

Example 5: evaluation of the Effect of inducing intestinal memory T cells Using different rectal boosting strategies

The mouse immunization program was nose drop + rectal group immunization as described in example 1. The prime strategy was an immunogen OVA and adjuvant CT nasal drip vaccination, the boost strategy was a rectal vaccination with different immunogens (see table 4 for details), and the immunization strategy was evaluated 4 weeks after completion of immunization (immune memory phase) for the effect of inducing tissue specific memory T cells. The experimental procedure was as follows:

(1) c57BL/6 mice 6-8 weeks old were randomly divided into 5 groups, named according to different rectal boost strategies: OVA + aluminum adjuvant group (control group), OVA + CXCL10 group, OVA + CT group, and LM-OVA group.

(3) Mice of each experimental group were immunized by nasal drip with immunogen OVA and adjuvant CT, and control group was primed by nasal drip with adjuvant CT. The anaesthesia and inoculation doses were as described in example 1.

(4) Mice were immunized 7 days after priming using different rectal boost strategies, and the specific immunization program is shown in table 4. Wherein, the LM-OVA is a recombinant Listeria vector vaccine for expressing OVA. The control group was rectally boosted with the adjuvant CT. Wherein the LM-OVA inoculation dose is 1 multiplied by 10 per mouse⁷CFU, the inoculation amount of OVA is 10 micrograms per mouse, the inoculation volume ratio of the aluminum adjuvant to the OVA is 1:1, the inoculation mass ratio of cholera toxin to the OVA is 1:10, and the inoculation dose of CXCL10 is 3 micrograms per mouse.

(5) Detecting different rectum boosting immunization strategies to induce OVA by flow cytometry at the 4 th week after the last injection immunization_257-264Effect of specific rectal memory T cells. Flow assay OVA_257-264The method of specific T cells (i.e., CD45.1+ cells) is described in example 1.

TABLE 4 mouse experiments with different rectal booster strategies to induce tissue memory T cells

Different rectum booster immunity strategies induce rectum OVA_257-264The ratio of specific memory T cells is shown, for example, in fig. 4A: the control group did not detect CD45.1+ T cells at the rectal site. Compared with the control group, the OVA + aluminum adjuvant group, the OVA + CXCL10 group, the OVA + CT group and the LM-OVA group all have more CD45.1+ T cells, and show that the better rectal OVA is shown_257-264Specific T cell immune responses.

The experiment proves that the rectum colonizing memory T cells can be well induced by adopting a combined strategy of combining the parenteral primary immunization with the intestinal tract boosting immunization and carrying out the intestinal tract boosting immunization on the mice by using different adjuvants and different vaccine carriers.

Example 6: evaluation of toxic challenge protection effect of different rectum booster immunity strategies

Mice were immunized according to Table 4, and after four weeks of completion of immunization (immunomemory phase), challenged with L-OVA by rectal instillation at a dose of 1X 10 per mouse⁹CFU, CFU is a colony forming unit. And 3 days after the challenge, taking each group of rectal tissues for homogenization treatment, diluting the homogenate liquid in a gradient manner, dropwise adding the diluted homogenate liquid on a brain-heart infusion agar plate, and culturing until clear colonies grow out, wherein the number of the colonies represents the bacterial load of the rectal tissues.

The toxic challenge protective effect of different rectal boosting strategies is shown in fig. 4B: compared with a control group, the OVA + aluminum adjuvant group, the OVA + CXCL10 group, the OVA + CT group and the LM-OVA group can obviously reduce the rectal bacterial load.

The experiment proves that the combined inoculation method combining the parenteral primary immunization with the intestinal tract boosting immunization is adopted, and different adjuvants and different vaccine carriers are used for carrying out rectal boosting immunization on the mice, so that rectal pathogen infection can be effectively resisted.

