# US 10,864,260 B2 · Neospora vaccine composition

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- Number: US 10,864,260 B2
- Document ID: US10864260B2
- Status: Granted
- Kind: B2
- Application number: 15/771,832
- Filed: 2016-10-28
- Published: 2018-11-01
- Granted: 2020-12-15
- Earliest claimed: 2015-10-28
- Assignees: Universidad Complutense De Madrid
- Applicants: Universidad Complutense De Madrid
- Inventors: Javier Regidor Cerrillo; David Arranz Solís; Esther Collantes Fernández; Gema Álvarez García; Luis Miguel Ortega Mora
- Web page: https://patentgrep.com/patent/US10864260B2

## CPC

- A61K 39/012: Coccidia antigens
- A61K 39/002: Protozoa antigens
- A61P 33/02: Antiprotozoals, e.g. for leishmaniasis, trichomoniasis, toxoplasmosis
- C07K 14/44: from protozoa
- C12N 1/10: Protozoa; Culture media therefor
- A61K 2039/552: {Veterinary vaccine}
- A61K 2039/55577: {Saponins; Quil A; QS21; ISCOMS}
- A61K 2039/70: {Multivalent vaccine}
- Y02A 50/30: Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- Y02A: TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE

## Abstract

The present invention relates to new protein compositions, methods for producing said protein compositions, pharmaceutical compositions comprising said protein compositions and methods for treating infections caused by Neospora caninum. In particular, the present invention relates to a protein composition comprising the proteins specified in Table A in an amount of at least about 2 times (fold change) higher than the same protein present in the whole tachyzoite, as calculated by quantitative label-free liquid chromatography-tandem mass spectrometry (LC-MS/MS).

## Claims

### Claim 1

An enriched antigen extract composition, comprising:
the following proteins having the amino acid sequences of SEQ ID NOs: 45, 58, 60, 76, 80, 87, 120, 121, 140, 156, 176, 177, 185, 188, 229, 246, 247, 285, 289, 307, 315, 318, 322, 323, 347, 362, 382, 390, 397, 440, 453, 459, 463, 486, 491, 519, 540, 560, 570, 577, 633, 636, 680, 716, 769, 785, 886, 887, 895, 897, 899, 902, and 1022, wherein each protein is present in an amount at least about 2 fold change higher than the same protein present in a whole tachyzoite extract, as calculated by quantitative label-free liquid chromatography-tandem mass spectrometry (LC-MS/MS);
wherein the enriched antigen extract composition is produced according to a method comprising the following steps;
(a) centrifuging a hypertonic solution of Neospora cells to separate the soluble fraction and insoluble fraction, wherein the centrifugation takes place at a centrifugal force ranging from about 8.000×g to about 15.000×g, for a time ranging from about 40 minutes to about 90 minutes and at a temperature ranging from about 1° C. to about 10° C.;
(c) recovering the insoluble fraction from step (b);
(d) mixing the recovered insoluble fraction with a non-ionic surfactant; and
(e) homogenizing the mixture of step (d) to yield the enriched antigen extract;
wherein the whole tachyzoite extract is prepared by direct resuspension of 108 frozen tachyzoites of the Nc-Spain 7 isolate of N. caninum, with accession number CCAP 2051/1, in 100 μl of 1% Triton-X 100 (v/v);
and an adjuvant.

