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Title:
STIMULATION OF VACCINATION BY ANGIOTENSIN PEPTIDES
Document Type and Number:
WIPO Patent Application WO/2014/116587
Kind Code:
A1
Abstract:
The present invention provides angiotensin peptide compositions and methods for use of the compositions in vaccination.

Inventors:
LARSEN, Robert (USC Stevens Institute for Innovation, 1150 South Olive Street Suite 230, Los Angeles CA, 90015, US)
RODGERS, Kathleen, E. (4403 Galeano Street, Long Beach, CA, 90815, US)
DIZEREGA, Gere, S. (231 Bonetti Drive, San Luis Obispo, CA, 93401, US)
Application Number:
US2014/012332
Publication Date:
July 31, 2014
Filing Date:
January 21, 2014
Export Citation:
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Assignee:
UNIVERSITY OF SOUTHERN CALIFORNIA (1150 South Olive Street, Suite 2300Los Angeles, CA, 90015-2477, US)
International Classes:
A61K39/21
Domestic Patent References:
WO2011053789A2
WO2002006308A2
Other References:
DOWNHAM M R ET AL: "Evaluation of two carrier protein-angiotensin I conjugate vaccines to assess their future potential to control high blood pressure (hypertension) in man", BRITISH JOURNAL OF CLINICAL PHARMACOLOGY, BLACKWELL SCIENTIFIC PUBL, GB, vol. 56, no. 5, 1 November 2003 (2003-11-01), pages 505-512, XP002615427, ISSN: 0306-5251, DOI: 10.1046/J.1365-2125.2003.01926.X [retrieved on 2003-10-22]
PREM N GUPTA ET AL: "Development of liposome gel based formulations for intravaginal delivery of the recombinant HIV-1 envelope protein CN54gp140", EUROPEAN JOURNAL OF PHARMACEUTICAL SCIENCES, ELSEVIER, AMSTERDAM, NL, vol. 46, no. 5, 6 February 2012 (2012-02-06), pages 315-322, XP028489133, ISSN: 0928-0987, DOI: 10.1016/J.EJPS.2012.02.003 [retrieved on 2012-02-15]
Attorney, Agent or Firm:
HARPER, David, S. (Mcdonnell Boehnen Hulbert & Berghoff LLP, 300 South Wacker Drive Suite 310, Chicago IL, 60606, US)
Download PDF:
Claims:
We claim

1. A pharmaceutical composition, comprising

(a) an amount effective of an immunogen sufficient to elicit an immune response in a mammal;

(fa) an amount effective of an angiotensin peptide or salt thereof to stimulate a local immune response in a mammal at a site of application of the composition: and

(c) a pharmaceutically acceptable carrier.

2. The pharmaceutical composition of claim 1, wherein the immunogen comprises an immifflogen selected from the group consisting of a killed viral immunogen, a killed bacterial immunogen, one or more viral proteins or antigenic fragments thereof, and one or more bacterial proteins or antigenic fragments thereof or combinations thereof.

3. The pharmaceutical composition of claim 1 or 2, wherein the immunogen comprises an immunogen selected from the group consisting of a killed human immunodeficiency virus (HIV) immunogen, a killed feline immunodeficiency virus (FTV) immunogen, one or more HIV proteins or antigenic fragments thereof and one or more FIV proteins or antigenic fragments thereof or combinations thereof,

4. The pharmaceutical composition of claim 1 or 2„ wherein the immunogen comprises a an immunogen selected from the group consisting of HIV-gpl20 or antigenic fragments thereof and FTV-p24 or antigenic fragments thereof or combinations thereof.

5. The pharmaceutical composition of any one of claims 1-4, wherein the composition is formulated for mucosal administration.

6, The pharmaceutical composition of any one of claims l-5„ wherein the

pharmaceutically acceptable carrier comprises between about 0.5% and about 4%

liydroxyethyl cellulose (HEC).

7. The pharmaceutical composition of any one of claims 1-5, wherein the

pharmaceutically acceptable earner comprises about 2% HEC.

8. The pharmaceutical composition of any one of claims 1-7, wherein the angiotensin peptide is present at between about 0.01 mg/mi and about 30 mg/ml.

9. The pharmaceutical composition of any one of claims 1 -8, wherein the angiotensin peptide is angiotensin 1-7 (A(l-7)) or a salt thereof,

10. The pharmaceutical composition of any one of claims 1-8, wherein the angiotensin peptide comprises at leas four contiguous amino acids of

(a) groups R!-RS in the sequence of general formula I

R^- -R^-R^R8 (SEQ ID NO: 1) wherein R* is selected from tlie group consistin of H, Asp, Glu, Asn, Acpc (1-aminocyclopentane carboxyiie acid), Ala, Me~GIy, Pro, Bet, G3u{ H2), Gly, Asp( ¾) and Sue, or is absent,

is selected from the group consisting of Arg, L s, Ala, Cit, Om. Ser(Ac). Sar, D- Arg and D-Lys,

R3 is selected from the group consisting ofVal, Ala, Leu, aorLeu. lie, Gly, Lys, Pro, Aib, Acpc and Ί yi .

R4 is selected from the group consisting of Tyr, Tyr(P03)2, Thr, Ser, homoSer, azaTyr and Ala;

R3 is selected from the group consisting of lie, Ala, Leu, norLeu, Val and Gly;

R° is selected from the group consisting of His, Arg or 6-N¾-Phe;

R7 is selected from the grou consisting of Pro or Ala; and

R8 is selected from t e group consisting of Plie, Phe(Br) He and Tyr, excluding sequences including R4 as a terminal Tyr group; or

(b) groups R2-R8 in the sequence of general formula II R2-R3-R4-R5-R°-R7-R8 (SEQ ID NO: 34)

in which R2 is selected from the group consisting of H, Arg, Lys, Ala, Om, Citron, Sei Ac), Sar, D-Aig and D-Lys;

R^-R3 are as defined above, and

excluding sequences including R4 as a terminal Tyr group.

11, A method of vaccination, comprising administering to a subject in need of vaccination:

(a) vaccine; and

(b) a amount effective of a angiotensin peptide or salt thereof to stimulate a local immune response in a mammal at a site of application of the composition ;

wherein the vaccine and the angiotensin peptide are administered topically to a mucosa of die subject.

12, The method of vaccination of claim 11, wher ein the vaccine and the angiotensin peptide are administered as a single pharmaceutical composition.

13 , The method of any one of claims 11-12, wherein the vaccine comprises an

immunogen selected from the group consisting of a killed vir al immunogen, a killed bacterial immunogen, one or more vir al proteins or antigenic fragments thereof, and one or more bacterial proteins or antigenic fragments thereof, or combinations thereof

14. The method of any one of claims 11-12, wherein the vaccine comprises an inimifflogeii selected from the group consisting of a killed human nniunodefkiency virus (HIV) imniimogen, a killed feline immimodeficiency vims (FIV) inunuuogen, one or more HIV proteins or antigenic fragments thereof, and one or more FW proteins or antigenic fragments thereof or combinations thereof.

15. The method of any one of claims 11-12, wherein the vaccine comprises an imimmogen selected from the grou consisting of HiV-gpl 20 or antigenic fragments thereof and FTV-p24 or antigenic fr agments thereof or combinations thereof.

16. The method of any one of claims 11-15, wherein the vaccine and the angiotensin peptide are administered in a formulation comprising between about 0.5% and about 4% iiydroxyetiryi cellulose (HEC).

17. The method of any one of claims 11-15, wherein the vaccine and the angiotensin peptide are administered in a formulation comprising about 2% HEC.

18. The method of any one of claims 11-1 , wherein the vaccine and the angiotensin peptide are topically administered at an oral mucosa of the subject.