Example 7: evaluation of Effect of different vaccination programs on inducing memory T cells at mucosal sites

C57/BL6 mice were immunized using different immunization programs. The immunogen was OVA and the adjuvant was CT (both from Sigma). Four weeks after completion of immunization (immunological memory period), evaluation of different vaccination programs induced rectal, reproductive tract, pulmonary mucosa OVA_257-264Effects of specific memory T cells.

The experimental procedure was as follows:

(1) mouse grouping, OT-I cell adoptive and immunization protocols were as described in example 1.

(2) At 4 weeks after the end of the last immunization, different vaccination programs were used to induce OVA in rectal, reproductive and pulmonary lavage fluids by flow cytometry_257-264Specific memory T cells (i.e., CD45.1+ cells) account for the proportion of CD8+ T cells. Flow assay OVA_257-264The specific T cell method was as described in example 1.

The results of the ratio of different vaccination programs inducing specific memory T cells for each mucosal tissue are shown in fig. 5A: the specific T cell proportion of rectum, genital tract and lung in rectum and rectum group is lower than 3%; the proportion of CD45.1+ T cells at the rectal part of the nasal drip and nasal drip group and the nasal drip and reproductive tract group is lower than 2%, and memory T cells with higher proportion can be induced in the lung and reproductive tract; the nasal drip + rectum group and the nasal drip + rectum group can induce memory T cells with higher proportion in the lung and can improve the proportion of CD45.1+ T cells in the rectum part; the response of the nasal drop + rectum group in rectum and genital tract is stronger than that of the nasal drop + rectum group, which shows that the nasal drop + rectum group can simultaneously and effectively induce TRM of rectum, genital tract and pulmonary mucosa.

The experiment proves that the combined inoculation method of primary immunization of the respiratory tract and boosting immunization of the intestinal tract can induce specific memory T cells in the intestinal tract, the reproductive tract and the respiratory tract simultaneously.

Example 8: evaluation of Effect of different vaccination programs for inducing endogenous memory T cells in various sites

C57/BL6 mice were immunized using different immunization programs. The immunogen was OVA and the adjuvant was CT (both from Sigma). Four weeks after completion of immunization (immunomic memory period), evaluation of different vaccination programs induced rectal, pulmonary mucosa, spleen OVA_257-264Effects of specific memory T cells. The experimental procedure was as follows:

(1) c57BL/6 mice 6-8 weeks old were randomly divided into 5 groups, and named as control group and experimental group (including nasal drop + nasal drop group, nasal drop + rectal group and rectal group) according to different immunization programs.

(2) Mice were immunized. Mice in each group were immunized with different immunization programs at week 0, week 1, and week 2, respectively, and the immunogens in the experimental groups were both OVA and CT, while the control group was CT only. Specific immunization protocols are shown in table 5. The anesthesia procedure, the inoculation dose, and the inoculation volume were as described in example 1.

Mice in this example were not adoptive with OT-i cells to assess the formation of endogenous specific memory T cells.

TABLE 5 mouse experiments with different vaccination programs to induce endogenous memory T cells in various locations

ND means no treatment.

(3) At 4 weeks after the last immunization, the OVA of rectum, lung mucosa and spleen is evaluated by adopting an enzyme-linked immunospot assay_257-264Response profile of specific memory T cells. The ELISA assay was as described in example 2. This example uses OVA_257-264Lymphocytes were stimulated with a stimulation concentration of 5 μ g/ml of peptide.

Inducing endogenous OVA of each part by different inoculation procedures_257-264The effect of specific memory T cells is shown in fig. 5B: no positive spots were detected in the control group; the number of the positive spots of the nose drop and the nose drop group at the rectal part is less than 20/1 multiplied by 10⁶Lymphocytes, effective in inducing memory T cell responses in the lung and spleen; compared with a nasal drop group and a nasal drop group, the nasal drop group and the rectal drop group can obviously increase the number of positive spots of the rectal part, the number of the positive spots of spleen and lung mucosa is slightly reduced, and the TRM of the system, the lung mucosa and the rectum can be effectively induced by the nasal drop group and the rectal drop group.