### Claim 2 (depends on 1)

The enriched antigen extract composition according to claim 1, wherein the enriched antigen extract composition comprises the following proteins in an amount having the following fold change as compared to the amount of the same protein that is present in the whole tachyzoite extract:
NCLIV_018120 (SEQ ID NO:177) at about 4.15 fold change higher;
NCLIV_003410 (SEQ ID NO:886) at about 3.35 fold change higher;
NCLIV_058550 (SEQ ID NO:347) at about 3.33 fold change higher;
NCLIV_042610 (SEQ ID NO:897) at about 3.06 fold change higher;
NCLIV_032910 (SEQ ID NO:382) at about 2.99 fold change higher;
NCLIV_024830 (SEQ ID NO:519) at about 2.7 fold change higher;
NCLIV_015180 (SEQ ID NO:323) at about 2.52 fold change higher;
NCLIV_066970 (SEQ ID NO:453) at about 2.48 fold change higher;
NCLIV_025730 (SEQ ID NO:560) at about 2.47 fold change higher;
NCLIV_006640 (SEQ ID NO:289) at about 2.42 fold change higher;
NCLIV_003470 (SEQ ID NO:887) at about 2.39 fold change higher;
NCLIV_01900) (SEQ ID NO:390) at about 2.37 fold change higher;
NCLIV_054510 (SEQ ID NO:785) at about 2.36 fold change higher;
NCLIV_066350 (SEQ ID NO:577) at about 2.35 fold change higher;
NCLIV_057020 (SEQ ID NO:902) at about 2.32 fold change higher;
NCLIV_056680 (SEQ ID NO:246) at about 2.3 fold change higher;
NCLIV_044290 (SEQ ID NO:463) at about 2.29 fold change higher;
NCLIV_032030 (SEQ ID NO:680) at about 2.27 fold change higher;
NCLIV_052240 (SEQ ID NO:362) at about 2.27 fold change higher;
NCLIV_008730 (SEQ ID NO:318) at about 2.22 fold change higher;
NCLIV_061830 (SEQ ID NO:120) at about 2.22 fold change higher;
NCLIV_002770 (SEQ ID NO:440) at about 2.18 fold change higher;
NCLIV_014760 (SEQ ID NO:636) at about 2.17 fold change higher;
NCLIV_054520 (SEQ ID NO:322) at about 2.17 fold change higher;
NCLIV_033810 (SEQ ID NO:716) at about 2.16 fold change higher;
NCLIV_043330 (SEQ ID NO:121) at about 2.16 fold change higher;
NCLIV_000430 (SEQ ID NO:486) at about 2.15 fold change higher;
NCLIV_030860 (SEQ ID NO:176) at about 2.14 fold change higher;
NCLIV_048570 (SEQ ID NO:58) at about 2.14 fold change higher;
NCLIV_004190 (SEQ ID NO:76) at about 2.13 fold change higher;
NCLIV_019450 (SEQ ID NO:459) at about 2.13 fold change higher;
NCLIV_027160 (SEQ ID NO:185) at about 2.13 fold change higher;
NCLIV_044600 (SEQ ID NO:633) at about 2.13 fold change higher;
NCLIV_000300 (SEQ ID NO:307) at about 2.12 fold change higher;
NCLIV_046830 (SEQ ID NO:540) at about 2.12 fold change higher;
NCLIV_004280 (SEQ ID NO:315) at about 2.11 fold change higher;
NCLIV_006720 (SEQ ID NO:229) at about 2.1 fold change higher;
NCLIV_046800 (SEQ ID NO:188) at about 2.1 fold change higher;
NCLIV_051960 (SEQ ID NO:769) at about 2.1 fold change higher;
NCLIV_010600 (SEQ ID NO:45) at about 2.09 fold change higher;
NCLIV_015920 (SEQ ID NO:87) at about 2.09 fold change higher;
NCLIV_012830 (SEQ ID NO:285) at about 2.08 fold change higher;
NCLIV_0376 (SEQ ID NO:1022) at about 2.08 fold change higher;
NCLIV_024420 (SEQ ID NO:140) at about 2.06 fold change higher;
NCLIV_036130 (SEQ ID NO:570) at about 2.06 fold change higher;
NCLIV_036400 (SEQ ID NO:156) at about 2.06 fold change higher;
NCLIV_006780 (SEQ ID NO:397) at about 2.04 fold change higher;
NCLIV_046940 (SEQ ID NO:899) at about 2.04 fold change higher;
NCLIV_001370 (SEQ ID NO:491) at about 2.03 fold change higher;
NCLIV_001300 (SEQ ID NO:60) at about 2.02 fold change higher;
NCLIV_015380 (SEQ ID NO:80) at about 2.02 fold change higher;
NCLIV_038990 (SEQ ID NO:895) at about 2 fold change higher; and
NCLIV_043110 (SEQ ID NO:247) at about 2 fold change higher.