19. The method of any one of claims i 1-16, wherein the angiotensin peptide is administered at between about 1 mg/ml and about 10 mg/ml.

20. The method of any one of claims 11-19, wherein the angiotensin peptide is A(l-7) or a salt thereof.

21. The method of any one of claims 11-19, wherein the angiotensin peptide comprises at least four contiguous amino acids of

(a) groups R5-RS in the sequence of general formula I

RLR2-R^R R5-RS-R7 Rs (SEQ ID O: i)

wherein R1 is selected from the group consisting of H, Asp, Glu, Asn, Acpc (1-aminocyclopentane carboxylic acid), Ala, Me'Gly, Pro, Bet, Glu{ ¾), Gly, Asp(NH2) and Sue, or is absent

Rr is selected from the group consisting of Arg, Lys, Ala, Cit, Oni, Ser(Ac), Sar, D- Arg and D-Lys,

Rs is selected from the group consisting ofVal, Ala, Leu, norLeu. lie, Gly, Lys, Pro, Aib, Acpc and Tyr;

R4 is selected fr om the group consisting of Tyr, Τντ(Ρ<¾)2, Tnr, Ser, homoSer, azaTyr. and Ala;

R5 is selected from the group consisting of lie, Ala, Leu, norLeu, Val and Gly;

R& is selected from the grou consisting of His, Arg or 0- ¾-Phe; R' is selected from the group consisting of Pro or Ala; and

Rs is selected from the group consisting of Phe, Phe(Br). He and Tyr. excluding sequences including R4 as a terminal Tyr group; or

(b) groups R2-R8 in the sequence of general formula II R2-R -R4-Rs-Rs-R" -Rs (SEQ ID NO: 34)

in which ^ is selected from the group consisting of L Arg, Lys, Ala, Om, Citron, Ser(Ac), Sar, D-Arg and D-Lys;

RJ-RS are as defined above, and

excluding sequences including R4 as a terminal Tyr group.

Description:
Stimulation of vaccination by angiotensin peptides

Cross Reference

This application claims priority to U.S. Provisional Patent Application Serial No. 61/755710 tiled January 23„ 2013, incorporated by reference herein in its entirety.

Background

The acquired immune deficiency syndrome (ADDS) epidemic is sustained largely through sexual transmission of human immunodeficiency virus (HIV). Often, HTV infection appears to he established with just a very few founder viruses. As the virus breaches the mucosal barrier there is a vigorous host immune response. Augmenting the mucosal immuno!ogica! barrier prior to exposure to HTV by mucosal immunization may prevent transmission through immune exclusion.

Summary of the Invention

In a first aspect, the invention provides pharmaceutical compositions, comprising

(a) an amount effective of an imimmogen sufficient to elicit an immune response in a mammal;

(b) an amount effective of an angiotensin peptide or salt thereof to stimulate a local immune response in a mammal at a site of application of the composition: and

(c) a phai iiaceutically acceptable carrier.

In one embodiment, the immunogen comprises an imimmogen selected from the group consisting of a killed viral immunogen, a killed bacterial immunogen, one or more viral proteins or antigenic fragments thereo and one or more bacterial proteins or antigenic fragments thereof, or combinations thereof In another embodiment, the immunogen comprises an immunogen selected from the group consisting of a killed human

immunodeficiency virus (HTV) immunogen, a killed feline immunodeficiency virus (FIY) immunogen, one or more HTV proteins or antigenic fragments thereof and one or more FIV proteins or antigenic fragments thereof, or combinations thereof In a still further" embodiment, the vaccine comprises an immunogen selected from the group consisting of HIV-g l20 or antigenic fragments thereof and FIV-p24 or antigenic fragments thereof, or combinations thereof.

In another embodiment, the composition is formulated for mucosal administration.

In a second aspect, the invention provides methods of vaccination, comprising administering to a subject in need of vaccination:

(a) a vaccine; and

(b) an amount effective of an angiotensin peptide or salt thereof to stimulate a local immune response in a mamma! at a site of application of the composition :

wherein the vaccine and the angiotensin peptide are administered topically to a mucosa of the subject I one embodiment, the vaccine and the angiotensin peptide are administered as a single pharmaceutical composition. In another embodiment, vaccine comprises an imrnunogen selected from the group consisting of a killed viral irnniunogen, a killed bacterial iminmiogen, one or more viral proteins or antigenic fragments thereof, and one or more bacterial proteins or antigenic, fragments thereof, or combinations thereof. In a further embodiment, the vaccine comprises an inimunogen selected from the group consisting of a killed human immunodeficiency virus (HIV) inimunogen, a killed feline

immunodeficiency virus (FTV) immunogen, one or more HIV proteins or antigenic fragments thereof, and one or more FIV proteins or antigenic fragments thereof, or combinations thereof. In a still further embodiment, the vaccine comprises an immunogen selected from the group consisting ofHIV-gpl2G or antigenic fragments thereof and FIV-p24 or antigenic fragments thereof, or combinations thereof.

Detailed Description of the Invention

As used her ein, the singular forms "a," "an" and "the" include plural referents unless the context clearly dictates otherwise. "And" as used herein is interchangeably used with "o " unless expressly stated otherwise.

Ail embodiments within and between different aspects of the invention can be combined unless the context clearly dictates otherwise.

As used herein, the term "about" means + - 5% of the relevant measurement or unit

In a first aspect, the present invention provides pharmaceutical composition, comprising

(a) a amount effective of an immunogen sufficient to elicit an immune response in a mammal; (b) an amount effective of an angiotensin peptide or salt thereof to stimulate a local immune response in a mammal at a site of application of the composition; and

(c) pharmaceutically acceptable carrier

The inventors have demonstrated that angiotensin peptides are broadly applicable as vaccine adjuvants, and thus the recited pharmaceutical compositions have broad use, for example, in vaccinations. The pharmaceutical compositions of the invention may include any suitable angiotensin peptide, such as those described in detail below. In one embodiment, the angiotensin peptide comprises or consists of A(l-7), with an amino acid sequence of Asp-Arg- Val-Tyr-Ile-His-Pro (SEQ ID NO: 4).

In one embodiment, the peptides for use in the invention comprise or consist of a sequence of at least four contiguous amino acids of groups R ! -R s in the sequence of general formula I

R 1 -R 2 -R 3 -R 4 -R 5 -R 5 -R 7" R 8 (SEQ ID NO: i)

wherein R 1 is selected from the group consisting of H, Asp, Glu, Asn, Acpc (1-aminocyclopentane carboxylic acid), Ala, Me 2 Gly, Pro, Bet, Glu NH 2 ), Gly, Asp(N¾) and Sue, or is absent,

R ~ is selected from the group consisting of Arg, Lys, Ala, Cit, Qui, Ser(Ac). Sar, D- Arg and D-Lys,

R 3 is selected from the group consisting ofVa , Ala, Leu, norLeu, He, Gly, Lys, Pro, Aib, Acpc and Tyr;

R 4 is selected from the group consisting of Tyr, Tyr(P0 3 ) 2 , Thr, Ser, bomoSer, azaTyr, and Ala;

R 3 is selected from the gr oup consisting of lie, Ala, Leu, norLeu, Val and Gly;

R 6 is selected from the group consisting of His. Arg or 6-NH 2 -Phe;

R' is selected from the group consisting of Pro or Ala: and

R 8 is selected from the group consistin of P e, Phe(Br), He and Tyr, excluding sequences including R 4 as a terminal Tyr group.

Exemplary AT2 agonists useful in the practice of the invention include the All analogues set forth above subject to the restriction that R 6 is p-NLL-Pfae.

