CN105709219A - AIDS vaccine based on symmetrical-conformation immunogen and preparation method of AIDS vaccine - Google Patents

Abstract

The invention belongs to the field of vaccines, and relates to an AIDS vaccine based on a symmetrical conformational immunogen and a preparation method thereof. According to the immunogenic characteristics of the MPE conserved epitope in the near-membrane region of HIV-1 transmembrane protein gp41, the present invention designs a novel symmetrical conformational immunogen containing a long sequence through protein structure simulation technology; the experimental results show that the MPER epitope in the immunogen Form a stable helical structure, and its binding ability to the human broad-spectrum neutralizing antibody 10E8 that recognizes this epitope is significantly higher than that of the undesigned MPER immunogen, and the serum and antibodies produced after immunizing animals can bind HIV-1 more strongly The natural conformation of the envelope protein Env, the antibody produced by immunization can efficiently inhibit the infection of HIV-1 virus to target cells in the cell model, and the inducible antibody can mediate significant antibody-dependent cell-mediated cytotoxicity; As an AIDS vaccine, the symmetrical conformation immunogen 4sMPER of the present invention can overcome the membrane-near conformation defect of the original epitope, and has significant advantages.

Description

A kind of AIDS vaccine based on symmetrical conformational immunogen and its preparation method

technical field

The invention belongs to the field of vaccines, and in particular relates to an AIDS vaccine based on a symmetrical conformational immunogen and a preparation method thereof.

Background technique

The prior art discloses that AIDS (AIDS) is acquired immunodeficiency syndrome (AIDS) caused by human immunodeficiency virus (Humanimmunodeficiencyvirus, HIV) infection. Since the discovery of the first AIDS case in 1981, AIDS has spread rapidly around the world for more than 30 years. The disease has become a global health crisis and one of the major challenges facing the world; it seriously jeopardizes social progress and economic growth. According to reports, at present, more than 60 million people in the world are infected with HIV, and 30 million of them die from AIDS and related diseases caused by AIDS. In view of this, how to effectively prevent HIV-1 infection and reduce morbidity and mortality is an urgent task facing our country and the world. Studies have shown that vaccines are an effective means of controlling the spread and infection of HIV-1. However, an insurmountable difficulty has been encountered in the development of AIDS vaccines—that is, HIV-1 is highly variable; the sites selected by many vaccines are mainly natural Most of these epitopes are the sites with the highest mutation frequency of HIV-1. Therefore, it is difficult to construct vaccines using these antigens to overcome concurrent HIV-1 subtypes. How to utilize and select the conserved regions of HIV-1 to design vaccines has become an important problem in the field of AIDS vaccine research.

MPER is HIV-1 gp41 proximal membrane region (amino acid sequence number: 660-683; reference strain is HXB2) is a tryptophan-rich conserved region on gp41. MPER plays an important role in the process of viral membrane fusion (1). Research experiments have shown that MPER-deleted HIV strains lose the ability to infect target cells. Many broad-spectrum neutralizing antibodies, such as 10E8, 4E10, 2F5, etc., all recognize this site. Among them, 10E8 is the second-generation broad-spectrum neutralizing antibody isolated from patients. Its crystal structure shows that the binding conformation of the MPER region is a helical conformation (2), further suggesting that the natural conformation of the MPER region may be a helical conformation, which is consistent with the previous research results, so this suggests that MPER is a good choice for designing vaccines as an immunogen. However, designing vaccines based on MPER regions as antigens faces great challenges. Studies have shown that in the natural state, MPER has a helical structure and is rich in tryptophan and many other hydrophobic amino acids. Many important broad-spectrum neutralizing antibody recognition amino acids in the MPER region are buried in the cell membrane due to hydrophobic interactions, such as L669, W670, W672 , F673, I675, W678, L679, Y681, I682 and K683 etc. (3). This may also be one of the reasons why the MPER region is less immunogenic as an immunogen. Therefore, it is difficult to induce neutralizing antibodies in vaccines currently designed using MPER as an antigen. However, studies have shown that using the MPER region as an antigen to immunize monkeys to achieve protection, but it has not been able to induce neutralizing antibodies that recognize the MPER region. The analysis found that the antibody-dependent cell-mediated cytotoxicity (Antibody-dependent cell-mediated cytotoxicity, hereinafter referred to as ADCC) induced by the MPER region is the main reason for the protective effect. Therefore, whether it can induce a stronger ADCC effect is also critical in vaccine design. In summary, how to design an immunogen based on the MPER region so that it can mimic the natural conformation and expose its main amino acid sites in the cell membrane environment, so as to better produce antibodies, form neutralization and ADCC effects, is to use MPER A key technical issue in designing HIV vaccines. In order to solve the above technical difficulties, the inventors of the present application designed a novel symmetric conformational immunogen 4sMPER with a long sequence through protein structure simulation technology. Orientation, so that part of it is exposed to the membrane, HIV vaccines based on it induce more antibodies that recognize the native conformation, and enhance neutralization and ADCC.