The experiment proves that the combined inoculation method of primary immunization of the respiratory tract and boosting immunization of the intestinal tract can induce endogenous specific memory T cells in the intestinal tract, the respiratory tract and the spleen simultaneously.

The foregoing shows and describes the general principles, essential features, and advantages of the invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are described in the specification and illustrated only to illustrate the principle of the present invention, but that various changes and modifications may be made therein without departing from the spirit and scope of the present invention, which fall within the scope of the invention as claimed. The scope of the invention is defined by the appended claims and equivalents thereof.

Claims

1. A method of inducing the formation and proliferation of mucosal resident memory T cells, comprising at least one primary immunization at a site other than the gut and at least one booster immunization at a site in the gut such that mucosal resident memory T cells are formed and proliferate.

2. The method of claim 1, wherein the mucosal resident memory T cells are rectal mucosal resident memory T cells.

3. The method of claim 1, wherein the primary immunization of the parenteral area comprises but is not limited to intramuscular, intradermal, subcutaneous, nasal drip, aerosol inhalation, sublingual, and mucosal resident memory T cell formation and proliferation.

4. The method of claim 1, wherein the intestinal site is boosted by means including but not limited to rectal vaccination, and small intestinal vaccination by oral enteric vaccine.

5. The method of claim 1, wherein the immunogen used in the primary and booster immunizations includes but is not limited to HIV, herpesvirus, poliovirus, coxsackievirus, echovirus, neoenterovirus, rotavirus, calicivirus (norovirus and sapovirus), astrovirus, enteroadenovirus, hepatitis virus, human papillomavirus, Listeria, helicobacter, Vibrio cholerae, Vibrio parahaemolyticus, Escherichia, Shigella, Salmonella, Klebsiella, Proteus, Enterobacter, Serratia, Citrobacter, Morganella, and Morganella.

6. The method of claim 1, wherein adjuvants including but not limited to aluminum adjuvant, cholera toxin and its subunits, oligodeoxynucleotides, manganese ion adjuvant, freund's adjuvant, MF59 adjuvant, QS-21 adjuvant are used in the priming and boosting.

7. The method of claim 1, wherein the primary immunization of the site outside the intestinal tract is a nasal drop or aerosol inhalation vaccination with a frequency of 2 vaccinations.

8. The method of claim 1, wherein the number of vaccinations in the rectal booster immunisation is 2 or more.

9. The method of claim 1, wherein the parenteral vaccination comprises but is not limited to recombinant plasmid vaccines, recombinant protein subunit vaccines, recombinant bacterial vector vaccines, recombinant viral vector vaccines, virus-like particle vaccines, nanoparticle vaccines, live attenuated vaccines, inactivated viral and bacterial vaccines, and combinations of different vaccine forms.

10. The method of claim 1, wherein the vaccination modality of the intestinal site booster vaccination includes but is not limited to recombinant plasmid vaccines, recombinant protein subunit vaccines, recombinant bacterial vector vaccines, recombinant viral vector vaccines, virus-like particle vaccines, nanoparticle vaccines, attenuated live vaccines, inactivated virus and bacterial vaccines, and combinations of different vaccine modalities.

11. The method of claim 1, wherein the mucosal resident memory T cell is an HIV-1 specific rectal mucosal resident memory T cell, and the method comprises the steps of: immunizing animals by using immunogen gp120 and adjuvant CT, inoculating the animals by nasal drip for primary immunization and inoculating the animals by rectum for boosting immunization, wherein the immunization interval of the vaccines is 1 week, obtaining rectal lymphocyte suspension of the immunized animals at 4 weeks after the immunization is finished, and separating HIV-1 specific rectal mucosa colonizing memory T cells.

12. The method of claim 1, wherein the method comprises a primary immunization with a single nasal drop and a booster immunization with two rectal cavity inoculations, and the method can simultaneously induce the formation and proliferation of mucosal resident memory T cells in rectum, lung and genital tract.