In a further preferred embodiment of each of the above embodiments (SEQ ID NO:

15),

R 1 is selected from the gr oup consisting of Asp and Glu, or is absent:

R 2 is selected from the group consistin of Arg, Lys, and Ala; is selected from the group consisting ofVal, Ala, Leu, norLeu, He, Gly, Lys, and

Pro;

R 4 is selected from the group consisting of Tyr and homoSer;

R 5 is selected from the group consisting of lie, Ala, Leu, norLeu, Val and Gly;

R 6 is selected from the group consisting of His and Arg;

R "' is selected from the group consisting of Pro or Ala; and

R 8 is selected from the group consisting ofPhe. lie, or is absent.

In alternate embodiments, the peptides comprise or consist of at least five, six, seven, or eight contiguous amino acids of groups R J -R 8 in the sequence of general formula I. In a further alternative, the polypeptides consis essentially of a sequence of at least four, five, six, seven, or eight contiguous amino acids of groups R ! -R s in the sequence of general formula I.

Particularly preferred combinations tor R 1 and R 2 are Asp-Arg, Asp-Lys, Ghi-Arg and Glu-Lys. Particularly preferred embodiments of this class include the following: AHI or AII(2-8). Arg-Val-Tyr-De-His-Pro-Phe [SEQ ID NO:2]; AH(3-8), also known as desl -AIII or AIV, Val-Tyr-Ue-His-Pro-Phe [SEQ ID NO:3]; AH(l-7), Asp-Arg-Val-Tyr-Ile-His-Pro [SEQ JD NO:4]; AII(2-7). Ai-g-Val-Tyr-ne-His-Pro [SEQ ID NO:5]; ΑΠ(3-7), Val-Tyi-Ile-His-Pro [SEQ ID O:6]; AII(5-S), lie-His-Pro-Phe [SEQ ID NO:?]; AH(l-6), Asp-Arg-Val-lyr-Ile- His [SEQ ID NO:8j; AII(l-5), Asp-Arg- Vai-Tw-Ile [SEQ ID NO:9]; AII(l-4), Asp-Arg- Val- TyT [SEQ ID NO: 10]; and AH(l-3). Asp-Arg-Val , Other preferred embodiments include: Arg-noiLeu-Tyi-Ile-His-PiO-Phe [SEQ ID NO: 11] and Arg-Val-Tyr-norLeu-His-Pro-Phe [SEQ ID NO: 12]. Still another preferred embodiment encompassed within the scope of the invention is a peptide having the sequence Asp-Arg-Pro-Tyr-IIe-His-Pro-Ph [SEQ ID NO:13],

Other preferred embodiments comprise or consist of

Asp-Arg- Val-Tyr-Val-His-Pro-Phe SEQ ID NO; 18

Asn-Arg-Val-Tyr-Val-His-Pro-Phe SEQ ID NO: 19

Ala-PiO-Gly-Asp-Aig-I e-Tyr-Val-His-Pro-Phe SEQ ID NO: 20

Glu-Arg-Val-Tyr-Ile-His-Pro-Phe SEQ ID NO: 21

Asp-Lys-Val-Tyr-Ile-His-Pro-Phe SEQ ID NO: 22

Asp-Aig-Ala-Tyr-Ile-His-Pro-Phe SEQ ID NO: 23

Asp-Arg- Val-Hn--Ile-His-Pro-Phe SEQ ID NO: 24

Asp-Arg- Val-Tyr-Leu-His-Pro-Phe SEQ ID NO; 25

Asp-Arg-Val-Tyr-Ile-Arg-Pro-Plie SEQ ID NO: 26

Asp-Arg-Val-Tyr-rie-His-Ala-Phe SEQ ID NO: ?'7 Asp-Ai-g-Val-Tyr-Ile-His-Pro-Tyr SEQ ID NO: 28

Pio-^g-Vai-Tyr-Ile-His-Pi -Pbe SEQ ID NO: 29

Asp-Arg-PiO-Tyr-Ile-His-PiO-Phe SEQ ID NO; 13

Asp-Arg^ r al-Ty (P0 3 )2-Ile-His-Pro-P e SEQ ID NO; 30

Asp- Arg-norLeu-Tyr-IIe-His-Pro-Phe SEQ ID NO : 31

Asp-Arg-Val-Tyr-norLeii-His-Pro-Plie SEQ ID NO: 32

Asp-Arg-Val-homoSer-T i-Ile-His-Pro-Phe SEQ ID NO: 33

Another class of peptides of particular interest in accordance with the present invention are those of the general formula II:

R 2 -R 3 -R 4 -R S -R*-R 7 -R S (SEQ ID NO: 34)

in which R 2 is selected from the group consisting ofH, Arg, Lys, Ala, Ora, Citron, Se i Ac), Sar, D-Arg and D-Lys;

R 3 -R s are as defined above, and

excluding sequences including R 4 as a terminal Tyr gr oup, ,

A particularly preferred subclass of the compounds of general formula II has the formul

HI of the formula Arg-Val-Tyr-Ile-His-Pro-Phe [SEQ ID NO:2], Other preferred compounds include peptides having the structures Arg-Val-Tyr-Gly-His-Pro-Phe [SEQ ID NO; 36] and Arg-Val-Tyr-Aia-His-Pro-Phe [SEQ ID NO:37].

In the above formulas, the standard three-letter abbreviations for amino acid residues are employed. Other residues are abbreviated as follows:

TABLE 1

Abbreviation for Amnio Acids

Me 2 GIy NJ^-dimethylgiycyi

Bet. 1 -carix>xy-N,N.N-¾imemyliHethanamimum hydroxide inner salt

(betaine)

Sue Succinyi

Phe(Br) p-broffio-L-phenylalanyl

azaTyr aza- * -homo-L-tyrosyl

Acpc 1-aiimioeyclopentane carboxyli acid Aib 2-ammoisobutyiic acid

Sar N-methylglycyl (sarcosine)

Cit Citron

Ora Ornithine

NorLeu (N e) NorLeucine

HomoSer HotnoSeiiiie (isotheiouine)

It has been suggested that AH and its analogues adopt either a gamma or a beta turn (Regoli, et aL, Pharmacological Reviews 26:69 (1974)}, In general, it is believed that neutral side chains iu position R\ R* and R' may be involved in maintaining the appropriate distance between active groups in positions R 4 , R 's and R* primarily responsible for binding to receptors and or intrinsic activity. Hydrophobic side chains in positions R\ R* and R 8 may also play an important role in the whole conformation of the peptide and/or contribute to the formation of a hypothetical hydrophobic pocket.

AppiOpiiate side chains on the amino acid in position R * may contribute to affinity of the compounds for target receptors and/or play an important role in the conformation of the peptide. For mis reason, Arg and Lys are particularly preferred as R A . Alternatively, Ri may be H. Ala, Oni, Citron, Ser(Ac), Sar, D-Arg, or D-Lys.

Fo purposes of the present invention, it is believed that R J may be involved in the formation of linear or nonlinear hydrogen bonds with R 5 (in the gamma turn model) or R 6 (in the beta turn model). R 3 would also participate in the first turn in a beta antiparallel structure (which has also been proposed as a possible structure). In contrast to other positions in general formula I, it appears that beta and garnma branching are equally effective in this position. Moreover, a single hydrogen bond may be sufficient to maintain a relatively stable confonnation. Accordingly, R 3 may suitably be selected from Lys. Val. Ala, Leu, norLeu, He, Gly, Pro, Aib, Acpc and Tyr.

With respect to R 4 , conformational analyses have suggested that the side chain in this position (as well as in R* and R y ) contribute to a hydrophobic cluster believed to be essential for occupation and stimulation of receptors. Thus. R 4 is preferably selected from Tyr, Thr, Tyr (P0 3 )2- homoSer, Ser and azaTyr In this position, Tyr is particularly preferred as it may form a hydrogen bond with the receptor site capable of accepting a hydrogen horn the phenolic hydroxy! (Regoli, et al. (1 74), supra). It has also been found that R 4 can be Ala, and that it can be used for cyclization of angioteiision peptides. In position R ' , an amino acid witli a β aliphatic or alicyelic chain is particularly desirable. Therefore, while Gly is suitable in position R 5 , it is preferred that the amino acid in this position be selected from lie, Ala, Leu, norLeit, and Val.

