# US 12,109,258 B2 · Synthetic malaria immunogens, combinations thereof, and their use to prevent and treat malaria infections

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- Number: US 12,109,258 B2
- Document ID: US12109258B2
- Status: Granted
- Kind: B2
- Application number: 16/302,718
- Filed: 2017-05-19
- Published: 2019-10-10
- Granted: 2024-10-08
- Earliest claimed: 2016-05-19
- Assignees: The Trustees of the University of Pennsylvania; Ahmed S.I. Aly; Inovio Pharmaceuticals, Inc.; The Wistar Institute of Anatomy and Biology
- Applicants: The Trustees of the University of Pennsylvania; Inovio Pharmaceuticals, Inc.; The Wistar Institute of Anatomy and Biology; Ahmed S. I. Aly
- Inventors: David Weiner; Emma Reuschel; Bernadette Ferraro; Ahmed S. I. Aly
- Web page: https://patentgrep.com/patent/US12109258B2

## CPC

- A61K 39/015: Hemosporidia antigens, e.g. Plasmodium antigens
- A61P 33/06: Antimalarials
- C07K 14/445: Plasmodium
- A61K 2039/53: {DNA (RNA) vaccination}
- A61K 2039/55527: {Interleukins}
- A61K 2039/55538: {IL-12}
- C07K 2319/02: containing a signal sequence
- C07K 2319/20: containing a tag with affinity for a non-protein ligand
- C07K 2319/42: containing a HA(hemagglutinin)-tag
- C12N 2800/107: for mammalian
- C12N 2800/22: Vectors comprising a coding region that has been codon optimised for expression in a respective host
- 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

Provided herein are sequences of Plasmodium spp. liver stage exported proteins as well as expression constructs expressing the sequences. Also provided herein are methods for generating an immune response against malaria using the expression constructs provided herein.

## Claims

### Claim 1

A nucleic acid molecule comprising one or more nucleic acid sequence that encodes one or more consensus Plasmodium spp. liver stage (LS) immunogen selected from the group consisting of EXP1 (export protein 1), EXP2 (export protein 2), EXP23 (export protein 23), ICP (inhibitor of cysteine protease), TMP21 (transmembrane protein 21), UIS3 (upregulated in infective sporozoites-3), UIS10 (upregulated in infective sporozoites-10), SPECT1 (sporozoite microneme protein essential for cell traversal 1), SPECT2 (sporozoite microneme protein essential for cell traversal 2) and RON2 (rhoptry neck protein 2), wherein the nucleic acid sequence is selected from the group consisting of:
a) a nucleotide sequence encoding an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 10, SEQ ID NO: 13, SEQ ID NO:17, SEQ ID NO: 20, SEQ ID NO: 23, SEQ ID NO: 29, SEQ ID NO: 32, SEQ ID NO: 35, SEQ ID NO: 38, and SEQ ID NO: 50;
b) a nucleic acid sequence that encodes an amino acid sequence comprising a fragment comprising at least 90% of the full length of the amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 10, SEQ ID NO: 13, SEQ ID NO: 17, SEQ ID NO: 20, SEQ ID NO: 23, SEQ ID NO: 29, SEQ ID NO: 32, SEQ ID NO: 35, SEQ ID NO: 38, and SEQ ID NO: 50;
c) a nucleic acid sequence that encodes the amino acid sequence comprising at least 90% homology to the amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 10, SEQ ID NO: 13, SEQ ID NO: 17, SEQ ID NO: 20, SEQ ID NO: 23, SEQ ID NO: 29, SEQ ID NO: 32, SEQ ID NO: NO: 35, SEQ ID NO: 38, and SEQ ID NO: 50;
d) the nucleotide sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 54, and SEQ ID NO: 55;
e) a nucleotide sequence comprising a fragment comprising at least 90% of the full length of the nucleotide sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 54, and SEQ ID NO: 55; and
f) a nucleotide sequence comprising at least 90% identity to the nucleotide sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 54, and SEQ ID NO: 55.

### Claim 2 (depends on 1)

The nucleic acid molecule of claim 1, wherein the nucleic acid sequence encodes an IgE leader sequence of SEQ ID NO: 16.

### Claim 3 (depends on 1)

The nucleic acid molecule of claim 1, wherein said nucleic acid molecule is a plasmid.

