Ebola virus composition/vaccine

Abstract

The present invention is directed to a composition and/or plasmid and/or vector vaccine which encodes four fragments of the GP1 Ebola protein attached to the extracellular domain of the potent immunostimulatory protein CD40 ligand, in the configuration of two compositions and/or vaccines that are mixed together, to respectively increase the levels of antibodies and CD8 effector T cells, against the lethal Ebola virus.

Claims

1. A pharmaceutical composition comprising a mixture of at least two vectors for generating a humoral and cellular response against an Ebola virus in an individual, comprising: a first expression vector containing a first transcription unit encoding a first fusion protein comprising (a) three fragments SEQ ID NOS 1-3, (b) a first extracellular domain of a secretable CD40 ligand, (c) a linker connecting said three fragments to an amino terminal end of the extracellular domain of the secretable CD40 ligand; a second expression vector containing a second transcription unit encoding a second fusion protein comprising (a) a single fragment SEQ ID NO. 4, (b) a second extracellular domain of a secretable CD40 ligand, (c) a linker connecting said single fragment to an amino terminal end of the extracellular domain of the second CD40 ligand; and said first and second fusion proteins each configured for promoting an antibody response against the Ebola virus and a CD8 effector T cell response against the Ebola virus.

2. A pharmaceutical composition of claim 1, wherein said fragments are all from the protein pEbolaGP1.

3. A pharmaceutical composition of claim 1, wherein said composition is subcutaneously administered as a single dose or as multiple doses.

4. A pharmaceutical composition of claim 1, wherein said expression vector is a plasmid DNA or viral vector.

5. The composition of claim 1, wherein said expression vector is an adenoviral vector.

6. The composition of claim 1, wherein the size of said single fragment or said three fragments each linked to an extracellular domain of the CD40 ligand, is sufficiently small so as not to disrupt the assembly of the CD40L trimer.

7. The composition of claim 1, wherein the CD40 ligand is a human CD40 ligand.

8. The composition of claim 1, wherein each of said three fragments SEQ ID NOS. 1-3, has an epitope recognized and bound by Class I and Class II MHC, and said one fragment SEQ ID NO. 4 has an epitope recognized and bound by Class I MHC and Class II MHC.

9. The composition of claim 1, wherein said antibody response is a neutralizing antibody response.

10. A method to help protect an individual against the Ebola virus by administering to the individual one or more pharmaceutical compositions of claim 1.

Description

BRIEF DESCRIPTION OF THE DRAWING

(1) FIG. 1 illustrates an overview of TAA/ecdCD40L vaccine platform for application in infectious diseases, in which a fragment of the target associated antigen (TAA) is attached to extracellular domain (ecd) of the potent immunostimulatory antigen CD40L.

DETAILED DESCRIPTION OF THE INVENTION

(2) Location of Amino Acid Fragments of the Ebola GP1 Protein Proposed for Ebola Composition and/or Vaccine.

(3) Fragments Which Bind Antibodies Which Protect Mice Against Lethal Challenge with a Mouse Adapted Ebola Zaire Virus: Wilson et al (5) identified the three lines peptides from GP1 which bound to antibodies which protected two strains of mice (BALB/c and C57BL/6) from a lethal challenge by the Zaire strain of Ebola virus:

(4) a. Peptide 1: AA 401-417 (SEQ ID No. 1-ATQVEQHHRRTDNDSTA)

(5) b. Peptide 2: AA 389-405 (SEQ ID NO. 2-HNTPVYKLDISEATQVE)

(6) c. Peptide 3: AA 477-493 (SEQ ID NO. 3-GKLGLITNTIAGVAGLI)

(7) These three peptides primarily bind antibodies and are from the same GP1 protein, and they are each relatively short (each approximately 16 amino acids in length). These three peptides are fused together with ecdCD40L as part of a first composition and/or vaccine, whereas SEQ ID NO 4 (addressed below) is separately fused with ecdCD40L as a second composition and/or vaccine which is a relatively long amino acid listing of 60 amino acids which primarily contains contact sites for the cellular receptor.