### Claim 3 (depends on 1)

The enriched antigen extract composition according to claim 1, wherein the composition comprises the following proteins: NCLIV_018120 (SEQ ID NO:177), NCLIV_003410 (SEQ ID NO:886), NCLIV_058550 (SEQ ID NO:347), NCLIV_042610 (SEQ ID NO:897), NCLIV_032910 (SEQ ID NO:382), NCLIV_024830 (SEQ ID NO:519), NCLIV_015180 (SEQ ID NO:323), NCLIV_066970 (SEQ ID NO:453), NCLIV_025730 (SEQ ID NO:560), NCLIV_006640 (SEQ ID NO:289), NCLIV_003470 (SEQ ID NO:887), NCLIV_019000 (SEQ ID NO:390), NCLIV_054510 (SEQ ID NO:785), NCLIV_066350 (SEQ ID NO:577), NCLIV_057020 (SEQ ID NO:902), NCLIV_056680 (SEQ ID NO:246), NCLIV_044290 (SEQ ID NO:463), NCLIV_032030 (SEQ ID NO:680), NCLIV_052240 (SEQ ID NO):362), NCLIV_008730 (SEQ) ID NO:318), NCLIV_061830 (SEQ ID NO:120), NCLIV_002770 (SEQ ID NO:440), NCLIV_014760 (SEQ ID NO:636), NCLIV_054520 (SEQ ID NO:322), NCLIV_033810 (SEQ ID NO:716), NCLIV_043330 (SEQ ID NO:121), NCLIV_000430 (SEQ ID NO:486), NCLIV_030860 (SEQ ID NO:176), NCLIV_048570 (SEQ ID NO:58), NCLIV_004190 (SEQ ID NO:76), NCLIV_019450 (SE ID NO:459), NCLIV_027160 (SEQ ID NO:185), NCLIV_044600 (SEQ ID NO:633), NCLIV_000300 (SEQ ID NO:307), NCLIV_046830 (SEQ ID NO:540), NCLIV_004280 (SEQ ID NO:315), NCLIV_006720 (SEQ ID NO:229), NCLIV_046800 (SEQ ID NO:188), NCLIV_051960 (SEQ ID NO:769), NCLIV_010600 (SEQ ID NO:45), NCLIV_015920 (SEQ ID NO:87), NCLIV_012830 (SEQ ID NO:285), NCLIV_0376 (SEQ ID NO:1022), NCLIV_024420 (SEQ ID NO):140), NCLIV_036130 (SEQ ID NO:570), NCLIV_036400 (SEQ ID NO:156), NCLIV_006780 (SEQ ID NO:397), NCLIV_046940 (SEQ ID NO:899), NCLIV_001370 (SEQ ID NO:491), NCLIV_001300 (SEQ ID NO:60), NCLIV_015380 (SEQ ID NO:80), NCLIV_038990 (SEQ ID NO-895), NCLIV_043110 (SEQ ID NO:247), NCLIV_004750 (SEQ ID NO:416), NCLIV_025000 (SEQ ID NO:511), NCLIV_041790 (SEQ ID NO:299), NCLIV_058420 (SEQ ID NO:740), NCLIV_015430 (SEQ ID NO:25), NCLIV_023620 (SEQ ID NO:327), NCLIV_049050 (SEQ ID NO:127), NCLIV_029420 (SEQ ID NO:179), NCLIV_010320 (SEQ ID NO:95), NCLIV_006060 (SEQ ID NO:380), NCLIV_032810 (SEQ ID NO:894), NCLIV_004860 (SEQ ID NO:226), NCLIV_007260 (SEQ ID NO:24), NCLIV_026590 (SEQ ID NO:893), NCLIV_054540 (SEQ ID NO:629), NCLIV_0153 (SEQ ID NO:889), NCLIV_030420 (SEQ ID NO:107), NCLIV_051920 (SEQ ID NO:872), NCLIV_065970 (SEQ ID NO:407) NCLIV_026430 (SEQ ID NO:457), NCLIV_061160 (SEQ ID NO:78), NCLIV_070010 (SEQ ID NO:1), NCLIV_036300 (SEQ ID NO:760), NCLIV_057950 (SEQ ID NO:158), NCLIV_038320 (SEQ ID) NO:254), NCLIV_040600 (SEQ ID NO:553), NCLIV_030820 (SEQ ID NO:228), NCLIV_034990 (SEQ ID NO:442), NCLIV_037520 (SEQ ID NO:154), NCLIV_054120 (SEQ ID