In another embodiment, in pq¾ides of particular interest in accordance wi th the present invention R 6 is His, Arg or 6-NH 2 -Phe, The unique properties of the imidazole ring of histidine (e.g., ionization at physiological pH, ability to act as proton donor or acceptor, aromatic character) are believed to contribute to its particular utility as R 6 . For example, conformational models suggest that His may participate in hydroge bond formation (in the /·><· ; .<; model) or in the second tarn of the antiparaile! structure by influencing the orientation of R' . Similarly, it is presently considered that R' should be Pro or Ala in order to provide the most desirable orientation of R s . In position R s , both a hydrophobic ring and an anionic carboxyl terminal appeal- to be particularly useful in binding of the analogues of interest to receptors; therefore, Tyr, lie, Phe(Br), and especially Phe are preferred for purposes of the present invention.

Analogues of particular interest include the following:

TABLE 2

Angiotensin II Analogues

All Amino Acid Sequence Sequence

Analogue Identifier Name

Analogue 1 Asp-Arg-Val-Tyr-Val-His-Pro-Phe SEQ ID NO: 18

Analogue 2 Asn-Arg-Val-Tvr-Val-His-Pro-Phe SEQ ID NO: 19

Analogue 3 Ala-Pro-Gly-Asp-Arg-Ile-Tyr-Val-His-Pro-Phe SEQ ID NO: 20

Analogue 4 Glu-Arg-Val-Tyr-Ile-His-Pro-Phe SEQ ID NO: 21

Analogue 5 Asp-Lys-Val-Tw-Ile-His-PiO-Plie SEQ ID NO: 22

Analogue 6 Asp-Arg-Ala-Tyr-Ile-His-Pro-Phe SEQ ID NO: 23

Analogue 7 Asp-Aig-Val-Tiff-Ile-His-PiO-Phe SEQ ID NO: 24

Analogue 8 Asp-Arg-Val-Tyr-Leu-His-Pro-Phe SEQ ID NO: 25

Analogue 9 Asp-Aig-Val-Tyr-IIe-Aig-PiO-Phe SEQ ID NO: 26

Analogue 10 Asp-Aig-Val-Tyr-Iie-His-Ala-Phe SEQ ID NO: 27

Analogue 1 1 Asp-Ai " g-Val-Tyr-Ile-His-PiO-Tyi " SEQ ID NO: 28

Analogue 12 Pro-Arg-Val-Tyr-Ile-His-Pro-Phe SEQ ID NO: 29 Analogue 13 Asp-Arg-PiO-Tw-Iie-His-Pro-P e SEQ ID NO 13

Analogue 14 Asp-Arg-Val-Tyr(P0 3 ) 2 --le-His-Pro-Phe SEQ ID NO 30

Analogue 15 Asp-Arg-norLeu-Tyr-Ile-His-Pro-Phe SEQ ID NO 3

Analogue 16 Asp-Arg-Val-Tyr-iiorLeu-His-Pro-Phe SEQ ID NO 32

Analogue 17 Asp-Arg-Vai-homoSer-Tyr-Ile-His-Pro-Phe SEQ ID NO 33

Other particularly preferred embodiments include:

1GD Ala4-Aii(l-7) DRVAIHP SEQ ID NO.-38

2GD PI 3-AH(1-7) DRPYIHP SEQ ID O:39

5GD Lys3-AII(l-7) DRKYIHP SEQ ID O:40

9GD NorLeu-AII(i-7) DR(iior)YIHP SEQ I NO:41

GSD 28 ile 8 -AII DRVYIHPI SEQ ID NO: 42

Ala3ainmoPhe6 All: RVAIHPF SEQ ID O-.43

Ala3-AIH RVAIHPF SEQ ID NO.-44

Gly 1 -AII GRVYIHPF SEQ ID NO.-45

NorLe« 4 -AIII -RVYnLHPF SEQ ID O:46

Aepc 3 -AII DR(Acpe)YIHPF SEQ ID O:47

GSD 37B Om 2 -AII D(Om)VYIHPF SEQ ID O:48

GSD38B Citroii 2 -AII D(CitiOn)YYIHPF SEQ ID NO:49

3GD Pi 3 Ala 4 -An(I-7) DRPAIHP SEQ ID NO:50

8GD Hydroxy-Pro 3 -AII(l -7) DRP(OH)AIHP SEQ ID NO.-51

In another embodiment, the peptides may be any of those disclosed in

US20100055146, incoiporaied by reference herein in its entirety. In various embodiments, the polypeptide is:

a 4,7-cyclized analog of Angiotensin II (Ang(l-S), or any of its analogues disclosed herein:

a 4,7-cyclized analog of Angiotensin IH (Ang(2-8)), or any of its analogues disclosed herein;

a 4,7-cyclized analog of Angiotensin TV (Ang(3-8)). or any of its analogues disclosed herein; or

a 4,7-cyclized analog of Ang(l-7), or any of its analogues disclosed herein.

In another embodiment , the methods comprise administering a agonist of the MAS receptor. Any suitable polypeptide or non-polypeptide agonist of the MAS receptor may be used, including but not limited to A(i -7) and analogues thereof, A779 (D-Ala A(l-7):

available fiom Sigma Chemical Co.) and AVE0991, (see, for example, Pinheiro et al.

Hypertension. 2004 Oct:44(4):490-6. Epub 2004 Aug 23).

The polypeptides for use in the present invention may he lineal- or cyclized in any suitable manner, such as those described in WO2008/018792, including but not limited to polypeptides comprising thioether bridge between positions 4 and 7, or other positions.

The polypeptides may be recombinantly expressed or chemically synthesized using any suitable techniques, which are well within the level of those of skill in the art.

Any suitable irmnunogen or combination of immunogens can be used in the compositions of the invention. The immunogen(s) must be capable of stimulating an immune response in any suitable mammal, including but not limited to humans, cattle, pigs, goats, dogs, cats, horses and any other mammalian pet or food animal such as chickens, turkeys, Cornish game hens, pheasants, or ducks.

For example, the immunogen can be a whole or partial product of a virus, bacteria, fungal organism, mycobacteria, parasite (single cell or multiple cell), or derived fiom a plant, fermentation product (generally produced by yeasts and/or molds) or engineered through chemical synthesis (for example a DNA vaccine antigen). Additionally, the immunogen could be derived from human cells, including but. not limited to cancer cells or parte thereof, which could enhance host responses to destroying cancer cells.

In one non-limiting embodiment, the immunogen is a bacteria or antigenic portion thereof. In one embodiment, the bacteria may include, but is not limited to. at least one of: Neisseria meningitides; N. gonotrheoeae Legionella pneumonia; Vibrio cholera e; Vibrio parahemolyticus. Vibrio volnificans. Streptococcal species including Group A streptococci; Staphylococcus aureus: Staphylococcus epidermidis; Pseudomonas aeruginosa;

Coryriobacferia diphtheriae; Clostridium spp.including C. perfringens: Eschericia coli;

Bacillus anthracis; Bartonella henselae; Bartonella quintana; Coxielki burnetii; Chlamydia pneumonia, C. trachomatis; Mycobacterium leprae; M. tuberculosis. Non-tuberculous mycobacteria; Salmonella species including S. typhi and non-t phoidal strains; Shigella sonnie and other Shigella species: Helicobacter pylori; H, influenza, H. parainfluenzae, Yersinia enierocolhica; pseudotuberculosis; Listeria monocytogenes; Mycoplasma pneumonia; M. urifyticum; Treponema pallidum; L eptospira species; Francisella ndarensis; Brucella melitensis; B, aborus, B. suis, Campylobacter jejuni; Enterobacter aerogenes and other enterobacteraeea; Proteus mirabilis and other Proteus species; and Klebsiella pneumonia and other Klebsiella species, or combinations thereof. In another non-limiting embodiment the immunogen is a vaccine already in general use but would be enhanced by addition of an angiotensin peptide. In one embodiment vaccine the vaccine in use could be the polyvalent influenza vaccine either the killed vims or live vims; the polyvalent pneumococcal vaccine: the polyvalent meningococcal vaccine; the polio vaccine; the multiple vaccine combination measles, mumps and rubella vaccine; or the tetanus and diphtheria vaccine, or combinations thereof.

