A method of generating an HIV-specific cytotoxic T-cell response in a host involves an initial administration of a T-helper molecule to the host to prime T-helper cells of the immune system of the host and a subsequent administration to the host of a mixture of the T-helper molecule and a T-cell inducing HIV-derived molecule to generate an HIV-specific T-cell response in the host.

SUMMARY OF THE INVENTION

In accordance with one aspect of the present invention, there is provided a method of generating an HIV-specific cytotoxic T-cell (CTL) response in a host, which comprises:

administering to the host a T-helper molecule to prime T-helper cells of the immune system of the host, and

subsequently administering to the host a mixture of said T-helper molecule and a T-cell inducing HIV-derived molecule to generate an HIV-specific T-cell response in the host.

Accordingly, the immune system of the host, which may be a human host, is primed by any convenient T-helper molecule and then there is subsequently administered the T-helper molecule in admixture with a T-cell inducing molecule. In this way, an HIV-specific T-cell response is obtained.

The T-helper molecule may be any of the materials well known to provide such MHC class II-helper activity in the immune system, including T-cell human DP, DR, DQ-specific T-cell epitopes. The material used as the T-helper molecule in the experimentation described herein is a peptide which corresponds to a portion of the hepatitus B virus nucleocapsid antigen, identified as CLP-243 (SEQ ID NO: 10). The T-helper molecule may be administered with an adjuvant, if desired.

The T-cell inducing HIV-derived molecule generally includes a peptide corresponding to a portion of a HIV-1 antigen and containing at least one T-cell epitope. In particular, the peptides may correspond to sequences of the Rev protein of HIV-1, particularly corresponding to amino acids 52 to 116 (SEQ ID NO:9) (Table 2) of HIV-1 (LAI) Rev (CLP-164). The amino acid sequence of Rev protein is that of the LAI isolate. The invention includes the use of corresponding peptide sequences from Rev proteins from other HIV-1 isolates, including primary isolates.

In the experimentation described herein, the peptide was effective in the protocol described herein when provided in the form of a lipopeptide, particularly when the lipid is palmitoyl or cholesterol. Two particular lipopeptides used herein are CLP-175 and CLP-176 being the palmitoyl and cholesterol derivatives, respectively, of CLP-164.

The mixtures of the T-helper molecule and T-cell inducing HIV-derived molecule may be administered with a suitable adjuvant.

The present invention further provides, in another aspect, certain novel peptides derived from the Rev protein of HIV-1. Accordingly, in this aspect of the invention, there is provided a peptide having an amino acid sequence corresponding to amino acids 52 to 116 (SEQ ID NO:9) of the sequence of the Rev protein of HIV and containing T-cell epitopes within amino acids 65 to 75 (SEQ ID NO:3), 78 to 87 (SEQ ID NO:5) and 102 to 110 (SEQ ID NO:8) (Table 1). Such peptide may be provided in the form of a lipopeptide including CLP-175 or CLP-176. The specific amino acid sequences of the peptide having SEQ ID NO:9 is that for the LAI isolate of HIV-1. Included within the scope of the invention is the corresponding peptide and corresponding T-cell epitope sequences of the Rev protein of other HIV-1 isolates, including primary isolates.

Advantages of the present invention include: an immunization procedure to induce a T-cell response in a host immunogenic peptides for use in such procedure.

DETAILED DESCRIPTION OF INVENTION

The inventors have found that two nanomer peptides, designated CLP-177 (SEQ ID NO:2) and CLP-72 (SEQ ID NO:8), a hexamer designated CLP-178 (SEQ ID NO:3), and a 12-mer designated CLP-182 (SEQ ID NO:7) of the HIV-1 (LAI) Rev protein (the amino acid sequences of the respective peptides appear in Table 1), were individually able to bind and stabilize membrane-bound Human Major Histocompatibility Complex (HLA) class 1 molecules, HLA-A2, which is the predominant HLA class 1 subtype found in caucacians. The inventors have also found that a long peptide (SEQ ID NO:9), encompassing the amino acid residues 52 to 116 of the HIV-1 (LAI) Rev protein, and constructed by having a single cholesterol or palmitoyl moiety attached to its amino-(N-) terminus via a KSS linker to form lipopeptides, CLP-176 and CLP-175 respectively, is also capable of eliciting CTL as well as antibody responses in HLA-A2 transgenic mice.