NO:231), NCLIV_061210 (SEQ ID NO:496), NCLIV_010650 (SEQ ID NO:730), NCLIV_026340 (SEQ ID NO:669), NCLIV_013150 (SEQ ID NO:200), NCLIV_055360 (SEQ ID NO:6), NCLIV_066600 (SEQ ID NO:189), NCLIV_015790 (SEQ ID NO:492), NCLIV_055730 (SEQ ID NO:245), NCLIV_067140 (SEQ ID NO:26), NCLIV_007800 (SEQ ID NO:40), NCLIV_008850 (SEQ ID NO:74), NCLIV_028110 (SEQ ID NO:581), NCLIV_065470 (SEQ ID NO:86), NCLIV_040650 (SEQ ID NO:896), NCLIV_041830 (SEQ ID NO:368), NCLIV_050590 (SEQ ID NO:81), NCLIV_051010 (SEQ ID NO:389), NCLIV_058800 (SEQ ID NO:903), NCLIV_003190 (SEQ ID NO:427), NCLIV_042820 (SEQ ID NO:3), NCLIV_058840 (SEQ ID NO:438), NCLIV_020980 (SEQ ID NO:892), NCLIV_032050 (SEQ ID NO:244), NCLIV_053290 (SEQ ID NO:248), NCLIV_060220 (SEQ ID NO:211), NCLIV_061940 (SEQ ID NO:455), NCLIV_003650 (SEQ ID NO:436), NCLIV_006290 (SEQ ID NO:888), NCLIV_016800 (SEQ ID NO:49), NCLIV_034130 (SEQ ID NO:68), NCLIV_052350 (SEQ ID NO:129), NCLIV_054570 (SEQ ID NO:249), NCLIV_055850 (SEQ ID NO:160), NCLIV_015480 (SEQ ID NO:381), NCLIV_015950 (SEQ ID NO:890), NCLIV_030620 (SEQ ID NO:283), NCLIV_041210 (SEQ ID NO:536), NCLIV_047860 (SEQ ID NO:30), NCLIV_054250 (SEQ ID NO:371), NCLIV_058890 (SEQ ID NO:37), NCLIV_070060 (SEQ ID NO-284), NCLIV_003580 (SEQ ID NO:539), NCLIV_005620 (SEQ ID NO:166), NCLIV_036700 (SEQ ID NO:62), NCLIV_056570 (SEQ ID NO:901), NCLIV_059730 (SEQ ID NO:904), NCLIV_008230 (SEQ ID NO:334), NCLIV_015410 (SEQ ID NO:97), NCLIV_032390 (SEQ ID NO:91), NCLIV_033780 (SEQ ID NO:321), NCLIV_036610 (SEQ ID NO0194), NCLIV_045300 (SEQ ID NO:898), NCLIV_049830 (SEQ ID NO:557), NCLIV_055760 (SEQ ID NO:155), NCLIV_058440 (SEQ ID) NO:72), NCLIV_064840 (SEQ ID NO:398), NCLIV_038360 (SEQ ID NO:48), NCLIV_043760 (SEQ ID NO:227), NCLIV_052270 (SEQ ID NO:794), NCLIV_027850 (SEQ ID NO:547), NCLIV_011960 (SEQ ID NO:667), NCLIV_018530 (SEQ ID NO:125), NCLIV_022690 (SEQ ID NO:758), NCLIV_024630 (SEQ ID NO:122), NCLIV_027530 (SEQ ID NO:337), NCLIV_045600 (SEQ ID NO:618), NCLIV_001970 (SEQ ID NO:18), NCLIV_011410 (SE II NO:10), NCLIV_014360 (SEQ ID NO:198), NCLIV_019110 (SEQ ID NO:2), NCLIV_030070 (SEQ ID NO:652), NCLIV_014430 (SEQ ID NO:485), NCLIV_042410 (SEQ ID NO:96), NCLIV_043930 (SEQ ID NO:199), NCLIV_061560 (SEQ ID NO:533), NCLIV_025670 (SEQ ID NO:39), NCLIV_027930 (SEQ ID NO:564), NCLIV_028540 (SEQ ID NO:251), NCLIV_064260 (SEQ ID NO:906), NCLIV_069590 (SEQ ID NO:145), NCLIV_013360 (SEQ ID NO:630), NCLIV_040540 (SEQ ID NO:297), NCLIV_040970 (SEQ ID NO:293), NCLIV_056670 (SEQ ID NO:215), NCLIV_067010 (SEQ ID NO:116), NCLIV_004140 (SEQ ID NO:528), NCLIV_028750 (SEQ ID NO:240), NCLIV_031780 (SEQ ID NO:31), NCLIV_035190 (SEQ ID NO:578), NCLIV_050470 (SEQ ID NO:101), NCLIV_051560 (SEQ ID NO:384), NCLIV_054800 (SEQ ID NO:220), NCLIV_064950 (SEQ ID NO:130), NCLIV_011700 (SEQ ID NO:192), NCLIV_012920 (SEQ ID NO:69), NCLIV_024030 (SEQ ID NO:305), NCLIV_030890 (SEQ ID NO:281), NCLIV_032780 (SEQ ID NO:181), NCLIV_060140 (SEQ ID NO:253), NCLIV_065210 (SEQ ID NO:44), NCLIV_069460 (SEQ ID NO:717), NCLIV_001660 (SEQ ID NO:580), NCLIV_016540 (SEQ ID NO:230), NCLIV_028680 (SEQ ID NO:149), NCLIV_032830 (SEQ ID NO:792), NCLIV_055490 (SEQ ID NO:7), NCLIV_056560 (SEQ ID NO:852), NCLIV_066840 (SEQ ID NO:16), NCLIV_031770 (SEQ ID NO:147), NCLIV_047810 (SEQ ID NO:139), NCLIV_060660 (SEQ ID NO:208), NCLIV_006490 (SEQ ID NO:326), NCLIV_020840 (SEQ ID NO:21), NCLIV_028090 (SEQ ID NO:303), NCLIV_040440 (SEQ ID NO:296), NCLIV_044200 (SEQ ID NO:146), NCLIV_051110 (SEQ ID NO:611), NCLIV_053940 (SEQ ID NO:567), NCLIV_055720 (SEQ ID NO:151), NCLIV_068850 (SEQ ID NO:90), NCLIV_019520 (SEQ ID NO:891), NCLIV_020720 (SEQ ID NO:163), NCLIV_022950 (SEQ ID NO:260), NCLIV_031670 (SEQ ID NO:84), NCLIV_032110 (SEQ ID NO:201), NCLIV_056300 (SEQ ID NO:377), NCLIV_063370 (SEQ ID NO:425), NCLIV_007770 (SEQ ID NO:157), NCLIV_036570 (SEQ ID NO:601), NCLIV_051840 (SEQ ID NO:403), NCLIV_052390 (SEQ ID NO:115), NCLIV_062890 (SEQ ID NO:449), NCLIV_003050 (SEQ ID NO:19), NCLIV_004810 (SEQ ID NO:698), NCLIV_021080 (SEQ ID NO:92), NCLIV_000940 (SEQ ID NO:221), NCLIV_010730 (SEQ ID NO:88), NCLIV_014950 (SEQ ID NO:466), NCLIV_020920 (SEQ ID NO:632), NCLIV_037190 (SEQ ID NO:98), NCLIV_048380 (SEQ ID NO:900), NCLIV_049900 (SEQ ID NO:5), NCLIV_056430 (SEQ ID NO:124), NCLIV_060730 (SEQ ID NO:46), NCLIV_062940 (SEQ ID NO:271), NCLIV_064490 (SEQ ID NO:907), NCLIV_012230 (SEQ ID NO:516), NCLIV_015260 (SEQ ID NO:606), NCLIV_017840 (SEQ ID NO:640), NCLIV_023790 (SEQ ID NO:332), NCLIV_057710 (SEQ ID NO:656), NCLIV_061040 (SEQ ID NO:264), NCLIV_025600 (SEQ ID NO:761), NCLIV_0260 (SEQ ED NO:402), NCLIV_033250 (SEQ II) NO:206), NCLIV_048040 (SEQ ID NO:499), NCLIV_059960 (SEQ ID NO:905), NCLIV_066020 (SEQ ID NO:17), NCLIV_070170 (SEQ ID NO:908), NCLIV_012400 (SEQ ID NO:386), NCLIV_036830 (SEQ ID NO:109), NCLIV_041120 (SEQ ID NO:311), NCLIV_053880 (SEQ ID NO:298), NCLIV_025920 (SEQ ID NO:59), NCLIV_033680 (SEQ ID) NO:262), NCLIV_043270 (SEQ ID NO:42), NCLIV_064530 (SEQ ID NO:394), NCLIV_015210 (SEQ ID NO:118), NCLIV_019830 (SEQ ID NO:501), NCLIV_025450 (SEQ ID NO:287), NCLIV_048050 (SEQ ID NO:649), NCLIV_049080 (SEQ ID NO:797), NCLIV_058450 (SEQ ID NO:638), NCLIV_003310 (SEQ ID NO:142), NCLIV_026600 (SEQ ID NO:306), and NCLIV_040040 (SEQ ID NO:863).