(8) The amino acids, for example, in bold font in SEQ ID NO. 1 are considered the most important component of each peptide for binding to the protective antibodies. Each of the antibodies binding these peptides would protect mice from a lethal Ebola challenge when 100 micrograms of the antibody were administered 24 hours before challenge. The percentage of mice protected decreased as the dose of antibody was decreased to 25 micrograms. The antibody binding to peptide 2 (SEQ ID NO. 2) neutralized the infectivity of Ebola whereas the antibodies binding to peptides 1 and 3 (SEQ ID NOS. 1 and 3) did not. Although the antibodies binding to peptides 1-3 (SEQ ID NO. 1-SEQ ID NO.3) bound GP1 from both Ebola strains from Zaire, they did not bind Ebola strains from the Ivory Coast nor from Sudan. The region of GP1 from which these 3 peptides are derived is poorly conserved among Ebola strains and are not shared by any region in the soluble form of GP1. On the basis of these peptides being recognized by neutralizing antibodies to Ebola, the peptides 1-3 (SEQ ID NO. 1-SEQ ID NO. 3) are proposed to be attached to ecdCD40L to generate Composition and/or Vaccine 1.

(9) Fragment from the Region of Ebola GP1 which Contains Contact Sites for the Cellular Receptor:

(10) The amino acid regions between amino acids 54-201 has been shown to contain the base and head regions of GP1 which are thought to play a role in attachment of the Ebola virus to its cellular receptors (6-9). Amino acid residues K114, K115, K140, G 143, P146 and C147 have been proposed as important contact sites for the GP1 with its cellular receptor (6). All the amino acids in the proposed receptor binding region (AA 54-201) are highly conserved among all Ebola strains (6). Therefore, the amino acids between AA100 and AA160 (see reference 8) are proposed as the fourth peptide (SEQ ID NO. 4 below) to attach to ecdCD40L to generate Composition and/or Vaccine II. As noted above the composition and/or second vaccine II is distinct in its structure from the first composition and/or vaccine in that the first composition and/or vaccine comprises the three peptides comprising SEQ ID NOS 1-3. A mixture of these two vaccines/compositions I and II, would serve as the composition and/or vaccine, for individuals infected with and/or to prevent being infected with, the Ebola virus. Although not wishing to be bound by any theory, it is believed that the cells infected in the vicinity of the site of injection, the antigen/CD40 ligand secretory which is taken up by antigen presenting cells (e.g. DCs) in the vicinity of the infected cells would be that the fewer the compositions and/or vaccines mixed would enhance the injected particles to be taken up and connect to the CD40 port on dendritic cells.

(11) AA100-AA160 of GP1: (EAGEWAENCYNLEIKKPDGSECLPAAPDGIRGFPRCRYVHKVSGTGPCAGGFAFHKEG AFF-SEQ ID NO. 4)

(12) TAA/ecdCD40L Composition and/or Vaccine Induces High Titers of Neutralizing Antibodies.

