CN110624106A

CN110624106A - Combined application of PD-1 signal channel antagonist and AIDS vaccine

Info

Publication number: CN110624106A
Application number: CN201810653522.XA
Authority: CN
Inventors: 孙彩军; 陈凌; 潘恩祥; 闫洪滨
Original assignee: GENOR BIOPHARMA CO Ltd; Guangzhou Institute of Biomedicine and Health of CAS
Current assignee: GENOR BIOPHARMA CO Ltd; Guangzhou Institute of Biomedicine and Health of CAS
Priority date: 2018-06-22
Filing date: 2018-06-22
Publication date: 2019-12-31

Abstract

The invention relates to combined application of a PD-1 signal channel antagonist and an AIDS vaccine. Specifically, the present invention discloses a composition comprising: (A) a prophylactically and/or therapeutically effective amount of a first active ingredient that is an antagonist against the PD-1 signaling pathway; (B) a prophylactically and/or therapeutically effective amount of a second active ingredient which is an acquired immunodeficiency syndrome vaccine. The invention relates to an active ingredient combination, a medicine box, a medicine composition and application thereof in preparing medicines for preventing and/or treating acquired immunodeficiency syndrome. The composition, the active ingredient combination and the kit of the present invention have an excellent effect of preventing and/or treating acquired immunodeficiency syndrome.

Description

Combined application of PD-1 signal channel antagonist and AIDS vaccine

Technical Field

The invention relates to the field of medicinal preparations, in particular to combined application of a PD-1 signal channel antagonist and an AIDS vaccine, and particularly application in preventing AIDS virus infection.

Background

Human Immunodeficiency Virus (HIV), the first discovered to have passed for over thirty years to date, is the fourth leading cause of death in humans today, the causative agent of acquired immunodeficiency syndrome (AIDS). Currently, there are tens of millions of HIV virus carriers worldwide, and the most effective treatment strategy is cocktail therapy proposed in 1996, but the antiretroviral therapy (ART) cannot clear viral genes integrated into the host genome, so that the curative function cannot be achieved. Therefore, they must inhibit the rebound of the virus through lifetime treatment, but long-term administration of the drug also causes the drug-resistant strains to become serious worldwide, and simultaneously causes intangible pressure on the research of HIV, so that the research of effective HIV prevention strategies is imperative. Based on the previous research results, the single vaccine immunization strategy cannot well control HIV virus infection, and no preventive AIDS vaccine is on the market.

Therefore, there is a great need in the art to develop drugs and methods for effectively preventing and treating aids.

Disclosure of Invention

The invention aims to provide a medicine and a method for effectively preventing and treating AIDS.

The invention aims to provide a composition, a kit and a combination of active ingredients for preventing and/or treating AIDS, and application thereof in preparing medicaments for preventing and/or treating AIDS, wherein the synergistic effect of the two active ingredients is utilized to enhance the effect of preventing and/or treating AIDS.

In a first aspect of the present invention, there is provided a composition comprising:

(A) a prophylactically and/or therapeutically effective amount of a first active ingredient that is an antagonist against the PD-1 signaling pathway;

(B) a prophylactically and/or therapeutically effective amount of a second active ingredient which is an acquired immunodeficiency syndrome vaccine;

wherein the antagonist is PD-1 antibody, the acquired immunodeficiency syndrome vaccine is live vector virus vaccine, the mass of the PD-1 antibody is C1 mg, the number of virus particles of the live vector virus vaccine is C2, and the ratio of C1 to C2 is 1:10⁷-1:10¹⁰Preferably, 1:10⁸-1:10⁹。

In another preferred embodiment, the antagonist against the PD-1 signaling pathway is effective to block the PD-1 signaling pathway.

In another preferred embodiment, the antagonist against the PD-1 signaling pathway is selected from the group consisting of: small molecule compounds, nucleic acid molecules (e.g., antisense nucleic acids), polypeptides or biological macromolecules (e.g., antibodies), small molecule ligands, criprpr agents, or combinations thereof.

In another preferred embodiment, the nucleic acid molecule is selected from the group consisting of: miRNA, shRNA, siRNA, ribozyme, or a combination thereof.

In another preferred embodiment, the antagonist against the PD-1 signaling pathway is selected from the group consisting of: PD-1 antibodies, small molecule inhibitors of PD-1, RNA interference agents (such as miRNA, shRNA, siRNA, etc.) that down-regulate PD-1 expression, or combinations thereof.

In another preferred embodiment, the PD-1 antibody is a monospecific antibody, bispecific antibody, trispecific antibody, or multispecific antibody having the antigen binding region of the PD-1 antibody.

In another preferred embodiment, the PD-1 antibody is a diabody or a single chain antibody.

In another preferred embodiment, the antibody is selected from the group consisting of: an antibody of animal origin, a chimeric antibody, a humanized antibody, or a combination thereof.

In another preferred embodiment, the PD-1 antibody further comprises a fragment, derivative or analog of the antibody.

In another preferred embodiment, the antibody derivative is a single chain antibody and/or antibody fragment, such as: fab, Fab ', (Fab')₂Or other antibodiesAnd (3) derivatives.

In another preferred embodiment, the PD-1 antibody is an antibody or immunologically active fragment thereof selected from the group consisting of: pembrolizumab, nivolumab, humanized PD-1 antibody 7A4, humanized PD-1 antibody 7A4D, humanized PD-1 antibody 13F1, or a combination thereof.

In another preferred embodiment, the fragment of the antibody having immunological activity is selected from the group consisting of: an scFv, an Fab fragment, an Fab 'fragment, an F (ab)' 2 fragment, or a combination thereof.

In another preferred embodiment, the pembrolizumab is a marketed PD-1 antibody drugThe heavy and light chain sequences are described in The monoclonal antibody database (mAb-DB) of The international immunogenetic information system (IMGT), with an antibody accession number (mAb-DB ID) of 472.

In another preferred embodiment, the nivolumab is a marketed PD-1 antibody drugThe heavy and light chain sequences are described in the monoclonal antibody database of IMGT, under antibody accession number 424.

In another preferred example, the heavy chain sequence of the humanized PD-1 antibody 7A4 is SEQ ID No. 1 and the light chain sequence thereof is SEQ ID No. 2.

In another preferred example, the humanized PD-1 antibody 7A4D has the heavy chain sequence SEQ ID No. 3 and the light chain sequence SEQ ID No. 4.

In another preferred example, the humanized PD-1 antibody 13F1 has a heavy chain sequence of SEQ ID No. 5 and a light chain sequence of SEQ ID No. 6.

In another preferred embodiment, the acquired immunodeficiency syndrome vaccine is an inactivated virus vaccine, an attenuated live virus vaccine, a polypeptide vaccine, a subunit vaccine, a live vector virus vaccine, a viroid vaccine, a DNA vaccine, or a combination thereof for preventing and/or treating human immunodeficiency syndrome.

In another preferred embodiment, the inactivated virus vaccine or live attenuated virus vaccine is an inactivated virus or an attenuated strain of an immunodeficiency virus, preferably a Human Immunodeficiency Virus (HIV) or Simian Immunodeficiency Virus (SIV), that induces immunodeficiency syndrome.

In another preferred embodiment, the polypeptide vaccine or subunit vaccine is a recombinant viral membrane protein monomer or polypeptide from HIV or SIV, comprising non-infectious particles of one or more HIV or SIV proteins.

In another preferred embodiment, the live vector viral vaccine uses a non-HIV or SIV expression system or viral vector for expression of immunogenic fragments from HIV or SIV. Preferably, the expression system or viral vector is selected from the group consisting of: a vaccinia virus vector system, a baculovirus vector system, an adenovirus vector system, a yellow fever virus vector, a venezuelan equine encephalitis virus vector, a poliovirus vector, a vesicular stomatitis virus vector, a herpes simplex virus vector, or a combination thereof.

In another preferred embodiment, the DNA vaccine uses plasmid DNA directly integrated into the immunogenic fragment expression sequence from HIV or SIV, and the immunogenic polypeptide is synthesized by expression processing of the host cell.

In another preferred embodiment, the immunogen of the acquired human immunodeficiency syndrome vaccine is selected from the group consisting of:

(a) a protein or polypeptide encoded by a structural gene from HIV or SIV; and/or

(b) A protein or polypeptide encoded by a regulatory gene derived from HIV or SIV.

In another preferred embodiment, the structural gene is selected from Gag, Pol, ENV, or a combination thereof.

In another preferred example, the structural gene Gag encodes a matrix protein, a capsid protein, a nucleocapsid protein, a P6 protein, a pre-polymeric protein P55, P27, etc.; the structural gene Pol encodes protease, reverse transcriptase and integrase (e.g., P66/51, P31, P10, etc.); and/or the structural gene ENV encodes a surface glycoprotein and a transmembrane protein (e.g., gp160, gp120, gp41, etc.).

In another preferred embodiment, the regulatory gene is selected from Tat, Rev, Nef, Vif, Vpu, Vpr, or combinations thereof.