In another non-limiting embodiment, the immunogen is a fungi or antigenic portion thereof. In one embodiment, the fungi may include, but is not limited to Candida species, such as C. albicans; Aspergilus species; Histoplasma capsulatunr, Bastomycosis dermatiditis'. Coccklioides immitis or the capsular antigen of Cryptococcus neoformans, or combinations thereof

In a further non-limiting embodiment, the immunogen is a virus or antigenic portion thereof. In one embodiment, the virus may include, but is not limited to adenovirus;

coxsackievirus; Enterocytopathic human orphan viruses (ECHO): hepatitis A, B, C, D or X viruses alone or in combination; the human herpes virus family currently composed of

Epstein-BaiT virus; herpes simplex; type 1: herpes simplex; type 2; human cytomegalovirus; human herpesvirus; type 8: varicella-zoster virus; human immvinodeficiency vims (HIV); influenza viruses; measles virus; mumps virus; parainfluenza vims; respiratory syncytial virus; papillomaviruses; rabies virus; and encephalitis viruses including but not limited to St. Louis encephalitis virus, West Nile virus, Japanese encephalitis virus, and Equine encephalitis viruses, or combinations thereof.

In another non-limiting embodiment, the immunogen is a par asite or antigenic portion thereof. In one embodiment, the parasite may include, but is not limited to trypanosome; haemoprotozoa and parasites capable of causing malaria; amoeba, enteric and systemic cestode; taeniid cestod; enteric coccidian; enteric flagellate protozoa; filarial nematode: gastrointestinal and systemic nematode and hookworm.

In one embodiment, the immunogen is one derived from a retrovirus, including but not limited to a whole retrovirus or portion thereof. Exemplary and non-limiting such retroviruses are human immitnodeficiency virus (HIV), feline immunodeficiency vims (FIV). feline leukemia vims (FeLV), bovine leukemia virus, human T-lymphotropic virus (HTLV), and as yet undiscovered retroviruses. Preferably, the immunogens are selected from the grou consisting of a killed human immimodeficiency virus (HIV) immunogen, a killed feline immunodeficiency vims (FIV) immunogen, one or more HIV proteins or antigenic fragments thereof, and one or more FW proteins or antigenic fragments thereof, or combinations thereof.

When the immunogeii comprises a viral imniimogen, any suitable inunimogen Scorn. the virus can be used. In one non-limiting embodiment where the immimogeii is an HIV i nniunogen, the inmiunogen may, for example, comprise gp 1 antigen, s l20 antigen, p24 antigen, or g l60 ants gen, or combinations thereof.

The pharmaceutical compositions can be formulated for delivery by any suitable route, including but not limited to mucosal, deraial topical applications, or injection by intradermal, transdermal, subcutaneous, intramuscular, intraperitoneal, intraarticular, epidural, intrathecal injection, ingested or taken through a feeding tube, passed or applied by an endoscope to the upper airway, throat, esophagus, upper or lower intestinal track, anus or by vaginal colposcopy, intravenous or intra-arterial infusion or bolus injection, or absorption through epithelial or mucocutaneous linings, hi one non-limiting embodiment, the composition is formulated for mucosal administration.

The carrier or diluent may include time delay material, such as glyceryl monostearate or glyceryl distearate alone or with a wax, or other materials well known in the art

In some embodiments, the pharmaceutical compositions are formulated as a gel. In these embodiments, the polypeptide, or salt thereof, may be present in the composition at a concentration of about 0.001 % to about 3% on a. weight (mg)< volume (ml) basis, or on a weight/weight (nig) basis. In various further embodiments, the polypeptide, or salt thereof, is administered in a pharmaceutical formulation at a concentration of about 0.005% to about 3%: about 0.01% to about 3%: about 0.05% to about 3%: about 0.01% to about 3%: about 0.5% to about 3%; about 1% to about 3%; about 2% to about 3%: about 0.005% to about 2%; about 0.01% to about. 2%; about 0.05% to about 2%; about 0.01% to about 2%; about 0.5% to about 2%; about 1% to about 2%: about 0.005% to about 1%: about 0.01% to about 1%: about 0.05% to about 1%; about 0.01% to about 1%; about 0.5% to about 1%: about 0.005% to about 0.75%; about 0.01% to about 0.75%; about 0.005% to about 0.75%; about 0.01% to about 0.75%: about 0.03% to about 1%: about 0.03% to about 0.75%; about 0.03% to about 0.5%: about 0.03% to about 0.25%; about 0.03% to about 0.1%: about 0.03% to about 0,075%: about 0.03% to about 0.05%; and about 0.03%; all on a weight (mg)/volume (ml) basis, or on a weight/weight (nig) basis.

In another embodiment of all aspects of the invention, the pharmaceutically acceptable carrier comprises about 0.5% to about 4% hydroxyethyl cellulose (HEC) on a. weight (mg)/volume (ml) basis, or on a weight/weight (nig) basis. In various further

I I embodiments, the pharmaceutically acceptable carrier may comprise about 1% to about 3% HEC, or about 2% HEC, on a weight {mg) volume (ml) basis, or on a weight/weight (mg) basis.

Any suitable amount effective of the angiotensin peptide (such as A(l -7)) may be used in the compositions,, as appropriate for a given use. In various non-limiiing

embodiments, the angiotensin peptide is present at between about 0.01 mg/ml to about 30 mg ml; about 0.01 mg/ml to about 10 mg/ml; about 0.1 mg/ml to about 30 mg/ml; about 0.1 mg ml to about 10 mg ml; about 1 mg ml to about 30 mg/ml; and about i mg/ml to about 10 mg/mi.

In all aspects of the invention, the polypeptides, or salt thereof may be administered (or present in the pharmaceutical compositions) together with one or more (a) a lyoprotectant; (fa) a surfactant; (c) a bulking agent; (d) a tonicity adjusting agent: (e) a stabilizer: (t) a preservative and/or (g) a buffer. In some embodiments, the buffer in the pharmaceutical composition is a Tris buffer, a histidiiie buffer, a phosphate buffer, a citrate buffer" or an acetate buffer. The peptides may be administered with a lyoprotectant, e.g. sucrose, sorbitol or trehalose, hi certain embodiments, the peptides may be administered with a preservative e.g. benzalkonium chloride, benzethom ' um, chlorohexidiue, phenol, m-cresol. benzyl alcohol, methylparaben, propylparaben, chlorobutanol, o-cresoL p-cresol, chlorocresol,

phenyhnercuric nitrate, thimerosal benzoic acid, and various mixtur es thereof. In other embodiments, the peptides may be administered with a bulking agent, like glycine, hi yet other embodiments, the peptides may be administered with a surfactant e.g., polysorbace-20, polysorbate-40, polysorfaate- 60, polysorbate-65, polysorbate-SO polysorbate-85, po!oxamer- 188, sorbitan monolaurate, sorbitan monopalmitate, sorbitan monostearate, sorbitan

mouooleate, sorbitan trilaurate, sorbitan tristearate, sorbitan tiioleaste, or a combination thereof The peptides may be adroinistered with a tonicity adjusting agent, e.g., a compound that renders the formulation substantially isotonic or isoosmotic with human blood.