The prior art relevant to the present invention has:

1. Apellániz, B., E. Rujas, P. Carravilla, J. Requejo-Isidro, N. Huarte, C. Domene, and J. L. Nieva. 2014. Cholesterol-dependent membrane fusion induced by the Gp41MPER-TMD connection suggests samechanism for broad HIV-1 neutralization. JVirol. 15; 88 (22):13367-77..

2. Jinghe Huang, G.O., Leo Laub, Mark K. Louder, Nicole A. Doria-Rose, Nancy S. Longo, Hiromi Imamichi, B.C. Robert T. Bailer, Shailendra K. Sharma, S. Munir Alam, Tao Wang, Yongping Yang, Baoshan Zhang, and R.W. Stephen A . Migueles, Barton F. Haynes, Peter D. Kwong, John R. Mascola & Mark Connors. 2012. Broad and potent neutralization of HIV-1byagp41-specific human antibody. Nature 15; 491(7424): 406-12.

3. Sun, Z.-Y.J., K.J.Oh, M.Kim, J.Yu, V.Brusic, L.Song, Z.Qiao, J.-h.Wang, G.Wagner, and E.L.Reinherz.2008 . HIV-1 broadly neutralizing antibody extracts its sepitope from akinked gp41 ectodomain region on the viral membrane. Immunity 28:52-63.

Contents of the invention

The purpose of the present invention is to solve the weak ability of inducing antibodies to recognize the natural conformation of the MPER polypeptide due to the conformation problem, and to provide an AIDS vaccine based on a symmetrical conformational immunogen and a preparation method thereof. It specifically relates to an amino acid sequence that can be used as an immunogen. The amino acid sequence includes a 4-repeat sequence of HIV-1gp41 near the membrane region (Membraneproximalexternalregion, MPER), and the amino acid sequence number is 660-683 (HXB2 is a reference strain), which can Better simulate the natural state of HIV-1 virus gp41 before the fusion of the MPER region. The HIV-1 MPER region is very conserved, which is closely related to its participation in the membrane fusion process and plays an important role (1); the broad-spectrum neutralizing antibody 2F5 that can recognize this epitope can neutralize about 67% of the virus, while another The broad-spectrum neutralizing antibody 10E8 that recognizes this epitope can neutralize 98% of clinical strains at a very low concentration (2), suggesting that this region is a good target for vaccine design; but the immunogenicity of the MPER region is relatively weak , especially with the whole virus as an immunogen; therefore, it is difficult to induce specific antibodies that recognize the MPER region by using pseudoviruses or VLPs. In addition, the natural conformation of MPER is a helical structure (2), while the synthesized and expressed MPER epitope monomer is essentially a random coil. In addition, many key epitopes in the MPER region antigen in the natural conformation are inserted into the cell membrane due to hydrophobic interaction, which adds a lot of difficulties to the MPER region as an immunogen to design vaccines. In order to solve the weak immunogenicity and conformation problems of MPER, the present invention uses protein structure simulation technology to design a new immunogen 4sMPER of tandem long-epitope MPER. The key amino acids (W672 and F673) of its epitope are located in different directions (as shown in Figure 1), which can solve the above-mentioned key problem of hiding important amino acids in the cell membrane after the MPER region binds to membrane lipids. The experimental results It also proves that the designed 4sMPER immunogen has a helical structure (as shown in Figure 2 and Figure 3), which can better bind to broad-spectrum neutralizing antibodies (Figure 4), and it can also be better exposed on the cell membrane. The previously hidden key binding site of the 10E8 antibody (Figure 5) can induce more specific antibodies that recognize the natural conformation (Figure 6), and these antibodies have neutralizing activity and ADCC effect (Table 1, Figure 7).