In another preferred embodiment, the acquired immunodeficiency syndrome vaccine is a live vector virus vaccine, and the expression system is preferably an adenovirus vector system, more preferably an adenovirus vector system type 5; more preferably, the GenBank accession number of the type 5 adenovirus vector sequence is AY339865.1, the type 5 adenovirus vector is a replication-deficient recombinant adenovirus vector, and the replication deficiency refers to deletion of an E1 gene necessary for replication on the adenovirus vector; more preferably, the immunogen of the live vector virus vaccine is obtained by inserting a foreign gene into the gene region of E1 by a homologous recombination technology, and the promoter is CMV.

In another preferred embodiment, the immunogen of the acquired immunodeficiency syndrome vaccine is a complete polypeptide or a partially expressed polypeptide fragment encoded by a gene selected from the group consisting of: gag, Pol, ENV, nef, vpx, vif, vpr, rev, tat, or combinations thereof.

In another preferred embodiment, the acquired immunodeficiency syndrome vaccine comprises one or more immunogen expression sequences encoding an immunogen selected from the group consisting of: gag, Pol, ENV, nef, vpx, vif, vpr, rev, tat, or combinations thereof. Preferably, the immunogen amino acid sequence or the immunogen expression sequence optimized by various codons is selected from the following group:

immunogens	Optimized nucleic acid sequences	Polypeptide GenBank accession number
			Gag	SEQ ID No:7	AAA47632.1
Pol	SEQ ID No:8	AAA47633.2
			ENV	SEQ ID No.9	AAA47637.1
nef	SEQ ID No:10	AAC57428.1
			vpx	SEQ ID No:11	AAL55641.1
vif	SEQ ID No.12	AAL55640.1
			vpr	SEQ ID No.13	AAL55642.1
rev	SEQ ID No.14	AAA47631.1
			tat	SEQ ID No.15	AAA47630.1

In another preferred embodiment, the acquired immunodeficiency syndrome vaccine further comprises other genes for enhancing cellular immune response, and the other genes can be co-expressed (through gene fusion) with immunogen expression sequences of HIV or SIV under the action of the same promoter or can be separately expressed under the action of different promoters; such other genes include, but are not limited to, the LC3 gene; more preferably, the LC3 gene is the LC3b gene, an exemplary example of which is described in chinese patent application 201310419864.2.

In another preferred embodiment, the first active ingredient is humanized PD-1 antibody (e.g., humanized PD-1 antibody 7A4, humanized PD-1 antibody 7A4D, humanized PD-1 antibody 13F1, pamumab or nivolumab), and the second active ingredient is a live vector virus vaccine comprising a replication-deficient adenovirus type 5 vector carrying Gag, Pol and ENV structural genes; the humanized PD-1 antibody has the mass of C1 mg, the number of virus particles of the live vector virus vaccine is C2, and the mass ratio of C1 to C2 is 1:10⁷-1:10¹⁰Preferably, 1:10⁸-1:10⁹。

In another preferred embodiment, the composition is a pharmaceutical composition.

In another preferred embodiment, the pharmaceutical composition comprises:

(C) a pharmaceutically acceptable carrier, and a pharmaceutically acceptable carrier,

In another preferred embodiment, the composition is used for preventing and/or treating acquired immunodeficiency syndrome.

In another preferred embodiment, the composition further comprises other active substances for preventing and/or treating acquired immunodeficiency syndrome.

In another preferred embodiment, the further active substances for the prophylaxis and/or treatment of acquired immunodeficiency syndrome are selected from the group consisting of: an antiretroviral drug, an immunomodulatory drug, an anti-opportunistic infection drug, or a combination thereof.

In a second aspect of the invention, there is provided a kit comprising:

(A) a first formulation comprising an antagonist against the PD-1 signaling pathway;

(B) a second formulation comprising an acquired immunodeficiency syndrome vaccine; and

(C) instructions for use;

In another preferred embodiment, the active ingredient of the first formulation is a PD-1 antibody.

In another preferred embodiment, the active ingredient of the second formulation is an immunogen in an acquired immunodeficiency syndrome vaccine or vaccine, preferably an immunogen expressed by a live vector virus vaccine.

In another preferred embodiment, the antagonist against the PD-1 signaling pathway is a PD-1 antibody.

In another preferred embodiment, the concentration of the PD-1 antibody in the first formulation is 1mg/ml to 100mg/ml, preferably 1mg/ml to 25 mg/ml.

In another preferred embodiment, the vaccine for acquired immunodeficiency syndrome is a live vector virus vaccine.

In another preferred embodiment, the instructions specify that the first and second agents are to be used in combination to prevent and/or treat acquired immunodeficiency syndrome.

In another preferred embodiment, the instructions indicate that the first formulation is administered prior to the second formulation, preferably the first formulation and the second formulation are administered at a time interval of 1-2 weeks.

In another preferred embodiment, the first formulation, the second formulation is administered simultaneously, separately or sequentially in the prevention and/or treatment of acquired immunodeficiency syndrome.

In another preferred embodiment, the kit is for use in the prevention and/or treatment of acquired immunodeficiency syndrome.

In a third aspect of the invention, there is provided a combination of active ingredients, the combination comprising or consisting of:

(A) a first active ingredient that is an antagonist against the PD-1 signaling pathway;

(B) a second active ingredient which is an acquired immunodeficiency syndrome vaccine;

In another preferred embodiment, the combination is used for the preparation of a medicament or vaccine for the prevention and/or treatment of acquired immunodeficiency syndrome.

In a fourth aspect of the present invention, there is provided a use of the composition according to the first aspect of the present invention for the preparation of a medicament or vaccine for the prevention and/or treatment of acquired immunodeficiency syndrome.

In another preferred embodiment, the acquired immunodeficiency syndrome comprises human acquired immunodeficiency syndrome and/or monkey acquired immunodeficiency syndrome.

In another preferred embodiment, the pharmaceutical composition is administered before, simultaneously with or after the administration of a further active substance for the prophylaxis and/or treatment of acquired immunodeficiency syndrome.

In a fifth aspect of the invention, there is provided the use of an antagonist against the PD-1 signalling pathway for (a) enhancing the prophylactic effect of a vaccine, or (b) the manufacture of a formulation or composition for use in enhancing the prophylactic effect of a vaccine, wherein the antagonist is a PD-1 antibody and the vaccine is an acquired immunodeficiency syndrome live vector viral vaccine.

In a sixth aspect of the present invention, there is provided a method for preventing or treating acquired immunodeficiency syndrome, said method comprising the steps of: administering to a subject in need thereof a composition according to the first aspect of the invention, a kit according to the second aspect of the invention or an active ingredient combination according to the third aspect of the invention.

In another preferred embodiment, the acquired immunodeficiency syndrome is human acquired immunodeficiency syndrome.

In another preferred embodiment, the subject is a mammal (e.g., a human).

In another preferred embodiment, the first active ingredient or first formulation active ingredient is a PD-1 antibody.

In another preferred embodiment, the amount of the first active ingredient or the active ingredient of the first preparation administered is from 0.1mg to 1000mg per kg of body weight, preferably from 1 to 200mg per kg, more preferably from 3 to 20mg per kg.

In another preferred embodiment, the administration interval of the first active ingredient or the first formulation active ingredient is at least once every four weeks, preferably at least once every three weeks, more preferably at least once every two weeks.

In another preferred embodiment, the first active ingredient or the first formulation active ingredient is applied at least 1 time, preferably not less than 3 times, more preferably not less than 5 times.

In another preferred embodiment, the second active ingredient or the second formulation active ingredient is a live vector virus vaccine.

In another preferred embodiment, the amount of the second active ingredient or the active ingredient of the second preparation applied is 5 × 10⁹-2×10¹¹Viral particles/kg body weight (vp/kg), preferably 1X 10¹⁰-9×10¹⁰Viral particles per kg body weight, more preferably 1.5X 10¹⁰-5×10¹⁰Viral particles/kg body weight.

In another preferred embodiment, the administration interval of the second active ingredient or the active ingredient of the second formulation is at least once every eight weeks, preferably at least once every six weeks, most preferably at least once every four weeks.

In another preferred embodiment, the second active ingredient or the second formulation active ingredient is applied at least 1 time, preferably 2 times.

In another preferred embodiment, the total effective active dose per unit time is: the amount of vaccine inoculation per mg of antibody per kg of body weight was 10⁷-10¹⁰A viral particle; more preferably, it is 10⁸-10⁹A viral particle. The unit time is an inoculation period of the combined medicine capable of inducing the organism to generate a preventive immunity effect, and is not less than 1 year, 9 months, 6 months, 3 months, 2 months, 1 month, preferably 2 months.

The total effective active dose is the ratio of the total dose of the used antibody to the total number of virus particles of the inoculated live vector virus vaccine in one inoculation period (namely unit time), and the ratio is achieved so as to cause the effect of effective preventive immunity.

In another preferred embodiment, said administering comprises administering the first active ingredient and the second active ingredient sequentially, or simultaneously.

In another preferred embodiment, the administering comprises administering the first formulation and the second formulation sequentially, or simultaneously.