Exemplary tonicity adjusting agents include sucrose, sorbitol, glycine, methionine, rnannitol, dextrose, inositol, sodium chloride, arginme and argmine hydrochloride, hi other embodiments, the peptides may be administered with a stabilizer, e.g., a molecule which, when combined wim the peptide substantially prevents or reduces chemical and/or physical instability of the protein of interest in lyopbilized or liquid form. Exemplary stabilizers include sucrose, sorbitol, glycine, inositol, sodium chloride, methionine, argmine, and arginme hydrochloride, paraben, and combina tions of methyl paraben and propyl paraben. In all aspects and embodiments of the invention, suitable acids which are capable of forming salts with the polypeptides include, but are not limited to, inorganic acids such as hydrochloric acid, hydrobromic. acid, perchloric acid, nitric acid, thiocyaiiic acid, sulfuric acid, phosphoric acid and the like; and organic acids such as formic acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, malonic acid, succinic acid, maleic acid, fumaric acid, aiithranilic acid, cinnamic acid, naphthalene sulfonic acid, suifanilic acid and the like. Suitable bases capable of forming salts with the peptides include, but are not limited to, inorganic bases such as sodium hydroxide,, ammonium hydroxide, potassium hydroxide and the like; and organic bases such as mono-, di- and tri-alkyl and aryi amines (e.g., triethylamine, diisopropyl amine, methyl amine, dimethyl amine and the like) and optionally substituted ethaiiol-amines (e.g.. ethanolamme, diethanolamiue and the like).

The polypeptides or salts thereof can further be derivatized to provide enhanced haif- life, for example, by linking to polyethylene glycol. The peptides or salts thereof may comprise L-amino acids, D-amino acids (which are resistant to L-amino acid-specific proteases in vivo), a combination of D- and L-amino acids, and various "designer" amino acids (e.g.. β-methyi amino acids, Ca-methyl ammo acids, and N -methyl amino acids, etc.) to convey special properties.

The polypeptides may be the sole active agent i the pharmaceutical composition, or the composition may further comprise one or more other active agents suitable for the desired tieatment. The methods may be used in conjunction with other therapies suitable for treating the relevant disorder.

Ail embodiments of the compositions, and combinations thereof, can also be used in the methods of the invention.

hi a second aspect , the invention provides methods of vaccination, comprising administering to a subject in need of vaccination:

(a) a vaccine; and

(b) an amount effective of an angiotensin peptide or salt thereof to stimulate a local immune response at a site of its administration to the subject;

wherein the vaccine and the angiotensin peptide or salt thereof are administered topically to a mucosa of the subject.

The subject can be any suitable mammalian subject or food animal, as described herein. The subject is one that is at risk of, but not yet suffering from, a disorder caused by the pathogen that the vaccine is directed against. As used herein, "method of vaccination" means to limit development of a disorder caused by the pa thogen by causing an immune response prior to exposure to active pathogen, such that future exposure to the pathogen is less likely to result in an active disorder caused by the pathogen.

Any suitable angiotensin peptide or salt thereof may be used in the invention, as disclosed above. In one embodiment, the angiotensin peptide is A(l-7) or a salt thereof. In one embodiment the vaccine and the angiotensin peptide or salt thereof are administered as a single pharmaceutical composition; in this embodiment, the method may comprise administering a pharmaceutical composition according to any embodiment or combinatio of embodiments of the invention.

The vaccine may comprise any suitable immunogen as described for the

pharmaceutical compositions of the invention. The immimogen(s) must be capable of stimulating an immune response in any suitable mammal, including but not limited to humans, cattle, pigs, goats, dogs, cats, horses and any other mammalian pet or food animal such as chickens, turkeys, Cornish game ens, pheasants, or ducks.

For example, the immunogen can be a whole or partial product of a virus, bac teria, fungal organism, mycobacteria, parasite (single cell or multiple cell), or derived fiom a plant, fermentation product (generally produced by yeasts and/or molds) or engineered through chemical synthesis (for example a DNA vaccine antigen). Additionally, the immunogen could be derived from human cells, mcludiug but not limited to cancer cells or parts thereof, which could enhance host responses to destroying cancer cells.

In one non-limiting embodiment, the immunogen is a bacteria or antigenic portion thereof, hi one embodiment, the bacteria may include, but is not limited to, at least one of: Neisseria menmgitides; N. gonatrheoeae Legionella pneumonia; Vibrio cholera e; Vibrio parahemolyticus. Vibrio volnificans. Streptococcal species including Group A streptococci; Staphylococcus aureus: Staphylococcus epidermidis; Pseudomorias aeruginosa;

Coryriobacferia diphtheriae; Clostridium spp.including C. perfringens: Eschericia coli; Bacillus anthracis; Bartonella henselae; Bartonella quintana; Coxielki burnetii; Chlamydia pneumonia, C. trachomatis; Mycobacterium leprae; M. tuberculosis. Non-tuberculous mycobacteria; Salmonella species including S. typh and non-t phoidal strains; Shigella sonnie and other Shigella species: Helicobacter pylori; H, influenza, H. parainfluenzae. Yersinia enterocolhica; pseudotuberculosis; Listeria monocytogenes; Mycoplasma pneumonia; M. urilyticuin; Treponema, pallidum; Leptospira species; Franciselln ndarensis; Brucella melitensis; B, aborus, B. suis, Campylobacter jejuni; Enterobacter aerogenes and other enterobacieraeea; Proteus mirabilis and other Proteus species; and Klebsiella pneumonia and other Klebsiella species, or combinations thereof. In another non-limiting embodiment the immunogen is a vaccine already i general use but would be enhanced by addition of an angiotensin peptide. In one embodiment vaccine the vaccine in use could be the polyvalent influenza vaccine either the killed virus or live virus; the polyvalent pneumococcal vaccine; the polyvalent meningococcal vaccine; the polio vaccine; the multiple vaccine combination measles, mumps and rubella, vaccine; or the tetanus and diphtheria vaccine, or combinations thereof.

In another non-limiting embodiment, the immunogen is a fungi or antigenic portion thereof. In one embodiment, the fungi may include, but is not limited to Candida species, such as C. albicans; Aspergilus species; Histoplasma capsulatunr, Bastomycosis dermatiditis'. Coccklioides immitis or the capsular antigen of Cryptococcus neoformans, or combinations thereof

In a further non-limiting embodiment, the immunogen is a virus or antigenic portion thereof. In one embodiment, the virus may include, but is not limited to adenovirus;

coxsackievirus; Enterocytopathic human orphan viruses (ECHO): hepatitis A, B, C, D or X viruses alone or in combination; the human herpes vims family currently composed of

Epstein-Barr virus; herpes simplex; type 1; herpes simplex; type 2; human cytomegalovirus; human herpesvirus; type 8; varicella-zoster virus; human immvinodeficiency vims (HIV); influenza viruses; measles virus; mumps virus; parainfluenza vims; respiratory syncytial virus; papillomaviruses; rabies virus; and encephalitis viruses including but not limited to St. Louis encephalitis virus, West Nile vims, Japanese encephalitis vims, and Equine encephalitis viruses, or combinations thereof.