(13) Applicant has developed a TAA/ecdCD40L vaccine platform, as illustrated in FIG. 1, for application in infectious diseases, in which a fragment of the target associated antigen (TAA) is attached to extracellular domain (ecd) of the potent immunostimulatory antigen CD40L (10-14). The attachment of the TAA to the ecdCD40L is designed to promote presentation of TAA on Class I as well as Class II MHC and provide helper function thereby overcoming the defective response to vaccination in immunosuppressed, debilitated patients including those who are of advanced chronological age, and increasing the potency of antigens which are weak immunogens to increase the titer of neutralizing antibodies induced by the vaccine (10-14). The TAA needs to be sufficiently small so that it does not disrupt the natural assembly of a homotrimeric array trimeric of the native wild type CD40 ligand. The fusion protein is engineered to be secreted from vector infected cells by the addition of a signal sequence to the amino terminal end of the TAA protein and by deletion of the transmembrane and preferably cytoplasmic domains of the CD40 ligand. The TAA/ecdCD40L composition and/or vaccine platform has shown to induce robust immune responses against 7 different antigenic targets associated with both cancer cells as well as infectious agents. A phase I clinical trial of a TAA/ecdCD40L cancer vaccine is currently under way (14). A fusion protein vaccine comprised of 2 fragments from the region of the H5N1 influenza hemagglutinin protein which binds cellular receptors attached to the ecdCD40L induced neutralizing antibody titers for influenza virus of over 1/4500 (13). The robustness of both the humoral as well as the cellular immune response induced by the TAA/ecdCD40L vaccine platform, as well as the high titers of neutralizing antibodies induced against foreign antigens on infectious agents, suggest that a TAA/ecdCD40L composition and/or vaccine which targets epitopes in the GP1 glycoprotein would be valuable both to prevent infection as well as reduce the tissue damage and mortality of individuals already infected with Ebola.

(14) TAA/ecdCD40L Composition and/or Vaccine for Ebola.

(15) For Composition/Vaccine #1, we will construct 2 expression plasmid cDNA transcription units, one comprised of a CMV promoter linked to a secretory sequence (sig) which is linked to the following three fragments (SEQ NOS 1-3) of the Ebola GP1 protein (which were described above in Section 4) attached to a 9 AA linker which is attached to the ecdCD40L: For Composition/Vaccine #2, a second expression plasmid cDNA transcription unit would be comprised of a CMV promotor linked to a secretory sequence (sig) which is linked to the fragment SEQ ID NO. 4 of the GP1 protein

(16) a. Peptide 1: AA 401-417 (SEQ ID NO. 1)

(17) b. Peptide 2: AA 389-405 (SEQ ID NO. 2)

(18) c. Peptide 3: AA 477-493 (SEQ ID NO. 3)

(19) d. Peptide 4: AA 100-160 (SEQ ID NO. 4)

(20) The plasmid expression vectors which encodes each of the two fusion proteins generated by attachment of each one of these peptides to the ecdCD40L will be designated as follows:

(21) a. Composition and/or Vaccine I: pEbolaGP1.sub.401-417/ecdCD40L, and pEbolaGP1.sub.389-405/ecdCD40L, and pEbolaGP1.sub.477-493/ecdCD40L

(22) b. Composition and/or Vaccine II: pEbolaGP1.sub.100-160/ecdCD40L

(23) These two plasmid expression vectors will be employed by administration intramuscularly (IM) as a 1:1 mixture of these two plasmids on Days 1, 7 and 21. The employment of these two plasmid compositions and/or vaccines could establish the ability of the TAA/ecdCD40L platform as an effective immunotherapeutic solution for the Ebola virus.

(24) Summary of Advantages of TAA/ecdCD40L Composition/Vaccine:

(25) Based on the pre-clinical studies of the TAA/ecdCD40L in TAA transgenic mouse models, the following advantages can be identified for the TAA/ecdCD40L vaccine strategy as compared to existing vaccine strategies: 1. The TAA/ecdCD40L composition/vaccine only requires 3 weeks to administer in order to induce a potent response due to the linkage to the ecdCD40L, thereby making it useful for use in containment of outbreaks in a local geographical area; 2. TAA/ecdCD40L overcomes anergy that may arise in patients with chronic disease (10, 12); 3. TAA/ecdCD40L induces higher titers of which are either protective or neutralizing; 4. The safety concerns with the attenuated Ebola vaccine strains will not exist with the TAA/ecdCD40L vaccine proposed above; 5. The vaccine would work better in older subjects (greater than 55 years) in which age group vaccines do not induce a fully protective immune response (12); 6. Two vector injections produce a memory response for at least a year (10); 7. Vector vaccine stable when frozen for up to 7 years.

9. REFERENCES

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