### Claim 4 (depends on 1)

The nucleic acid molecule of claim 1 further comprising one or more nucleotide sequences encoding one or more malaria antigen selected from the group consisting of: CSP (circumsporozoite protein), LSA1 (liver stage antigen 1), TRAP (hrombospondin-related anonymous protein), CelTOS (cell traversal protein for Plasmodium ookinetes and sporozoites), and Amal (apical membrane antigen 1).

### Claim 5 (depends on 4)

The nucleic acid molecule of claim 4 wherein the nucleotide sequence is selected from the group consisting of: a nucleic acid sequence encoding an amino acid sequence selected from the group consisting of SEQ ID NO:56, SEQ ID NO:59, SEQ ID NO:62, and SEQ ID NO:65; and a nucleotide sequence selected from the group consisting of SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:66 and SEQ ID NO:67.

### Claim 6 (depends on 1)

An immunogenic composition comprising at least one nucleic acid molecule of claim 1.

### Claim 7 (depends on 6)

The immunogenic composition of claim 6 further comprising one or more nucleotide sequences encoding one or more malaria antigen selected from the group consisting of: CSP, LSA1, TRAP, CelTOS, and Ama1.

### Claim 8 (depends on 6)

The immunogenic composition of claim 6 further comprising a nucleotide sequences that encodes IL-12, IL-15, IL-28B, IL-33 or RANTES.

### Claim 9 (depends on 6)

The immunogenic composition of claim 6 wherein said nucleotide sequences are incorporated into one or more plasmids.

### Claim 10

A method of immunizing a mammal against malaria comprising the step of administering an immunogenic composition comprising at least one nucleic acid molecule comprising one or more nucleic acid sequence that encodes one or more consensus Plasmodium spp. liver stage (LS) immunogen selected from the group consisting of: EXP1 (export protein 1), EXP2 (export protein 2), EXP23 (export protein 23), ICP (inhibitor of cysteine protease), TMP21 (transmembrane protein 21), UIS3 (upregulated in infective sporozoites-3), UIS10 (upregulated in infective sporozoites-10), SPECT1 (sporozoite microneme protein essential for cell traversal 1), SPECT2 (sporozoite microneme protein essential for cell traversal 2) and RON2 (rhoptry neck protein 2) wherein the nucleic acid sequence is selected from the group consisting of:
a) a nucleotide sequence encoding the amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:10, SEQ ID NO:13, SEQ ID NO:17, SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:26, SEQ ID NO:29, SEQ ID NO:41, SEQ ID NO:32, SEQ ID NO:35, SEQ ID NO:38, SEQ ID NO:44, SEQ ID NO:47, SEQ ID NO:50, and SEQ ID NO:53,
b) a nucleic acid sequence that encodes an amino acid sequence comprising a fragment comprising at least 90% of the full length of the amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:10, SEQ ID NO:13, SEQ ID NO:17, SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:26, SEQ ID NO:29, SEQ ID NO:41, SEQ ID NO:32, SEQ ID NO:35, SEQ ID NO:38, SEQ ID NO:44, SEQ ID NO:47, SEQ ID NO:50, and SEQ ID NO:53,
c) a nucleic acid sequence that encodes the amino acid sequence comprising at least 90% homology to the amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:10, SEQ ID NO:13, SEQ ID NO:17, SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:26, SEQ ID NO:29, SEQ ID NO:41, SEQ ID NO:32, SEQ ID NO:35, SEQ ID NO:38, SEQ ID NO:44, SEQ ID NO:47, SEQ ID NO:50, and SEQ ID NO:53:
d) the nucleotide sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:54, and SEQ ID NO:55,
e) a nucleotide sequence comprising a fragment comprising at least 90% of the full length of the nucleotide sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:54, and SEQ ID NO:55, and
f) a nucleotide sequence comprising at least 90% identity to the nucleotide sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:54, and SEQ ID NO:55 to the tissue of the mammal.

### Claim 11 (depends on 10)

The method of claim 10 comprising the steps of:
a) administering the immunogenic composition to the tissue of the mammal, and
b) electroporating cells of the tissue with a pulse of energy at a constant current effective to permit entry of the nucleic acid molecules into the cells.

### Claim 12 (depends on 10)

The method of claim 10 wherein the immunogenic composition is administered by intramuscular or intradermal injection.