On the basis of the experimentation provided herein, there is provided hereby a novel immunization protocol for inducing a HIV-specific cytotoxic T-cell response in a host by initial administration of a T-helper molecule to prime the immune system of the host followed by administration of a mixture of the T-helper molecule and a T-cell epitope-containing peptide corresponding to a portion of an HIV antigen.

The invention is illustrated herein by using, as the T-helper molecule, a peptide which corresponds to a portion of the hepatitus B virus nucleocapsid antigen. However, other T-helper molecules may be employed, such as those providing MHC class II-helper activity in the immune system.

The invention is illustrated herein by using, as the HIV T-cell epitope containing peptide, certain lipopeptides derived from the Rev protein. However, HIV T-cell epitope containing peptides derived from any other HIV proteins may be employed.

One model has recently been used to predict human CTL antigenic determinants on the basis of the primary sequence (see references 1 to 3, throughout this specification, various references are referred to in parenthesis to more fully describe the state of the art to which this invention pertains. Full bibliographic information for each citation is found at the end of the specification, immediately following the claims. The disclosures of these references are hereby incorporated by reference into the present disclosure). It has been proposed that CTL epitopes which are most favoured to bind and lodge into the peptide-binding groove of the human MHC class 1 molecule, such as HLA-A2, is usually 9 amino acids long. However, peptides containing 8 to 13 amino acids able to interact with HLA class 1 molecules have also been reported. In the majority of cases, these peptides are found to contain a leucine (L) or methionine (M) residue at position 2, and either L or valine (V) at their carboxy-terminal ends.

Location of the potential CTL containing motifs of the HIV-1 (LAI) Rev protein has been predicted by the reported peptide-binding motif algorithms. Table 1 shows the amino acid sequences of such predicted peptides (SEQ ID NOS: 1 to 8). The ability of the peptides containing these motifs to bind and stabilize membrane-bound HLA-A2 molecule was assessed using the T2 cell line. The cell line has been well documented to have defective TAP transporter function resulting in the majority of the intracellularly generated peptides being unable to be transported into the endoplasmic reticulum to associate with the newly synthesized HLA class 1 molecules, i.e. HLA-A2 (see references 4, 5). The majority of the HLA-A2 molecules displayed on the surface of the T2 cells are, therefore, empty (i.e. contain no peptides) and are unstable. Upon interaction with suitable peptides introduced exogenously, the stability of the HLA-A2 molecules can be restored.

The results of in vitro HLA-A2 stabilization experiments conducted herein demonstrated that two nanomers, namely, CLP-177 (SEQ ID NO:2) and CLP-72 (SEQ ID NO:8); and a 11-mer and a 12-mer represented by the peptides, namely CLP-178 (SEQ ID NO:3) and CLP-182 (SEQ ID NO:4) respectively; were capable of binding to HLA-A2 on T2 cells. This result was shown by shifting of the respective fluorescent peaks to the right due to higher density of class 1 molecules displayed on the cells, as shown in accompanying FIG. 1. A comparison of the respective fluorescence indices revealed that the potency of the peptides is in the order of CLP-177>CLP-72>CLP-178>CLP-182.

The constructions of lipidated Rev peptides which were tested are shown in Table 2. The results depicted in FIG. 2 illustrate that lipidated Rev 52 to 116 (SEQ ID NO:9) peptides, CLP-175 and CLP-176; as well as their non-lipidated counterpart, CLP-164, were immunogenic, as determined by IgG titre, when injected three times at a dose of 100.0 .mu.g into the A2 Kb transgenic mice (ref. 6). High IgG antibodies directed against the Rev 52 to 116 peptide (CLP-164) were detected in animals administered with Incomplete Freund's Adjuvant (IFA)-formulated CLP-175, or CLP-176 or CLP-164 (Panels A, B and C). Mice tested under a different experimental setting by priming them with a dose of CLP-243 in IFA, followed by boosting twice with a mixture of IFA-formulated CLP-243+CLP-175, or CLP-243+CLP-176 or CLP-243+CLP-164, similarly elicited a high anti-CLP-164 antibody response (Panels D to F). CLP-243 is an I-A.sup.b-restricted peptide encompassing the amino acids residues 128 to 140 (TPPAYRPPNAPIL; SEQ ID NO:10) of the hepatitus B virus nucleocapsid antigen (ref. 6).