### Claim 4 (depends on 1)

A pharmaceutical composition, comprising the enriched antigen extract composition according to claim 1, one or more pharmaceutically acceptable carriers or diluents.

### Claim 5 (depends on 1)

The pharmaceutical composition according to claim 1, wherein the adjuvant is a saponin.

### Claim 6 (depends on 4)

The pharmaceutical composition according to claim 4, wherein the pharmaceutical composition comprises an amount ranging from 0.0001% to about 0.5% w/v of the enriched antigen extract composition.

### Claim 7 (depends on 4)

A method of treating or preventing infections caused by Neospora, comprising administering to a mammal in need thereof a pharmaceutical composition of claim 4.

### Claim 8 (depends on 7)

The method according to claim 7, wherein the mammal is selected from the group consisting of canidaes and ungulates.

### Claim 9 (depends on 7)

The method according to claim 7, wherein the pharmaceutical composition is administered to the mammal in an amount of about 0.001 μg to about 10 μg per kg of the mammal.

### Claim 10 (depends on 5)

The pharmaceutical composition according to claim 5, wherein the saponin is spijoside.

### Claim 11 (depends on 9)

The method according to claim 9, wherein the pharmaceutical composition is administrated to the mammal at least two times and at least about 14 to about 21 days between each of administration.

### Claim 12 (depends on 9)

The method according to claim 9, wherein the pharmaceutical composition is administered by a route selected from the group consisting of intranasally, intradermally, subcutaneously, by aerosol, intramuscularly, wing web, eye-drop administration, and any combination thereof.

### Claim 13 (depends on 1)

The enriched antigen extract composition according to claim 1, wherein the Neospora cells belong to the species Neospora caninum.

### Claim 14 (depends on 1)

The enriched antigen extract composition according to claim 1, wherein the hypertonic solution comprises sucrose and/or sorbitol and/or mannitol.

### Claim 15 (depends on 1)

The enriched antigen extract composition according to claim 1, wherein the centrifugation takes place for about 60 minutes at a temperature of about 4° C.

### Claim 16 (depends on 1)

The enriched antigen extract composition according to claim 1, wherein the non-ionic surfactant is selected from the group consisting of Polysorbate 80, Triton X-114, Triton X-100, and Tween 20.