The present invention includes the following technical solutions:

1. The 4sMPER immunogen structure obtained by conformational simulation technology,

Use computer simulation technology to simulate the antigen in the MPER region, and select the mimetic body with a central symmetric conformation as a helical conformation;

2. Circular dichroism spectrometer to detect the secondary structure of 4sMPER

Circular dichroism spectrometer (J-815, JascoInc, Japan) was used to determine the secondary structure of 4sMPER antigen and MPER monomer peptide by circular dichroism;

3. Thermal stability analysis of 4sMPER detected by circular dichroism spectrometer

Thermal denaturation analysis (Tm value) is used to evaluate the thermal stability of 4sMPER antigen;

4. Detection of the binding ability of broad-spectrum neutralizing antibodies 10E8 and 4E10 targeting MPER region epitopes to 4sMPER

The present invention utilizes 10E8 and 4E10 monoclonal antibodies to detect the conformation of 4sMPER;

5. High-content detection of key amino acid sites of 4sMPER can be more easily exposed outside the cell membrane;

Flow cytometry detection of Ab-4sMPER (4sMPER-specific antibody purified after immunizing animals with 4sMPER antigen) is more likely to recognize the natural HIV-1 envelope protein Env;

10Ab-4sMPER induces antibody-mediated cytotoxicity (ADCC);

7. Measure the inhibitory activity of Ab-4sMPER and Ab-MPER to pseudovirus.

In one embodiment of the present invention, the above-mentioned 4sMPER polypeptide immunogen can be directly or in the form of a part of the combined immunogen, used as a protein vaccine to induce antibodies that can recognize the natural conformation of the conserved epitope in the MPER region of the virus, thereby neutralizing the virus and ADCC effect to achieve the purpose of AIDS prevention and treatment.

In another embodiment of the present invention, the DNA sequence encoding the 4sMPER immunogen can be further cloned into other vectors, such as AAV, vacciniavirus, ALVAC, MVA, Ad5 as a vaccine to enhance its immunogenicity.

The HIV vaccine described in the present invention can be further used in conjunction with adjuvants, and adjuvants include Freund's adjuvant, aluminum hydroxide adjuvant, MF59, AS02A, QA-21, Corynebacterium pumilus, lipopolysaccharide, cytokines or alum, And oil adjuvants or water-based adjuvants.

The invented HIV vaccine can be used for intramuscular injection, subcutaneous injection, intradermal injection, intraperitoneal injection, nasal cavity administration and oral administration. Those skilled in the art can use the techniques known in the art to formulate it so that it is suitable for the corresponding administration mode.

Description of drawings

Figure 1. The structure of 4sMPER immunogen obtained by conformational simulation technology.

Figure 2. The secondary structure of 4sMPER detected by circular dichroism spectrometer.

Figure 3. Thermal stability analysis of 4sMPER detected by circular dichroism spectrometer.

Figure 4. Detection of 4sMPER binding ability of 10E8 (A) and 4E10 (B).

Figure 5. High-content detection of key amino acid sites of 4sMPER can be more easily exposed outside the cell membrane. Figure 5A is the fluorescence image results, and Figure 5B is the high-content fluorescence quantitative results.

Figure 6. Ab-4sMPER (purified antibody specific to 4sMPER after immunizing animals with 4sMPER antigen) is more likely to recognize the envelope protein Env in the natural conformation of HIV-1 as detected by flow cytometry.

Figure 7. Ab-4sMPER can better mediate ADCC effect.

detailed description

Example 1

The 4sMPER immunogen structure obtained by conformational simulation technology:

Use computer simulation technology to simulate the antigen in the MPER region, and select a mimetic body with a central symmetric conformation as a helical conformation; as shown in Figure 1, the antigen 4sMPER designed by the present invention is a helical conformation, wherein the key hydrophobic amino acid site 672W is green, 673F For pink,. However, the designed antigen has a centrosymmetric structure, and the directions of the key hydrophobic amino acids 672W and 673F are different, so as to solve the problem that the key site of the antigen in the MPER region is buried in the cell membrane;

Circular dichroism spectrometer detects the secondary structure of 4sMPER:

Circular dichroism spectrometer (J-815, JascoInc, Japan) was used to determine the secondary structure of 4sMPER antigen and MPER monomer peptide (as shown in Figure 2) by circular dichroism method. Both 4sMPER antigen and MPER monomer peptide were used 50mM PBS with pH 7.2 was diluted to a final concentration of 10μM for later use. PBS was added to a 0.1cm quartz sample cup as a blank control. The following parameters were used for sample detection: detection temperature 4°C, bandwidth 5nm, resolution 0.1nm, light The diameter is 0.1cm, the response time is 4.0s, the scanning speed is 50nm/min, and the wavelength range is from 190nm to 280nm. Finally, using the software that comes with the instrument, the CD signal is converted into molar ellipticity (molarellipticity, [ θ]), the results show that the 4sMPER antigen has strong absorption peaks at 208 and 222nM, and has a significant helical structure, while the MPER antigen is a random coil;