In another preferred embodiment, the administration is first administering the first active ingredient or the first formulation and then administering the second active ingredient or the second formulation, preferably the dosing interval of the first active ingredient and the second active ingredient (or the first formulation and the second formulation) is 1-2 weeks.

In another preferred embodiment, the administration is performed by first administering the second active ingredient or the second formulation and then administering the first active ingredient or the first formulation, preferably, the dosing interval between the second active ingredient and the first active ingredient (or between the first formulation and the second formulation) is 1-2 weeks.

In another preferred embodiment, said PD-1 antibody is administered simultaneously with said vaccination, the PD-1 antibody being administered at a dose of 3mg/kg to 20mg/kg, and the vaccine being administered in a single doseAccording to 5X 10⁹-2×10¹¹Administering the viral particles per kilogram of body weight, wherein the PD-1 antibody is administered in combination with the vaccine not less than 2 times; more preferably, the PD-1 antibody is administered at a dose of 20mg/kg and the vaccine is administered at a dose of 2X 10¹⁰The virus particles/kg are administered once every four weeks, 2 times in combination.

In another preferred embodiment, the PD-1 antibody is administered prior to vaccination, the PD-1 antibody is administered every four weeks at a dose of 3mg/kg to 20mg/kg, and the vaccine is administered in a single dose of 5X 10⁹-2×10¹¹Viral particles are administered per kilogram of body weight; more preferably, the PD-1 antibody is administered at a dose of 20mg/kg once every four weeks and the vaccine is administered at 2X 10 for one week after administration of the PD-1 antibody¹⁰The virus particles/kg dose were inoculated for a total of 2 inoculations.

In another preferred embodiment, the PD-1 antibody is administered after vaccination, the PD-1 antibody being administered once every four weeks at a dose of 3mg/kg to 20mg/kg, and the vaccine being administered in a single dose of 5X 10⁹-2×10¹¹Viral particles/kg administration; more preferably, the vaccine is administered at 2X 10¹⁰Viral particles/kg dose were inoculated once every four weeks for a total of 2 inoculations, and PD-1 antibody was administered at a dose of 20mg/kg one week after each vaccination.

In another preferred embodiment, the vaccination mode is selected from the group consisting of: a mucosal route, an intramuscular route, an intravenous route, a nasal route, a sublingual route, or a combination thereof.

In another preferred embodiment, the antagonist of the PD-1 signaling pathway is administered in a manner selected from the group consisting of: intravenously, subcutaneously, orally, or a combination thereof.

It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.

Drawings

Figure 1 shows a combined immunization protocol and a later challenge experiment schedule. Respectively a PD-1 antibody + SIV vaccine combined group, an SIV vaccine group and a vaccine control group, wherein the SIV vaccine is a live vector virus vaccine of a replication-deleted 5-type adenovirus vector carrying Gag, pol and ENV expression sequences; the vaccine control group is a vaccine of type 5 adenovirus vector carrying influenza protein with replication deletion.

Figure 2 shows that blocking PD-1 in vivo promotes vaccine-induced specific immune responses. After the combined immunization, the specific immune response level of PBMCs to SIV polypeptide was detected separately by IFN-gamma ELISOPT technology at different time points. (A) One week after vaccine immunization, (B) the second week after the first immunization, (C) the second week after the second immunization, (D) the fourth week after the second immunization. At 42 weeks post-immunization, (E) levels of specific responses against Gag polypeptide (F) Pol polypeptide and (G) ENV polypeptide. (H) Each group of macaques is directed against the immune response of three polypeptides, SIV Gag, Pol and ENV. Ordinate is per 10⁶Number of enzyme-linked spots generated in one Peripheral Blood Mononuclear Cell (PBMC) (SFC, spot forming cells)

Figure 3 shows that blocking the PD-1 signaling pathway promotes specific cytokine secretion and multifunctional CD8+ T cell production in vivo. (A) A flow type two-dimensional scatter diagram after vaccine immunization is used for respectively detecting the secretion conditions of the cell factors 15 days, 45 days and 90 days after immunization by using a flow type technology; (B) cytokine secretion at day 15 post-immunization; (C) cytokine secretion at day 45 post-immunization; (D) cytokine secretion at day 90 post immunization.

FIG. 4 shows that in vivo blocking of PD-1 enhances ENV-specific CD8+ T cell proliferation. On day 25 after the combined immunization, the proliferation capacity of cells between the PD-1+ vaccine group (A), the vaccine group alone (B) and the control group (D) was examined by CFSE technique, respectively; (D) are statistical plots based on three experimental groups.

FIG. 5 shows PD-1 blockade significantly prolongs the protective effect of the vaccine. The specific immune response induced by the vaccine was effectively prolonged by blocking with PD-1 (green) compared to the vaccine group alone (blue) and the control group (red). Wherein A is an immune response to Gag polypeptide, B is an immune response to Pol polypeptide, C is an immune response to Env polypeptide, and D is an immune response to Gag, Pol, and Env polypeptides. Ordinate is per 10⁶Number of enzyme-linked spots generated in one Peripheral Blood Mononuclear Cell (PBMC) (SFC, spot forming cells)

Figure 6 shows that combined immunization enhances macaque resistance to SIV virus. Wherein, a is a protection curve when TCID50 is 5000 and TCID50 is 10000, and B is a protection curve when TCID50 is 50000 and TCID50 is 100000.

Figure 7 shows that the PD-1 blocking combination vaccine enhances macaques against SIV infection and reduces viral load. One arrow in the figure represents one challenge. + represents the SIV virus dose (characterized by TCID50[ half tissue culture infectious titer ]): 1000 parts of; 5000 parts of ++; 10000, + + + +; 15000, + ++; 25000 of ++++; 50000 parts of ++++; +++++++:100000. Wherein, A (left column) is a combined immunization group of the PD-1 antibody and the SIV vaccine, B (middle column) is a single immunization group of the SIV vaccine, and C (right column) is a control group.

Figure 8 shows the level of immune response specific for the helper protein. Ordinate is per 10⁶The number of Spot Forming Cells (SFC) produced in each Peripheral Blood Mononuclear Cell (PBMC) was represented by numbers 120290, 124352, 120294, 120316, 130402, 120308, 080159, 080132, and 080085 on the abscissa from left to right.

Detailed Description

The inventor of the present invention has extensively and deeply studied and unexpectedly found that the PD-1 antibody can be combined with the acquired immunodeficiency syndrome vaccine to act synergistically, so as to effectively prevent and/or treat the acquired immunodeficiency syndrome. Experiments show that blocking the PD-1 signal pathway during vaccine immunization is beneficial to preventing HIV infection, and especially the PD-1 antibody and the live vector virus vaccine are combined in a specific ratio (1: 10)⁷-1:10¹⁰) Can obviously improve the effect of preventing the AIDS virus. In addition to this, the present invention is,the blocking of the PD-1 signal path can enhance the control capability of the PD-1 signal path on viruses and reduce the viral load in blood plasma. On the basis of this, the present invention has been completed.

Term(s) for

In order that the disclosure may be more readily understood, certain terms are first defined. As used in this application, each of the following terms shall have the meaning given below, unless explicitly specified otherwise herein. Other definitions are set forth throughout the application.

The term "about" can refer to a value or composition that is within an acceptable error range for the particular value or composition as determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined.

As used herein, the term "comprising" or "includes" can be open, semi-closed, and closed. In other words, the term also includes "consisting essentially of …," or "consisting of ….

The term "administering" refers to the physical introduction of the product of the invention into a subject using any of a variety of methods and delivery systems known to those skilled in the art, including intravenous, intramuscular, subcutaneous, intraperitoneal, spinal cord or other parenteral routes of administration, e.g., by injection or infusion.

A first active ingredient

The first active ingredient of the present invention is an antagonist against the PD-1 signaling pathway, preferably an antibody to PD-1.

The above antagonists against the PD-1 signaling pathway may be used alone or in combination of two or more kinds, and when used in combination, the mass ratio of each compound is not particularly limited on the premise that the therapeutic purpose is achieved.

In the present invention, there is no particular limitation on the method for obtaining the PD-1 antibody.

PD-1 antibodies

As used herein, the term "PD-1" includes variants (mutated hPD-1), isoforms, and species homologs of human PD-1(hPD-1), hPD-1, and analogs having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to hPD-1. The complete hPD-1 sequence can be found under GenBank accession No. U64863.

In a preferred embodiment of the invention, the first active ingredient is a PD-1 antibody.

In another preferred embodiment, the PD-1 antibody is as described in the first aspect of the invention.

In another preferred embodiment, the nivolumab is a marketed PD-1 antibody drugHeavy and light chains thereofThe chain sequence is recorded in the monoclonal antibody database of IMGT, antibody accession number 424.