In another non-limiting embodiment, th immunogen is a par asite or antigenic portion thereof. In one embodiment, the parasite may include, but is not limited to trypanosome; haemoprotozoa and parasites capable of causing malaria; amoeba, enteric and systemic cestode; taeniid cestod; enteric coccidian; enteric flagellate protozoa; filarial nematode: gastrointestinal and systemic nematode and hookworm

In one embodiment, the immunogen is one derived from a rettoviras, including but not limited to a whole retrovirus or portion thereof. Exemplary and non-limiting such retroviruses are human immitnodeficiency virus (HIV), ielme immunodeficiency vims (FIV). feline leukemia vims (FeLV), bovine leukemia virus, human T-lyrnphotropic virus (HTLV), and as yet undiscovered retroviruses. Preferably, the immunogens are selected from the grou consisting of a killed human immunodeficiency virus (HIV) immunogen, a killed feline immunodeficiency vims (FIV) immunogen, one or more HIV proteins or antigenic fragments thereof and one or more FIV proteins or antigenic fragments thereof or

combinations thereof

When the immunogen comprises a viral immunogen, any suitable inirnimogen Scorn. the virus can be used. In one non-limiting embodiment where the immunogen is an HIV ininiunogen, the ininiunogen may, for example, comprise gp41 antigen, g l20 antigen, p24 antigen, or gp!60 antigen, or combinations thereof.

In all embodiments of the methods of the invention, the peptide or salt ther eof may be administered by any suitable route, including but not limited to mucosal, dermal topical applications, or injection by mtrademial, transdermal, subcutaneous, intraniuscuiar, intraperitoneal, intraarticular, epidural, intrathecal injection, ingested or take through a feeding tube, passed or applied by an endoscope to the upper airway, throat, esophagus, upper or lower intestinal track, anus or by vaginal colposcopy, intravenous or infra-arterial infusion or bolus injection, or absorption through epithelial or mucocutaneous linings. In one non-limiting embodiment, the peptide is administered mucosaliy.

In one exemplary embodiment, a gastrointestinal route can b used in which, tor example, a gel is applied at the time of upper endoscopy (for example, H. pylori vaccination topically in the upper GI tract) or lower endoscopy (for example, tumor or any of the toxin diseases listed above or even colon cancer).

In one non-limiting embodiment, the inventors have discovered that injection of A(i- 7) with, for example, FFV antigens increased mucosal anti-FIY antibodies in the rectum. The injection of the A(l-7) had distal effects not necessarily to the site of administration. Thus, the compositions and formulations can be used, for example, for limiting development of sexually transmitted diseases where the vaccine is given as an injection in a muscl group like the arm or buttock.

In some embodiments, the angiotensin polypeptide, or salt thereof may be administered in a pharmaceutical formulation at a concentration of about 0.001 % to about 3% on a weight (mg)/voIunie (ml) basis, or on a weight/weight (rag) basis. In various further embodiments, the polypeptide, or salt thereof, is administered in a pharmaceutical

formulation at a concentration of abou 0.005% to about 3%; about 0.01% to abou 3%; about 0,05% to about 3%; about 0.01% to about 3%; about 0.5% to about 3%; about 1% to about 3%: about 2% to about 3%; about 0.005% to about 2%; about 0.01% to about 2%; about 0.05% to about 2%: about 0.01% to about 2%: about 0.5% to about 2%; about 1% to about 2%; about 0.005% to about 1%; about 0.01% to about 1%; about 0.05 to about 1%; about 0.01% to about 1%; about 0.5% to about 1%; about 0.005% to about 0.75%; about 0.01% to about 0.75%; about 0.005% to about 0.75%; about 0.01% to about 0.75%; about 0.03% to about 1%; about 0.03% to about 0.75%; about 0.03% to about 0.5%; about 0.03% to about 0.25%; about 0.03% to about 0.1%; about 0.03% to about.0.075%; about 0.03% to about. 0.05%; and about 0.03%; all on a weight (mg)/volume (ml) basis, or on a weight/weight (mg) basis.

In another embodiment of the methods of the invention, the pharmaceutically acceptable carrier comprises about 0.5% to about 4% hydroxyethyl cellulose (HEC) on a weight (mg) volume (ml) basis, or on a weight weight (mg) basis. In various further embodiments, the pharmaceutically acceptable carrier may comprise about 1% to about 3% HEC, or about 2% HEC, on a weight (mg)/volume (ml) basis, or on a weight/weight (mg) basis.

Any suitable amount effective of the angiotensin peptide (such as A(l-7)) may be used in the methods of the invention, as deemed appropriate for a given use by an attending physician. In various non-lnmfiiig embodiments, the angiotensin peptide is present at between about 0.01 mg/ml to about 30 mg ml; about 0.01 mg/ml to about 10 mg ml; about 0,1 mgml to about 30 mg/ml; about O.i mg ml to about 10 mg/ml; about i mg/ml to about 30 mg/ml; and about 1 mg/ml to about 10 mg/ml.

The methods may include any other embodiments as disclosed in the example that follows. Such embodiments may be used in any combination in the methods of the invention, unless the context clearly dictates otherwise.

Examples

The studies described below characterize the effect of angiotensin (1-7) when coadministered with the killed feline immunodeficiency virus (Fel-O-Vax FIY) vaccme on the generation of mucosal immunity. We demonstrated a dramatic increase in local secretory IgA immune responses.

Introduction

The AIDS epidemic is sustained largely through sexual transmission ofHTV. Often, HTV infection appears to be established with just a very few founder viruses. As the virus breaches the mucosal barrier there is a vigorous host immune response. Augmenting the mucosal immunological barrier prior to exposure to HIV by mucosal immunization may prevent transmission through immune exclusion. Local generation of secretory IgA antibodies (SIgA) constitutes the largest humoral immune system in the body. SIgA has been demonstrated to effectively block epithelial penetration of HIV and in individuals negative for HIV who live with a HIV-positive partner often appear to be protected by specific SIgA in their genital tract. SIgA in saliva has been shown to cleave gp!20 effectively neutralizing the ability of HIV to bind and enter host cells. Certain regions in the gp!20 surface protein are constant and required to maintain HTV infectious capability. Antibodies to the gp-120 region have broad neutralizing ability. SIgA antibodies to g l20 would disrupt the CD4 cell binding site complex which is essential for virus-host cell recognition steps and thus would prevent HTV entry into host cells and the resultant viral replication.

The feline immunodeficiency virus (FIV) is a retrovirus which occurs worldwide in domestic, cats and can lead to rmmunodeilciency similar to HIV. FIV shares multiple pathogenic properties with HIV. Both HIV and HY cause cytopathic effects in lymphocytes leading to CD4+ T-lyniphocyte cell depletion and opportunistic infections and both viruses infect T-lymphocytes, employ the CXCR4 co-receptor for cell entry (although this is not the primary binding receptor for HTV), and can be transmitted by blood, sexual secretions and through vertical transmission. Thus, the pathobiology of FIV in cats is not dissimiiar to that of HTV in humans. FIX ' infection has been established in a cat model system, making FIV a suitable candidate for evaluating local immune effects on the potential to interrupt "sexual" transmission of this retroviral agent. At present a killed FIV virus vaccine (Fel-O- Vax® FIV) has proven efficacy in preventing disease, but not infection.

We describe our experience employing angiotensin (1-7) in combination with a killed FIV vaccine used as the source of antigen in raising mucosal antibodies to FIV following topical application.

Methods

Animals: Eleven (11) female outbred cats 24 weeks old dial were free of FIV, feline leukemia virus (FeLV) and feline infectious peritonitis vims were employed. The animals were restrained for the application of test articles to mucous membranes and collection of blood and mucosal secretions. Sedation, if necessary, was induced by subcutaneous administration of ketamine hydrochloride (22 mg/kg) and acepromazine maleate (0.1 mg cg). Euthanasia was accomplished with a lethal overdose of bar biturates. All activities were approved by the Institutional Animal Care and Use Committee and supervised by veterinary staff.