Thermal stability analysis of 4sMPER detected by circular dichroism spectrometer:

Thermal denaturation analysis (Tm value) is used to evaluate the thermal stability of the 4sMPER antigen. The absorbance at 222nm is detected on a circular dichroism spectrometer (J-815, Jasco Inc, Japan), and the thermal stability of the polypeptide is detected with a temperature gradient of 5°C/min. Denaturation, the temperature setting range is scanned from 4°C to 99°C, and the midpoint temperature of the thermal dissociation transition is calculated using software, that is, the Tm value; the results show that the 4sMPER antigen has a very stable helical structure (as shown in Figure 3), and its Tm value About 95°C;

Detection of the binding ability of broad-spectrum neutralizing antibodies 10E8 and 4E10 targeting MPER region epitopes to 4sMPER:

In the present invention, 10E8 and 4E10 monoclonal antibodies are used to detect the conformation of 4sMPER, 5 μg/ml of MPER polypeptide and 4sMPER polypeptide are used for coating, overnight at 4°C, and 2% skimmed milk powder is used to seal for 2 hours. After sealing, the initial concentration of 20 μg is added. 10E8 and 4E10 in a 4-fold dilution series per ml, incubated at 37°C for one hour, washed 3 times with PBS containing 0.05% Tween 20, then added goat anti-human HRP-labeled secondary antibody (1:3000 dilution), incubated at 37°C Incubate for one hour, use TMB to develop color, and measure its OD450. The results show that with the increase of 10E8 and 4E10 antibody concentration, the OD450 value also increases. 10E8 can recognize 4sMPER peptide at a very low concentration, but MPER recognition is not Higher concentrations were required (as shown in Figure 4A), indicating that 10E8 binds 4sMPER more strongly; the 4E10 antibody showed similar results (as shown in Figure 4B); the results indicated that the conformation of the symmetric epitope was more readily bound by the native The conformational recognition of 10E8 and 4E10 mAbs suggests that its conformation is closer to the natural conformation;

The key amino acid sites of 4sMPER in high-content detection can be more easily exposed outside the cell membrane:

Use high content to detect whether the key amino acids of the 4sMPER antigen in the cell membrane environment can be exposed outside the cell membrane. 20μg/ml of 4sMPER and MPER are incubated with the cells at 37°C for 1 hour, and then 5μg/ml of a broad-spectrum epitope that recognizes the MPER region is added. Neutralizing antibody 10E8 (containing 1% BSA) was incubated at room temperature for 30 minutes, washed 2 times with PBS, then added secondary antibody goat anti-human IgG-FITC (10 μg/ml) and incubated at room temperature for 40 minutes, washed 2 times with PBS, and then Use high content (Opera, PE) to take pictures, and calculate the quantitative fluorescence value (as shown in Figure 5B), as shown in Figure 5A, in the cell membrane environment, MPER is difficult to be recognized by 10E8, and the binding ability of 4sMPER to 10E8 Significantly improved, in which only the secondary antibody was added to the cell control (Cell), and the same amount of 10E8 and secondary antibody were added to the cell antibody control (Cell-Ab) for parallel control. Figure 5B shows that the high-content instrument can quantitatively calculate the fluorescence intensity of a single cell , the results showed that, compared with the single epitope of MPER, 10E8 had a stronger binding to the symmetric antigen 4sMPER, and there was a significant difference between the two (P=0.012<0.05);

Flow cytometry detection of Ab-4sMPER (4sMPER-specific antibody purified after immunization of animals with 4sMPER antigen) is easier to recognize the natural HIV-1 envelope protein Env:

10μg/ml Ab-4sMPER and Ab-MPER (1% BSA) were incubated with H9/IIIB cells stably expressing HIV-1 envelope protein Env for 30 minutes at room temperature, washed twice with PBS, and then added 10μg/ml Goat anti-rabbit-FITC (containing 1% BSA) was incubated at room temperature for 30 minutes, washed 2 times with PBS, washed 2 times with PBS, and then detected by flow cytometry (AccuriC6, BD), as shown in Figure 6, Ab-4sMPER had better binding (30%) to the native conformation Env expressed on the surface of H9IIIB, while Ab-MPER had relatively weaker binding (18%);