SEQ ID No:1

QIQLVQSGSELKKPGASVKVSCKASGYTFTNFGMNWVRQAPGQGLKWMGWISGYTREPTYAADFKGRFVISLDTSVSTAYLQISSLKAEDTAVYYCARDVFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG

SEQ ID No:2

DIVLTQSPASLAVSPGQRATITCRASESVDNYGYSFMNWFQQKPGQPPKLLIYRASNLESGVPARFSGSGSRTDFTLTINPVEANDTANYYCQQSNADPTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

SEQ ID No:3

SEQ ID No:4

DIVLTQSPASLAVSPGQRATITCRASESVDNYGYSFMNWFQQKPGQPPKLLIYRASNLESGVPARFSGSGSRTDFTLTINPVEADDTANYYCQQSNADPTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

SEQ ID No:5

QVQLQESGPGLVKPSQTLSLTCTVSGYSISSDYAWNWIRQPPGKGLEWMAYISYSGYTSYNPSLKSRITISRDTSKNQFSLKLSSVTAADTAVYYCARSLDYDYGTMDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG

SEQ ID No:6

EIVLTQSPATLSLSPGERATLSCRANSSVSSMHWYQQKPGQSPEPWIYAISNLAFGVPARFSGSGSGTDYTLTISSLEPEDFAVYYCQQWSSRPPTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

The invention includes not only intact antibodies, but also fragments of antibodies with immunological activity or fusion proteins of antibodies with other sequences. Accordingly, the invention also includes fragments, derivatives and analogs of the antibodies.

In the present invention, antibodies include murine, chimeric, humanized or fully human antibodies prepared using techniques well known to those skilled in the art. Recombinant antibodies, such as chimeric and humanized monoclonal antibodies, including human and non-human portions, can be prepared using recombinant DNA techniques well known in the art.

The antibody of the present invention may be a double-chain or single-chain antibody, and may be selected from an animal-derived antibody, a chimeric antibody, a humanized antibody, more preferably a humanized antibody, a human-animal chimeric antibody, and still more preferably a fully humanized antibody.

The antibody derivatives of the present invention may be single chain antibodies, and/or antibody fragments, such as: fab, Fab ', (Fab') 2 or other antibody derivatives known in the art, and the like, as well as any one or more of IgA, IgD, IgE, IgG, and IgM antibodies or antibodies of other subtypes.

The antibodies of the invention may be murine, chimeric, humanized, CDR grafted and/or modified antibodies targeting human PD-1.

Any method suitable for producing monoclonal antibodies can be used to produce the PD-1 antibodies of the invention. For example, animals may be immunized with a linked or naturally occurring PD-1 homodimer or fragment thereof. Suitable immunization methods, including adjuvants, immunostimulants, repeated booster immunizations, and one or more routes may be used.

Any suitable form of PD-1 may be used as an immunogen (antigen) for the production of non-human antibodies specific for PD-1, which antibodies are screened for biological activity. The challenge immunogen may be full-length mature human PD-1, including a native homodimer, or a peptide containing a single/multiple epitope. The immunogen may be used alone or in combination with one or more immunogenicity enhancing agents known in the art. Immunogens can be purified from natural sources or produced in genetically modified cells. The DNA encoding the immunogen may be genomic or non-genomic in origin (e.g., cDNA). DNA encoding the immunogen may be expressed using suitable genetic vectors including, but not limited to, adenoviral vectors, adeno-associated viral vectors, baculovirus vectors, plasmids and non-viral vectors.

Humanized antibodies may be selected from any class of immunoglobulins, including IgM, IgD, IgG, IgA, and IgE. Optimization of the sequence of the essential constant domains to produce the desired biological activity is readily achieved by screening antibodies using the biological assays described in the examples below. Likewise, any type of light chain can be used in the compounds and methods herein. In particular, kappa, lambda chains or variants thereof are useful in the compounds and methods of the invention.

The sequence of the DNA molecule of the antibody or fragment thereof of the present invention can be obtained by a conventional technique, for example, by PCR amplification or genomic library screening. Alternatively, the coding sequences for the light and heavy chains may be fused together to form a single chain antibody.

Once the sequence of interest has been obtained, it can be obtained in large quantities by recombinant methods. This is usually done by cloning it into a vector, transferring it into a cell, and isolating the relevant sequence from the propagated host cell by conventional methods.

In addition, the sequence can be synthesized by artificial synthesis, especially when the fragment length is short. Generally, fragments with long sequences are obtained by first synthesizing a plurality of small fragments and then ligating them. The DNA sequence may then be introduced into various existing DNA molecules (or vectors, for example) and cells known in the art.

The invention also relates to a vector comprising a suitable DNA sequence as described above and a suitable promoter or control sequence. These vectors may be used to transform an appropriate host cell so that it can express the protein.

The host cell may be a prokaryotic cell, such as a bacterial cell; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as mammalian cells.

The steps described in the present invention for transforming a host cell with a recombinant DNA can be performed using techniques well known in the art. The obtained transformant can be cultured by a conventional method, and the transformant expresses the polypeptide encoded by the gene of the present invention. Depending on the host cell used, it is cultured in a conventional medium under suitable conditions.

Typically, the transformed host cells are cultured under conditions suitable for expression of the antibodies of the invention. The antibody of the invention is then purified by conventional immunoglobulin purification procedures, such as protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, ion exchange chromatography, hydrophobic chromatography, molecular sieve chromatography or affinity chromatography, using conventional separation and purification means well known to those skilled in the art.

A second active ingredient

The second active ingredient of the invention is an acquired immunodeficiency syndrome vaccine, preferably a live vector virus vaccine.

In another preferred embodiment, the immunogen of the acquired human immunodeficiency syndrome vaccine is as described in the first aspect of the invention.

SEQ ID No:7

SEQ ID No:9

SEQ ID No.10

SEQ ID No.11

SEQ ID No.12

SEQ ID No.13

SEQ ID No.14

SEQ ID No.15

Composition, kit, active ingredient combination and pharmaceutical composition

The composition of the present invention may be a pharmaceutical composition comprising:

The composition may further include a pharmaceutically acceptable carrier, if necessary. As used herein, the term "pharmaceutically acceptable" ingredient refers to a substance that is suitable for use in humans and/or animals without undue adverse side effects (such as toxicity, irritation, and allergic response), i.e., at a reasonable benefit/risk ratio.

As used herein, the term "effective amount" refers to an amount that produces a function or activity in and is acceptable to humans and/or animals.

As used herein, the term "prophylactically and/or therapeutically effective amount" refers to an amount that produces a prophylactic effect and/or therapeutic function or activity on a human and/or animal and is acceptable to a human and/or animal.

As used herein, the term "pharmaceutically acceptable carrier" refers to a carrier for administration of a therapeutic agent, including various excipients and diluents. The term refers to such pharmaceutical carriers: they are not essential active ingredients per se and are not unduly toxic after administration. Suitable carriers are well known to those of ordinary skill in the art.

The pharmaceutical composition of the present invention containing the first active ingredient, the second active ingredient, or their derivatives and metabolites may be in various forms suitable for oral administration, or may be various preparations for external administration or other preparations for parenteral administration. The external administration preparation can be further prepared into a preparation (including but not limited to) by adding auxiliary materials such as a surfactant, a transdermal absorption enhancer, a preservative, a solvent, an antioxidant, a humectant, a pH regulator, a colorant, perfume and the like: liniment, tincture, oil, ointment, plaster, cataplasm, plaster, patch, plastics, membrane, gel, cataplasma, acupoint application agent, spray, aerosol, implant, emulsion, etc. For the present invention, preferred dosage forms include: various dosage forms, implants and injections for oral administration.

The auxiliary materials added in the composition are common auxiliary materials in the field of preparation, and the type, the using method and the source of the auxiliary materials are well known by the technical personnel in the field.

The invention also provides a combination of active ingredients, which comprises the following components or is formed by combining the following components:

The invention also provides a kit comprising:

(C) instructions for use;

The instruction book indicates that the first preparation and the second preparation are combined together so as to prevent and/or treat the acquired immunodeficiency syndrome.

The compositions, active ingredient combinations, pharmaceutical compositions, kits of the invention can be prepared using conventional methods and equipment.

Composition, active ingredient combination, pharmaceutical composition, use of kit and administration mode

The invention provides application of the composition, the active ingredient combination and the pharmaceutical composition in preparing a medicament for preventing and/or treating acquired immunodeficiency syndrome.

In another preferred embodiment, the further active substances for the prophylaxis and/or treatment of acquired immunodeficiency syndrome are selected from the group consisting of: antiretroviral drugs, immunomodulatory drugs, and anti-opportunistic infection drugs.

In combination with the first and second formulations in the kit of the present invention, the first and second formulations may be administered simultaneously, separately or sequentially. The safe and effective dose of the active ingredient of the first preparation is generally 0.1mg to 1000mg/kg body weight, preferably 1 to 200mg/kg, more preferably 3 to 20mg/kg, and the safe and effective dose of the active ingredient of the second preparation is generally 5X 10⁹-2×10¹¹Viral particles/kg body weight, preferably 1X 10¹⁰-9×10¹⁰Viral particles/kg body weight, morePreferably 1.5X 10¹⁰-5×10¹⁰Viral particles/kg body weight. The administration mode comprises the following steps: when used in combination, the first formulation and/or the second formulation is administered in a manner selected from the group consisting of: intravenously, subcutaneously, orally, or a combination thereof.