Vaccine Preparation: Fel-O-Vax FIV (Fort Dodge Animal Health, Fort Dodge, Iowa) and A(l-7) was combined in a constant volume of 2% hydroxyethyl cellulose (2% HEC). The range of concentrations of A(l-7) ranged from 0 to 10 mg/riiL with a constant 0.33 mL of Fel-O-Vax FIV suspended i each mL of the gel applied for vaccination purposes. Two control animals received intramuscular Fel-O-Vax FIV at baseline, week 3 and week 5. One control animal had mucosal vaccination with Fel-O-Vax alone suspended in the 2% HEC without A(l-7) and one control animal received intramuscular Fel-O-Vax FIV with 0.3 mg/kg of A(l -7) at baseline, week 3 and week 5.

Delivery of arigiotensm(l-7j and FIV vaccine to mucosal surfaces: 2% HEC, a viscous gel, was used as the vehicle for the topical delivery of angiotensin 1 -7) and FIV vaccine. The topical vaccine was applied to the mucosal surface weekly for 6 consecutive weeks. 1.0 mL was applied to the muzzle for oral mucosal vaccination and 1.0 mL and 0.2 mL was instilled in the rectal and vaginal vaults, respectively for mucosal vaccination at these sites.

Measurement of Local Immune Response: The Weck-Cel Surgical Spear (Windsor Biomedical, Newton, NH) was used to obtain secretions. Once inserted, the sponge is allowed to remain hi place for 5 minutes. The secretions were extracted by centri&gation with 300 pL of a buffer solution. The buffer solution is prepared by mixing 50 uL of 100 - protease inhibitor cocktail and 250 uL of 10% Igepal (Sigma, St Louis, MO, LI.S.A.) with 4.7 nil of phosphate-buffered saline (PBS) containing 0.25% bovine serum albumin (BSA, Fraction V; Sigma). The 100* protease iniiibitor cocktail contains 100 pg/mL aprotmiu, 500 pg/mL leupeptin, 100 pg/niL bestatin (all Sigma), and 50 ^tg mL 4-(2-Aminoethyi)- beiizenesulfonyl fluoride hydrochloride (Roche Applied Sciences, U.S.A.) in phosphate buffered saline.

The local immune response in mucosal secretions was evaluated against the homologous FIV-p24 antigen (Cell Biolabs, Inc, San Diego, CA) or the heterologous HT - gpl20 antigen (ImmunoDiagnostics, Inc. Woburn, MA). Plates were prepared with 50 iig/nxL FFV-p24 antigen or with 500 ng/mL HFV-gpl20 antigen (Bachern Americans, Inc, Torrance, CA) incubated over night at 4°C, washed and the target sample added and incubated overnight again at 4 S 'C. The plates were washed and the indicator goat anti-feline IgG (Accurate Chemical & Scientific Corp., Westbury, NY, working titer 1; 10,000) or goat anti- feline IgA (Accurate Chemical & Scientific Corp., Westbury, NY, working titer 1.1,000) added and incubated for 24 hours at 4 '3 C, Following a final wash, substrate solution was added. The stop solution was added after 15 minutes for the IgG reaction and after 30 minutes for the IgA reaction. Plates were read at 450 ma. A positive titer had a relative absorbanee >0.100 above the control sample. Results

Intramuscular injection of Fel-O-Vax FIV resulted in hig serum IgG titers against FIV-p24 antigen at levels of 1 : 10,000 to 1 :20,00ο (Table 1), These responses were durable throughout the eight week study period. Topical vaccination using Fel-O-Vax FIV alone or in combination with A(l -7) produced only low semm lgG antibody titers to FiVp24 antigen in some animals (3/8, 3S%) with no discernible dose response effect from increasing dose levels of A(i-7).

In oral secretions, intramuscular injections of Fel-O-Vax FIV induced only low levels of SlgA-FIV -p24 antibodies. SlgA-HTV-gp 120 antibodies were found in high concentrations which increased when A( 1 -7) was combined with the intramuscular injections to even higher titers (Table 2). Comparable levels of SIgA-anti-HIV-g i20 antibody titers could be achieved with topical application of the FIV 7 antigen and A(l-7). The dose response observed with increasing A(l-7) concentrations in the topical vaccine was complex with an initial raise and then decline in SIgA-anti-HIV-gp 120 antibody titers as A(l -7) concentrations increased. IgG anti-HIV-gpl 20 antibodies in oral secretions were found in much lower concentrations than the SIgA-anti-HIV-gpl20 antibodies.

SIgA antibodies were not induced in vaginal secretions by intramuscular injection of Fel- O-Vax FIV in the absence of A(l -7) [Table 3]. Topical application of FI V antigen with A( i-7) again induced substantial SIgA-anti-HIV-gpl20 antibodies. At lower dose levels of A(l-7) IgG anti-HIV-pgl20 antibodies were also elicited, but as higher dose levels of A(l -7) were employed IgG antibody induction declined. Again, only low levels of SIgA against FIV -p24 were observed.

High levels of SIgA anti-HTV-gpl20 were found with either parenteral vaccination or topical application of Fel-O-Vax FIV with A(l-7) [Table 4]. Low levels of SlgA-anti- FI V-p24 were found in both methods of vaccination. Dismssion

Vaccines that act at mucosal surfaces offer several potential advantages: 1 ) virus could be trapped and inactivated at the mucosal surface prior to spread into the host's cells, 2) locally produced immune responses (external to the host) are expected to be more effective in preventing HTV Infection since it would effectively block access to the systemic immune system and avoid the consequences ofHTV replication in immune cells within the host, 3) mucosal application of a vaccine is painless, easily accomplished and can be widely

distributed to locations with limited medical resources and 4) with A(l-7) used as an adjuvant to stimulate local immune responses it has been possible to stimulate mucosal SIgA while having mimmal effect on IgG synthesis. This latter effect may be critical since it is believed that IgG may facilitate entry of HIV into host cells and IgA (the monomelic form) may inhibit IgG antibody-dependent cellular cytotoxicity.

Our studies have demonstrated that induction of SIgA antibody production at the mucosal surface following A( i-7) + FIV immunization is sustained for the two month period of observation. It is noteworthy that each mucosal surface (vaginal, oral and rectal) responded differently to the topical application of our vaccine materials suggesting that differing dose levels of A(l-7) and antigen may be used at each mucosal site. It is interesting that parenteral Fel-O-Vax FIV and A(l-7) appears to increase trafficking of cells to the vaginal mucosal surface.

Progress toward an effectiv HTV vaccine has been stymied for a quarter century. Combining A( i-7), as an adjuvant to enhance mucosal immune function is a novel approach.

1-J Table 3: Vaginal Secretory Antibody Titers

Vaccine SIgA Anfi-FIV-p24 i SIgA Aiiti- IgG Anti- Strategy HIV gpl20 2 HI gp!20 !

Week 2 Week 4 Week 6 Week 8 Week 8 Week 8

IM 1:8 1:4 Undiluie Undilute Negative 1:9,600

IM Negative Negative Negative Negative Negative 5 Not tested

IM+ A(l- Negative Undilute Undihite 1:4 1:9,600 Not tested

Topical 1:4 Negative Undilute Negative Negative 5 Negativ only

0.1 A(l-7) Undilute Undiluie 1:16 1:4 Negative Negativ

0.2 A(l-7) Undihite 1:4 1:8 Undilute 1:4,800 1:76,800

0.3 A(l-7) 1:64 1:8 Undihite Negative 1:2,100 1:4,800

0.4 A(l-7) 1:16 1:64 1:64 1:16 1:9,600 1:2,400

5.0 A( 1-7) Negative Negative Negative Negative Negative Negative

7.5 A(l-7) Negative Negative Negative Negative 1:1,200 Negative i0.0A(l- Negative 1:16 Negative Negative 1:38,400 Negative 7)

Undiluie was the lowest dilution tested.

1:4 was the lowest, dilution tested. Elution factor 300- fold

Sample tested was from week 6.