Ab-4sMPER can better induce antibody-mediated cytotoxicity (ADCC):

Spread 25 μl of H9IIIB (2.5×10 ⁴ ) cells into a 96-well plate, then dilute Ab-4sMPER and Ab-MPER gradients, add to H9/IIIB cells stably expressing HIV envelope protein (50 μl), and then Add 25 μl (1.5×10 ⁵ cells) of ADCC effector cells (promega) into H9/IIIB, and measure its luciferase after incubation at 37°C for 24 hours. The results are shown in Figure 7. Ab-4sMPER is stronger than Ab-MPER ADCC effect;

Inhibitory activity of Ab-4sMPER and Ab-MPER to pseudoviruses:

New Zealand big-eared rabbits were immunized with the designed conformational symmetric antigen 4sMPER peptide and MPER peptide. After 4 immunizations, the antibody titer was measured, and then the anti-4sMPER antibody (Ab-4sMPER) and anti-MPER Antibody (Ab-MPER) was purified, and the effects of Ab-4sMPER and Ab-MPER were measured by using pseudoviruses, including: 8,000 U87 cells per well, 100 μl system for plating, virus and 50 μl of serially diluted antibody system Mix, put it at 37°C, and incubate for 30 minutes. After incubation, add it to U87 cells. After incubating at 37°C for 72 hours, measure its luciferase. The experimental results are shown in Table 1. Ab-MPER-4 is effective against pseudovirus SC422661. 8. ZM109F.PB4, AE03, and SC19-15 have significant inhibitory activity, and their ICOs are 14.64±3.31, 5.69±2.10, 50.78±4.13, 31.92±6.19 (μg/ml), and the antibody Ab-MPER has no effect on all pseudo None of the viruses had significant inhibitory activity.

Table 1 shows the inhibitory activity of Ab-4sMPER and Ab-MPER (MPER-specific antibodies purified after immunizing animals with MPER peptides) against pseudoviruses.

Table 1

SEQUENCELISTING

<110> Fudan University

<120> An AIDS vaccine based on a symmetrical conformational immunogen and its preparation method

<130>20141124

<160>1

<170>PatentInversion3.3

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<212>PRT

<213> HIV

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AlaSerLeuTrpAsnTrpPheAsnIleThrAsnTrpLeuTrpTyrIle

151015

LysSerLeuTrpAsnTrpPheAsnIleThrAsnTrpLeuTrpTyrIle

202530

LysSerLeuTrpAsnTrpPheAsnIleThrAsnTrpLeuTrpTyrIle

354045

LysSerLeuTrpAsnTrpPheAsnIleThrAsnTrpLeuTrpTyrIle

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LysGly

65

Claims (5)

1. one kind based on the symmetrical immunogenic AIDS vaccine of conformation, it is characterized in that, it contains a kind of HIV (human immunodeficiency virus) (HIV) immunogen, including a kind of aminoacid sequence, described aminoacid sequence includes the 660-683 region amino acid sequence of HIVgp41 membrane-proximal region MPER and (numbers according to HXB2, referred to as MPER epi-position) 4 repeat tandem sequences, its architectural feature is for defining symmetrical conformation, wherein W672 and F673 is towards different directions in antigen, has certain symmetry.

2. AIDS vaccine as claimed in claim 1, it is characterised in that described HIV immunogen, wherein said aminoacid sequence is connected with its immunogenic aminoacid sequence that strengthens including Th cell epitope sequence further, to strengthen its immunogenicity.

3. AIDS vaccine as claimed in claim 2, it is characterized in that, described series connection aminoacid sequence, for series aiding connection, or reverse series connection, also or reverse in the same direction spaced series, its core is that in produced immunogen, MPER epi-position has symmetrical conformation, make it closer to native conformation so that it is key amino acid fully exposes.

4. AIDS vaccine as described in any one in claim 1-3, it is characterised in that described HIV immunogen, its coded DNA sequence can be cloned into other carrier, such as AAV, vacciniavirus, ALVAC, MVA, Ad5, give expression to the immunogen of correspondence in vivo and in vitro.

5. AIDS vaccine as described in any one in claim 1-3, it is characterized in that, described HIV vaccine, farther include adjuvant, described adjuvant is selected from Freund adjuvant, aluminum hydroxide adjuvant, MF59, AS02A, QA-21, coryne bacterium parvum, lipopolysaccharide, cytokine or Alumen and oil adjuvant or water type adjuvant.