In the process of drug combination, the interaction of the drugs is divided into additive action, synergistic action and antagonistic action according to the effect of the drugs when the drugs are used together, the synergistic action means that the effect of the drugs when the drugs are used together is more than times that when the drugs are used alone, the additive action means that the effect of the drugs when the drugs are used together is equivalent to that when the drugs are used alone, and the antagonistic action means that the effect of the drugs when the drugs are used together is less than that when the drugs are used alone.

The present invention also provides a method for preventing or treating acquired immunodeficiency syndrome, said method comprising the steps of: the compositions, active ingredient combinations, pharmaceutical compositions and kits of the present invention are administered to a subject in need thereof. The subject is a mammal, preferably a human.

In the prevention and/or treatment of acquired immunodeficiency syndrome, the mode of administration according to the invention comprises the sequential administration of the first active ingredient and the second active ingredient, or the simultaneous administration of the first active ingredient and the second active ingredient.

Such that upon administration of the composition, combination of active ingredients, or pharmaceutical composition of the present invention, a safe and effective amount of the composition, combination of active ingredients, or pharmaceutical composition of the present invention is administered to the mammal. The specific dosage will also take into account factors such as the route of administration, the health of the patient, etc., which are within the skill of the skilled practitioner. When the first active ingredient and the second active ingredient are administered sequentially, the interval between the administrations is not particularly required.

The compositions, active ingredient combinations, pharmaceutical compositions and kits of the present invention comprise a first active ingredient and a second active ingredient, each administered simultaneously or sequentially by the same or different routes, including but not limited to: oral administration, injection administration, intratumoral administration, implantation administration, intracavity administration, anal administration, transdermal administration and internal and external application;

preferred administration by injection includes: intravenous injection, intramuscular injection, subcutaneous injection, intracavity injection.

Compared with the prior art, the invention has the main advantages that:

(1) the use of the PD-1 antibody in combination with an AIDS vaccine can effectively enhance the immune response induced by the vaccine, prolong the immune response of the vaccine in vivo and improve the resistance to HIV and/or SIV virus infection. Below 50000 doses of TCID50, the PD-1 antibody in combination with the vaccine achieved full protection.

(2) Provides a strategy of AIDS vaccine which can be used for prevention, and fills the blank of the research field.

(3) The method for improving immune protection of the AIDS vaccine is provided, and the administration method of the PD-1 antibody and the AIDS vaccine is added, so that the immune stimulation effect of organisms is obviously improved.

(4) The immunogen expression sequence in the acquired immunodeficiency syndrome vaccine is a sequence optimized by codons and the like, so that the immunogen expression sequence is more suitable for being expressed in a human body, and has better preventive effect after being combined with a PD-1 antibody.

(5) The PD-1 antibody and the live vector virus vaccine of the invention are mixed in a specific ratio (1mg: 10)⁷vp-1mg:10¹⁰vp, more preferably 1mg:10⁸vp-1mg:10⁹vp) has the best effect of preventing the HIV when used together.

The features mentioned above with reference to the invention, or the features mentioned with reference to the embodiments, can be combined arbitrarily. All the features disclosed in this specification may be combined in any combination, and each feature disclosed in this specification may be replaced by alternative features serving the same, equivalent or similar purpose. Thus, unless expressly stated otherwise, the features disclosed are merely generic examples of equivalent or similar features.

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Experimental procedures without specific conditions noted in the following examples, generally followed by conventional conditions, such as Sambrook et al, molecular cloning: the conditions described in the laboratory Manual (New York: Cold Spring harbor laboratory Press,1989), or according to the manufacturer's recommendations. Unless otherwise indicated, percentages and parts are percentages and parts by weight.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In addition, any methods and materials similar or equivalent to those described herein can be used in the methods of the present invention. The preferred embodiments and materials described herein are intended to be exemplary only.

General experimental materials and methods

1. Experimental Material

1.1 Experimental animals

The Chinese rhesus monkey used in the application is purchased from Guangdong Landao biotechnology limited company and is raised in the Jiufer experiment base of animal experiment center of Guangzhou biological medicine and health research institute. All experimental animals and their operations were performed strictly under the Guangzhou institute of biomedical and health laboratory animal regulations.

1.2 viruses

Molecular cloning of SIVmac239 virus was performed by NIH AIDS Research & Reference Reagent Program under accession number M33262.1 in GenBank, and amplified and purified as necessary at the time of use.

1.3PD-1 antibodies

The PD-1 antibody used in this experiment was expressed and provided by the company jia and biopharmaceutical, inc, according to standard antibody production procedures, the antibodies 7A4,7A4D and 13F1 used were derived from the PD-1 antibody sequence described in chinese patent application 201540380001. X and were self-expressed according to the sequence of pamumab and nivolumab, and the antibodies used were dissolved at 25mg/ml in PBS buffer (pH 7.2).

1.4 vaccines and antigenic polypeptides

The Ad5-SIV-GPE, the Ad5-NA and the Ad5-M2H5 are independently constructed by the inventor, wherein the Ad5 is a 5-type adenovirus vector, the GenBank accession number of the sequence is AY339865.1, in the invention, an E1 gene necessary for virus replication is deleted, a foreign gene is inserted into the gene region of E1 by a homologous recombination technology, and the promoter is CMV. Ad5-SIV-GPE is a vaccine which is constructed by using a replication deletion type Ad5 adenovirus vector and can simultaneously express three antigens of SIV virus Gag, Pol and Env. In addition, the invention also independently constructs vaccines of single antigens such as Ad5-SIV-Gag, Ad5-SIV-Pol and Ad5-SIV-Env, and the like, wherein the immunogen is expressed by optimized nucleic acid; and Ad5-NA and Ad5-M2H5 are vaccines for expressing influenza proteins constructed based on Ad5 vectors, wherein the optimized NA nucleic acid sequence is SEQ ID NO: 16, the optimized M2H5 nucleic acid sequence is SEQ id no: 17. the vaccine dose is expressed as number of virus particles per ml of PBS buffer, pH 7.2.

SEQ ID No.16neuraminidase(NA)gene,

atgaaccccaaccagaagatcatcaccattggctccatctgcatggtgacaggcattgtctccctgatgctgcagattggcaacatgatctccatctgggtctcccactccatccacacaggcaaccagcatcagtctgagcccatctccaacaccaacctgctgacagagaaggctgtggcctctgtgaagctggctggcaactcctccctgtgccccatcaatggctgggctgtctactccaaggacaactccatcaggattggctccaagggcgatgtctttgtgatcagggagccattcatctcctgctcccatctggagtgcaggaccttcttcctgacccagggcgccctgctgaatgacaagcactccaatggcacagtgaaggacaggtccccccacaggaccctgatgtcctgccctgtgggcgaggccccatccccatacaactccaggtttgagtctgtggcctggtctgcctctgcctgccatgatggcacctcctggctgaccattggcatctctggccctgacaacggcgctgtggctgtgctgaagtacaatggcatcatcacagacaccatcaagtcctggaggaacaacatcctgaggacccaggagtctgagtgtgcctgtgtgaatggctcctgcttcacagtgatgacagatggcccatccaatggccaggcctcccacaagatcttcaagatggagaagggcaaggtggtgaagtctgtggagctggatgcccccaactaccactatgaggagtgctcctgctaccctgatgctggcgagatcacctgtgtctgcagggacaactggcatggctccaacaggccatgggtctccttcaaccagaacctggagtaccagattggctacatctgctctggcgtctttggcgacaaccccaggcccaatgatggcacaggctcctgtggccctgtctcctccaatggcgctggcggcgtgaagggcttctccttcaagtatggcaatggcgtctggattggcaggaccaagtccaccaactccaggtctggctttgagatgatctgggaccccaatggctggacagagacagactcctccttctctgtgaagcaggacattgtggccatcacagactggtctggctactctggctcctttgtgcagcatcctgagctgacaggcctggactgcatcaggccctgcttctgggtggagctgatcaggggcaggcccaaggagtccaccatctggacctctggctcctccatctccttctgtggcgtgaactctgacacagtgggctggtcctggcctgatgatgctgagctgccattcaccatcgacaagtaa

SEQ ID No.17M2H5

atgtccctgctgacagaggtggagacccccaccaggaatgagtgggagtgcaggtgctctgactcctctgaccccattgtggtggctgccaacatcattggcatcctgcatctgatcctgtggattctggacaggctgttcttcaagtgcatctacaggaggctgaagtatggcctgaagaggggccctgccacagctggcgtgcctgagtccatgagggaggagtacaggcaggagcagcagtctgctgtggatgtggatgatggccactttgtgaacattgagctggagtaa

SIV polypeptide powder was supplied by NIH AIDS Research & Reference Reagent Program, and then thawed with DMSO and stored in separate containers in the laboratory.

2. Experimental methods

2.1 IFN-. gamma.Elispot test the self-coating kit of Elispot from BD company was chosen, and after coating the cynomolgus IFN-. gamma.antibodies, the procedure was followed as described in the product instructions. And (3) detecting the IFN-gamma release amount of the PBMCs in the blood of the macaque.

1) With PBS: activating an Elispot plate of the PVDF membrane for 30 seconds by using a mixed solution of which the ratio of 75% ethanol is 1: 1;

2) quickly throwing away the activating solution, adding 200 mu L PBS and washing for 6 times;

3) adding 100 μ L diluted cynomolgus IFN- γ coated antibody (diluted 200 times with PBS), standing at 4 deg.C overnight;

4) removing the coating antibody, washing with PBS for 6 times, adding 200 μ L of R10 culture medium, and sealing at 37 deg.C in cell culture box for 2 h;

5) taking 10mL of anticoagulated blood of two SIV infected macaques respectively, separating PBMCs, and diluting the cell concentration to 30 ten thousand per 100 mu L by using an R10 culture medium according to the cell counting condition;

6) pouring off the sealing liquid, buckling the sterile paper to be dry, and uniformly spreading the cells on the plate;

7) DMSO, SIV-Gag polypeptide, positive stimulator ConA and corresponding concentrations of PD-1 antibody were added separately according to the previous layout.

8) Culturing at 37 deg.C with 5% CO2 for 24 h;

9) the cell sap was decanted, washed 6 times with PBST and dried on paper;

10) add 100. mu.L of cynomolgus IFN-. gamma.detection antibody (200-fold diluted with 5% inactivated FBS/PBST);

11) placing in a refrigerator at 4 ℃ overnight;

12) the detection antibody was decanted, washed 6 times with 200 μ L/well PBST and drained on paper;

13) diluting streptomycin-conjugated alkaline phosphatase with PBST containing 5% inactivated FBS at a ratio of 1:2500, adding 100 μ L of alkaline phosphatase to each well, and standing at 37 deg.C for 2 h;

14) washing with sterile PBST for 6 times, discarding the washing solution and drying on paper;

15) adding BCIP/NBT chromogenic substrate preheated at 37 ℃ for 30min, reacting for 9min in a dark place, wherein each well is 100 mu L;

16) discarding the substrate, washing with tap water for 1min, naturally air-drying overnight, and waiting for reading the plate;

17) and (4) reading the plate by using an enzyme-linked immunosorbent spot imager, analyzing the experimental result, and mapping.

2.2 intracellular multifactorial staining (ICS)

1) PBMC of monkeys were isolated by routine methods, recorded for isolation, and adjusted to 2X 10 cells per 200ul medium after cell counting⁶PBMCs added to a 48-well cell culture plate; the heavy suspension and the cell culture medium are both R10 culture medium;

2) adding corresponding SIV specific antigen peptide into the cell suspension, adding DMSO with corresponding concentration into a negative control hole, and adding PMA + ionomycin into a positive control;

3) culturing at 37 deg.C with 5% CO2 for 2 h;

4) after stimulating for two hours, adding diluted BFA (brefeldin) to block the secretion of cell factors to the outside of cells;

5) culturing at 37 deg.C with 5% CO2 for 8 hr;

6) blowing and beating the uniform cells, transferring the cells into a flow tube, adding 1mL of PBS washing liquor, uniformly mixing, and centrifuging at 350g of room temperature for 10 min;

7) discarding the supernatant, adding cell surface antibodies CD4-PE-CF594, CD8-APC-Cy7 and CD3-Pacific Blue into each tube respectively, and mixing the mixture evenly for 3 seconds by vortex oscillation, wherein each volume is 0.5 mu L;

8) dyeing for 30min at room temperature in dark place;

9) 1mL of PBS wash was added. Mixing, centrifuging at room temperature for 10min at 350 g;

10) washing is repeated once;

11) shaking the cells evenly, then adding 250 mu L of cytofix/cytoperm, and mixing evenly;

12) reacting at 4 ℃ in the dark for 20 min;

13) adding 1mL of 1 Xperm/wash (stock solution is 10 Xdiluted to 1 times with sterile distilled water before use), 350g, centrifuging for 8min, and discarding the supernatant;

14) repeating the previous step once;

15) discarding the supernatant, and adding intrabodies (IFN-. gamma. -PE and TNF-. alpha. -PE-Cy 7);

16) keeping away from light at 4 deg.C for 60 min;

17) adding 1mL of 1 Xperm/wash lotion, 350g, and centrifuging for 8 min;

18) the supernatant was discarded and 1mL of PBS wash was added. After mixing uniformly, 350g of the mixture is centrifuged for 10 minutes at room temperature;

19) discarding the supernatant, and adding 350 mu L PBS into each tube for resuspension;

20) and (5) performing detection and analysis on the machine.

2.3T cell proliferation assay (CFSE)

1) PBMCs were isolated by conventional methods. After cell counting, taking 1 x 10^6cells to a 15mL centrifuge tube, resuspending the cells by 8mL RPMI1640 culture medium, then centrifuging the cells for 5min at 350g, and resuspending the cells by 1mL DPBS of 2% FBS;

2) diluting the CFSE stock solution to 2 mu mol/L with RPMI1640, and performing light-shielding operation as much as possible;

3) mixing the cell suspension and the CFSE diluent in equal volume, and gently mixing the cell suspension and the CFSE diluent, wherein the cells are fragile at the moment, so that the movement needs to be gentle as much as possible;

4) mixing, standing at 37 deg.C for 10 min;

5) adding ice-cold DPBS (2% FBS) to 10mL, mixing, immediately transferring to ice, and performing ice bath for 5min to stop dyeing;

6) centrifuge at 350g for 5 minutes at 4 ℃. The supernatant was discarded and ice-cold R10 complete medium was added to rinse 2 times. Finally resuspension with appropriate R10 medium;

7) adding the cell suspension into a 24-hole culture plate according to the preset layout, wherein each hole is 500 mu L, SIV antigen peptide is added into the cell suspension, DMSO with corresponding concentration is added into a negative control hole, and phorbol ester and ionomycin are added into a positive control;

8) wrapping with tinfoil paper37℃,5％CO₂Culturing in dark for 6 days;

9) the homogenized cells were blown up and transferred to a flow tube (12X 75mm polystyrene tube). Each was rinsed once with 1mL PBS and transferred to the same flow tube. After mixing uniformly, centrifuging for 10min at room temperature with 300 g;

10) discard the supernatant (gentle operation, leave some residual supernatant);

11) after the cells are resuspended, cell surface antibodies CD3-Pacific Blue, CD4-PE-CF594 and CD8-APC-Cy7 are added, and the mixture is uniformly mixed and dyed for 30min in a dark place at room temperature;

12) washing twice with 1mL PBS, and discarding the supernatant;

13) resuspend with 350. mu.L PBS and test on the machine.

2.4 establishment of Low-dose infection model of Kiwi berry rectum

The experiment adopts SIVmac239 virus obtained by in vitro amplification and purification as stock solution, and the stock solution is diluted to 10 times of the virus attacking dose by using physiological saline containing 2% inactivated FBS during the infection period, placed on ice and brought to an animal room, and then diluted according to the virus attacking amount.

The toxic macaque needs to be fasted for 12 hours in advance, kept high in the hip after anesthesia, inserted into an anus for 4-7cm by a stomach tube for a child, connected with a syringe with viruses at the other end of the stomach tube, and injected into the macaque body after being inserted into a proper position and filled with a small amount of air. After the completion, the stomach tube of the child is taken out, so that the macaque continues to keep the hip high for 20min, and the virus liquid is prevented from flowing out.

The toxic counteracting dose is as follows: +:1000 TCID50 twice; ++:5000 TCID50 twice; +++:10000 TCID50 twice; ++++:15000 TCID50 once; +++++:25000 TCID50 once; ++++++:50000 TCID50 once; +++++++:100000 TCID50 once.

During the infection process, plasma was collected periodically and its plasma was tested for viral load, if viral load was detected 3 consecutive times and lg10 of the load was above test line 2, this indicates that the macaque was infected with SIV. The infected macaques will no longer be included in the challenge program, but will be continuously tracked for their viral load and level of immune response. However, infection with TCID50 at high doses continued in uninfected macaques.

Example 1 animal grouping and Combined immunization strategy

9 Chinese macaques are divided into 3 groups of 3 animals, and the animal information and the immunization strategy of each group are shown in table 1 and figure 1. Specific immunization times are shown in table 2. Samples are collected at regular time during the immunization period, and the change conditions of antigen-specific immune response level, multifunctional T cells, T cell proliferation and the like are detected. The antigen-specific immune response level is detected by using IFN-gamma ELISPOT technology, the change of the multifunctional T cells is evaluated by using ICS technology, and the proliferation capacity of the T cells is detected by using CFSE technology.

Table 1 details of animal groups

TABLE 2 animal Combined immunization schedules

Example 2 immune Effect of PD-1 antibody blocking Combined SIV vaccine immunopotentiating vaccine

Vaccine immunizations were performed according to the immunization protocol and strategy of example 1, using a 7A4D humanized antibody as the PD-1 antibody; the SIV vaccine selects replication-deficient adenovirus 5 vaccine (Ad5-GPE) simultaneously expressing three antigens of SIV Gag, Pol and Env to study in examples 2 to 7, wherein PD-1 antibody is administrated by intravenous drip at a dose of 20 mg/kg; first vaccine immunization Simplex intramuscular injections of 0.5ml (virus diluted to 10 concentration in PBS) were administered to both deltoids of monkeys¹¹Viral particles/ml). After the first vaccine immunization, the level of specific immune responses in the experimental animals was followed for a long time by the Elispot technique. One week after immunization, groups G1 and G2 with vaccine began to generate specific immune responses against SIV-ENV antigen polypeptide compared to the control group, which were not significantly different (P ═ 0)2449) (fig. 2A); after the second week, animals of groups G1 and G2 developed extremely high specific immune responses, with an average of 2987 points for the SIV-ENV polypeptide in group G1 and up to 2377 in group G2. Although the response level was higher in the G1 group after blocking with PD-1 antibody than in the vaccine group alone, and the difference between these two groups was gradually increased with respect to the first week, the P value was 0.2367 (fig. 2B); in the later tests, the difference between the specific response levels of the two groups was gradually increased (P. 0.0795), because the response level of the vaccine group was rapidly decreased, the average number of plaques was decreased from 2377 to 847, and though the PD-1 antibody + vaccine group was also decreased, the response remained high, and the number of plaques was 2788 against SIV-ENV stimulation (fig. 2C); in addition, the response of the vaccine alone group was reduced to 586 in the fourth week after the second immunization (the same method as the first immunization, i.e., single site intramuscular injection of deltoid muscle on both sides of monkey, 0.5ml per side injection), while the animal model after PD-1 antibody blocking still maintained an ultra-high specific response of SIV-ENV in vivo, with an average of 2556 (fig. 2D). The PD-1 antibody combination vaccine gave higher levels of immune responses in animals against SIV Gag, Pol and Env antigens after immunization compared to the immunization vaccine group alone and the control group (fig. 2E, F, G and H). The above data can demonstrate that the immune response of the SIV vaccine can be enhanced after blocking the PD-1 signaling pathway by the PD-1 antibody in vivo.

Example 3PD-1 antibody blockade in combination with the Effect of SIV vaccine immunization on T lymphocytes

Long-term follow-up and monitoring of multifunctional lymphocytes in vivo after immunization also showed a synergistic effect of PD-1 antibodies on vaccine immune responses. Single factor (IFN-gamma) secretion in the PD-1 antibody combination vaccine group 15 days after vaccine immunization⁺TNF-α^-，IFN-γ^-TNF-α⁺) CD8 (1)⁺0.5% of T cells, and IFN-gamma and TNF-alpha simultaneously secreting CD8⁺T cells were approximately 0.31% (FIG. 3A), whereas in the vaccine group, single factor secretion was approximately 0.88%, multifactorial secretion was approximately 0.21% (FIG. 3A), and at day 15 post-immunization, specific IFN-. gamma.s were present in both groups⁺TNF-α^-CD8⁺T cells did not differ significantly, but IFN-. gamma.^-TNF-α⁺CD8⁺And IFN-gamma⁺TNF-α⁺CD8⁺Were higher than the vaccine group alone (fig. 3B). At day 45, 4 weeks after booster immunization, the results of the test were very interesting, with the individual vaccine groups secreting single and dual factor CD8⁺In the case of T cell decrease (0.18% for single factor secreting cells and about 0.11% for double factor secreting cells), the immune response level after PD-1 antibody blocking was opposite to that of the vaccine group alone, and the antigen specificity was CD8⁺The proportion of T cells rose dramatically, with single factor secreting cells at about 1.86%, about 4-fold at 15 days. CD8 secreting dual factors⁺Approximately total T cell CD8⁺2.28% of T cells were 7-fold higher than in the initial immunization (fig. 3A and C). On day 90, the single-factor secreting cells in group G1 were 0.73%, IFN-. gamma.⁺TNF-α⁺CD8⁺T cells were 0.94%, while those of group G2 were 0.24% and 0.08%, respectively (fig. 3A and 3D). Although the response level was also reduced in group G1, the specific immune response level remained high compared to group G2, which was the vaccine immunized alone.

The combination of Elispot results shows that compared with Ad5-SIV-GPE vaccine immunized alone, the PD-1 antibody blocking combined Ad5-SIV-GPE vaccine remarkably enhances specific immune response in the body after boosting immunity, generates more CD8 secreting single factors and simultaneously secreting multiple cytokines⁺T cells. This also suggests that blocking the PD-1 signaling pathway in vivo may enhance the protective effect of the vaccine.

After one month of combined immunization, specific T cell proliferation against SIV-ENV antigen was also tested between three groups by CFSE assay. The results are shown in FIG. 4, with the PD-1 combination vaccine group directed against SIV-ENV, specific CD8⁺On average, T cells proliferated by 1.7%, whereas no proliferation was detected in the vaccine group and the control group alone. The results also demonstrate that blocking the PD-1 signaling pathway is effective in enhancing ENV-specific CD8⁺T cells proliferate.

Example 4 PD-1 blockade enhances specific responses of SIV vaccines

The level of immune response in vivo in animal models was monitored and it was observed whether the PD-1 antibody blocks the specific immune response that could prolong the induction of the vaccine. After 42 weeks from the first immunization, the IFN-gamma Elispot technology is adopted to detect the specific reaction strength of the antigen peptides against Gag, Pol and Env (the Ad5-SIV-GPE vaccine for immunization can express three proteins of Gag, Pol and Env of SIV). The results are shown in fig. 2, three chinese macaques in the G1 group all showed higher immune responses to Gag, Pol and Env three SIV antigen polypeptides, compared to the G1 group, only one macaque numbered 130402 showed responses in the G2 group, and only the other two macaques showed weak expression. 130402 responded, but at a lower level than the average level of the group G1. Whereas the G3 group, which had not been immunized, had little specific response against the three antigenic peptides.

Statistical analysis was performed with the results of specific immune responses against SIV-Env polypeptides by long-term follow-up monitoring (fig. 5). As a result, it was found that the level of immune responses of both the PD-1 antibody combination vaccine group and the vaccine alone group peaked at the second week after the first immunization, and were almost at the same level between the two groups, 2987 in the G1 group and 2377 in the G2 group (fig. 5C). However, after that, the responses of the two groups are reduced to different degrees, the G2 group is greatly reduced, and the G1 group has only slight change and is still equal to the highest point. By week 13, the average number of plaques in the G1 group was 1982, whereas the G2 group was 292, with a 6.7-fold difference between the two groups (fig. 5C); by 41 weeks, the mean number of points in the G1 group remained at a high level of 1211, whereas the G2 group was only 237, with the two groups differing in specific SFC by a factor of about 5.

It can be seen that in vivo blocking of PD-1 antibodies can significantly prolong vaccine-induced specific immune responses. The vaccine-induced response levels were greatly reduced by the seventh week without blocking the PD-1 signaling pathway, whereas the vaccine response was significantly enhanced by the PD-1 antibody used in this study, and the decline was slow at the later stage, and the specific immune response remained at a high level after 42 weeks in this group of animals, which was higher than that in the G2 group at the seventh week.

Example 5 PD-1 antibody combination vaccine immunization strategy enhances resistance of macaques to SIV Virus

This example deals with the toxicity attack of the macaque model described above. In order to better understand the infection dose that macaques can bear, a rectal low-dose infection mode is adopted. During challenge, the viral load of plasma in animal models was continuously monitored for a long period of time using real-time quantitative PCR techniques. Through long-term follow-up monitoring, all data are collated to draw a correlation graph of plasma viral load and allowable SIV challenge dose of macaques (figure 7). As can be seen from the results in the figure, only one macaque survived infection at infection doses up to 10 million TCID50 in the PD-1+ vaccine group, while the other two remained undetectable in viral load, indicating that both macaques successfully resisted infection by the SIV virus during challenge (7A and B). Although unfortunately infected with SIV virus, this macaque also showed strong resistance during infection with multiple prior doses of SIV virus (including 5 ten thousand TCID50) (fig. 6A and B), indicating that full protection was achieved with PD-1 antibodies in combination with the vaccine at doses below 50000TCID 50. Although this macaque was infected with SIV, its viral load continued to decrease after rising to the highest point (lg10 ═ 4.53), once at the lower limit of detection sensitivity (120294 in fig. 7). Whereas the SIV virus was less tolerated by the vaccine group alone compared to the PD-1 combination vaccine group. Two of the individual vaccine groups were infected with SIV (130402, 120308 in fig. 7), one of which was infected at 5000 TCIDs 50, whose viral load could be detected in plasma, and whose viral load could be detected for several weeks (130402 in fig. 7). Its viral load rose to about 10 a degree, although it was infected only at 10 ten thousand TCIDs 50 (120308 in FIG. 7)⁶Copy and remain at a high level of detection for a long period of time. The animals of the control group were more susceptible to infection with SIV virus than the first two groups, and it can be observed from the figure that they were infected with 5 thousand, 1 ten thousand and 5 ten thousand TCID50, respectively (fig. 6A and B). Two of the macaques infected at low doses showed a different trend in the viral load in their plasma compared to the first two groups, and the control group stably maintained the viral load in its plasma at the upper limit of the detection level after infection (fig. 7C). In the vaccine group, the viral load of the infected macaques at low dose begins to decrease after lasting for several weeks, and the macaques are detected for a long timeNot (fig. 7B). In 5 ten thousand TCID50 infected macaques, the plasma viral load is similar to the infection trend of a single vaccine group under high dose, and is stably maintained at 10 for a long time⁶Copies (080132, 080085 in fig. 7).

In addition, the helper proteins (nef, vpu, vif, vpr, rev and tat) are mainly products that are expressed by transcription and translation after the virus infects the host, and the SIV vaccine used previously does not express any helper protein, so that the specific immune response of the SIV virus helper protein is not expressed in the macaque without the SIV virus infection. Based on the basis, specific response to the auxiliary protein is detected by an Elispot technology, and the SIV infection condition in the macaques is further verified. The results showed that the response to SIV helper protein was barely detectable between groups before challenge (fig. 8A), and only three macaques did not produce specific responses, 120290 and 124352 for group G1 and 120316 for group G2, respectively (fig. 8B). This result is in full agreement with the results of real-time quantitative PCR detection of viral load in plasma.

In summary, the three macaques in the control group were all infected with SIV virus compared to the control group in the G1 and G2 groups, whereas only two of the G2 groups were infected, one of which was infected at the ultra-high dose. Therefore, the SIV vaccine used in the experiment can be preliminarily shown to enhance the body to resist the infection of the SIV virus to a certain extent. And compared with the groups G1 and G2, the combination of the PD-1 antibody and the SIV vaccine remarkably enhances the vaccine-induced protection effect, is favorable for improving the defense of the macaque against the SIV virus, and prevents the macaque from being infected by the SIV virus. Meanwhile, through analysis of the change condition of the viral load in the plasma of the macaques infected by the high-dose SIV, the result shows that PD-1 blocking is helpful for the macaques to effectively control the SIV viral load in vivo. Meanwhile, the inventor also tries the effect of inducing immune response and performing challenge test by simply using the PD-1 antibody, and the result shows that the animals cannot induce SIV specific immune response by only using the PD-1 antibody.

Example 6 study of the order of vaccination with PD-1 antibody and vaccine

The invention further researches the combined use of the PD-1 antibody and the vaccineIn order, the PD-1 antibody was dosed at 20mg/kg and the SIV vaccine was dosed at 2X 10¹⁰Animals immunized according to the immunization strategy of the following table, at the same PD-1 antibody and vaccine doses, three groups of animals all demonstrated the ability to resist challenge tests using the same challenge test protocol as in example 5, wherein animals of group G4 (PD-1 antibody first-administered group) exhibited a significantly better ability to resist viral infection than those of group G5 (simultaneous-administered group) and group G6 (PD-1 antibody later-administered group).

TABLE 3

Example 7 study of the proportional relationship between PD-1 antibody and vaccine

Based on the ratio of the test groups in example 1, the present inventors further studied the effect of a series of ratios of PD-1 antibody and SIV vaccine on the preventive effect of SIV vaccine, the specific dosage regimen and results are shown in Table 4, and the related tests were performed according to the immunization strategy of group G4 in Table 3, wherein PD-1 antibody was administered at a dose of 3mg/kg-20mg/kg, the amount of SIV vaccine inoculation was calculated on the basis of each mg of PD-1 antibody, and it can be seen that the ratio of PD-1 antibody to vaccine inoculation is closely related to the preventive effect obtained by the same challenge test protocol as in example 5, and the combination of both can obtain the preventive effect, and C1: C2 at 1:10 can obtain the preventive effect⁸–1:10¹⁰Satisfactory preventive effect can be obtained in all cases.

TABLE 4

EXAMPLE 8 Studies of other PD-1 antibodies in combination with vaccines

The invention also further researches other PD-1 antibodies, such as humanized antibodies (as described in Chinese patent application 201580040301. X) such as marketed pembrolizumab, nivolumab, 7A4, 13F1 and the like and related mouse antibody and chimeric antibody in combination with the type 5 adenovirus vector vaccine carrying Gag, pol and ENV expression sequences, which show the effect of promoting the preventive immune protection of the vaccine equivalent to 7A 4D.

EXAMPLE 9 study of other live vector viral vaccines in combination with PD-1 antibody

The invention also adopts the type 5 adenovirus vector to carry 1 or more expression sequences from structural genes and regulatory genes of SIV, and the combination immunization with 7A4D can show the enhancement of the immune activity of 7A4D antibody on the live vector virus vaccine of the type, and the preventive immune protection effect is observed.

Meanwhile, vaccines prepared by expressing immunogens by other types of live vector virus vaccines, such as adenovirus type 2 vector systems, vaccinia virus vector systems, baculovirus vector systems or adenovirus vector systems, yellow fever virus vectors, venezuelan equine encephalitis virus vectors, poliovirus vectors, vesicular stomatitis virus vectors and herpes simplex virus vector systems, have similar combined immune enhancement effects.

Discussion of the related Art

The application researches the protection effect of PD-1 and a vaccine immunization strategy and performs a challenge experiment on macaques. After 42 weeks of vaccine immunization, macaques were initially challenged with SIVmac239 rectal low dose multiple infections and subsequently monitored for viral load levels in their plasma for long periods. Summary of the data it is clear from the plots that only one of the three macaques in the PD-1 antibody + vaccine group was infected with SIV at an ultra-high dose of 10 ten thousand TCID50, but the viral load was not high after infection and gradually decreased after reaching the peak. Two of the three macaques in the vaccine group alone were infected compared to the G1 group, one at 5000TCID50 low doses and the other at 10 ten thousand TCID50, but the viral load in the plasma was always maintained at a higher level (approximately 106 copies per ml plasma). Three macaques in the control group were infected with SIV virus, and the viral load in the plasma of these three macaques remained above the detection sensitivity (lg10>2) from infection to the present, in sharp contrast to the first two groups. In order to exactly verify the condition that animals are infected with SIV virus in the challenge experiment, the specific response condition of the SIV auxiliary protein is detected by using an IFN-gamma Elispot technology. The results showed that no specific response to the helper protein was detected in only 120290 and 124352 of group G1 and 120316 of group G2 of the three cynomolgus PBMCs. This result is consistent with the results of the viral load in plasma, which also demonstrates the authenticity of the results of this experiment. Therefore, the use of the PD-1 antibody blocking combined vaccine can effectively protect macaques from infection of SIV (simian immunodeficiency virus) and improve the sensitivity of the macaques to the SIV, and in addition, an interesting problem is obtained by analyzing the change trend of the plasma viral load, namely the PD-1 blocking can increase the viral load in macaques control bodies. The content of this section will continue to be explored at a later stage.

The research proves that the use of the PD-1 antibody combined vaccine can effectively enhance the immune response induced by the vaccine, prolong the immune response of the vaccine in vivo and improve the resistance of macaques to SIV virus infection. Provides powerful scientific basis and data support for the prevention work of HIV/SIV virus, and also provides a new idea for the clinical application of other vaccines.

All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.

Claims

1. A composition, characterized in that the composition comprises:

wherein the antagonist is a PD-1 antibody, and the acquired immunity isThe vaccine for the immunodeficiency syndrome is a live vector virus vaccine, the mass of the PD-1 antibody is C1 mg, the number of virus particles of the live vector virus vaccine is C2, and the ratio of C1 to C2 is 1:10⁷-1:10¹⁰Preferably, 1:10⁸-1:10⁹。

2. The composition of claim 1, wherein the PD-1 antibody is an antibody or immunologically active fragment thereof selected from the group consisting of: pembrolizumab, nivolumab, humanized PD-1 antibody 7A4, humanized PD-1 antibody 7A4D, humanized PD-1 antibody 13F1, or a combination thereof.

3. The composition of claim 1, wherein the immunogen of the acquired human immunodeficiency syndrome vaccine is selected from the group consisting of:

4. The composition of claim 1, wherein the immunogen of the acquired immunodeficiency syndrome vaccine is an intact polypeptide or a partially expressed polypeptide fragment encoded by a gene selected from the group consisting of: gag, Pol, ENV, nef, vpx, vif, vpr, rev, tat, or combinations thereof.

5. The composition of claim 1, wherein the first active ingredient is a humanized PD-1 antibody (e.g., humanized PD-1 antibody 7A4, humanized PD-1 antibody 7A4D, humanized PD-1 antibody 13F1, pamumab, or nivolumab), and the second active ingredient is a live vector virus vaccine comprising a replication-deficient adenovirus type 5 vector carrying Gag, Pol, and ENV structural genes.

6. The composition of claim 1, wherein the composition is a pharmaceutical composition.

7. A kit, comprising:

(C) instructions for use;

8. An active ingredient combination, characterized in that the combination comprises or consists of the following components:

9. Use of a composition according to claim 1 for the preparation of a medicament or vaccine for the prevention and/or treatment of acquired immunodeficiency syndrome.

10. Use of an antagonist against the PD-1 signaling pathway for (a) enhancing the prophylactic effect of a vaccine, or (b) preparing a formulation or composition for enhancing the prophylactic effect of a vaccine, wherein said antagonist is a PD-1 antibody, and said vaccine is an acquired immunodeficiency syndrome live vector viral vaccine.