The results of the immunogenicity experiments demonstrating that the lipopeptides, CLP-176 and CLP-175, were CTL-inducing are shown in FIG. 3. A2 Kb transgenic mice primed subcutaneously with a dose of the I-A.sup.b-restricted peptide, CLP-243 in IFA, and boosted twice using the same immunization route with a mixture of the priming dose of CLP-243 and either 100.0 .mu.g of CLP-176 or CLP-175 in IFA were found to generate effector cells killing the Jurkat-A2 Kb target cells pulsed with the nanomer, CLP-177 (Panels A, B, E, F). The cytotoxic activity of the effectors were specific because Jurkat A2 Kb cells not loaded with CLP-177 were not killed (Panels C, D, G and H). In contrast, the A2 Kb transgenic animals injected similarly once with the CLP-243/IFA inoculum, then twice with CLP-243 plus CLP-164 in IFA, failed to elicit a significant CLP-177-specific effector response (Panels I, J, K, L).

The results of immunization experiments demonstrating that priming with the I-A.sup.b-restricted peptide, CLP-243, followed by boosting with a mixture of CLP-243 and CLP-176 or CLP-175, was more effective than immunization with the respective lipopeptide alone for the induction of CTL response are shown in FIG. 3. It was found that splenocytes of A2 Kb transgenic mice injected 3 times subcutaneously with a dose of 100.0 .mu.g of CLP-176, or CLP-175, or CLP-164 (the non-lipidated Rev 52 116) in IFA, and re-stimulated with CLP-177 pulsed Jurkat A2 Kb cells and exogenously added CLP-175 at a concentration of 15.0 .mu.g per ml did not result in the generation of effectors capable of killing Jurkat cells pulsed with the CLP-177 peptide (Panels M to X).

The results of the in vitro re-stimulation experiments showed that the simultaneous re-stimulation of the CLP-243-specific I-A.sup.b-restricted T-helper cells achieved by the addition of the CLP-243 peptide, and the CLP-177-specific effectors achieved by co-culturing them with CLP-177-pulsed Jurkat A2 Kb cells was required to augment the enrichment of the CLP-177-specific effectors to allow their detection in the in vitro CTL assay.

The components are administered in a manner compatible with the dosage formulation, and in such amount as will be therapeutically effective, immunogenic and protective in the immunization protocol. The quantity of material to be administered depends on the subject to be treated, including, for example, the capacity of the immune system of the individual to synthesize antibodies, and to produce a cell-mediated immune response. Precise amounts of active ingredient required to be administered depend on the judgement of the practitioner. However, suitable dosage ranges are readily determinable by one skilled in the art and may be of the order of micrograms to miligrams of material. The dosage may also depend on the route of administration and will vary according to the size of the host.

Immunogenicity can be significantly improved if the antigens are co-administered with adjuvants. Adjuvants enhance the immunogenicity of an antigen but are not necessarily immunogenic themselves. Adjuvant may act by retaining the antigen locally near the site of administration to produce a depot effect facilitating a slow, sustained release of antigen to cells of the immune system. Adjuvants can also attract cells of the immune system to an antigen depot and stimulate such cells to elicit immune response.

Immunostimulatory agents or adjuvants have been used for many years to improve the host immune responses to, for example, vaccines. Intrinsic adjuvants, such as lipopolysaccharides, normally are the components of the killed or attenuated bacteria used as vaccines. Extrinsic adjuvants are immunomodulators which are typically non-covalently linked to antigens and are formulated to enhance the host immune responses. Thus, adjuvants have been identified that enhance the immune response to antigens delivered parenterally. Some of these adjuvants are toxic, however, and can cause undesirable side-effects, making them unsuitable for use in humans and many animals. Indeed, only aluminum hydroxide and aluminum phosphate (collectively commonly referred to as alum) are routinely used as adjuvants in human and veterinary vaccines. The efficacy of alum in toxoids is well established and a HBsAg vaccine has been adjuvanted with alum.

A wide range of extrinsic adjuvants can provoke potent immune responses to antigens. These include aluminum phosphate, aluminum hydroxide, QS21, Quil A, derivatives and components thereof, calcium phosphate, calcium hydroxide, zinc hydroxide, a glycolipid analog, an octodecyl ester of an amino acid, a muramyl dipeptide, polyphosphazene, a lipoprotein, ISCOM matrix, DC-Chol, DDBA, and other adjuvants and bacterial toxins, components and derivatives thereof. Particularly advantageous combination are described in copending U.S. application Ser. No. 08/258,228 filed Jun. 13, 1994 and Ser. No. 08/483,856 filed Jun. 7, 1995, assigned to the assignee hereof and the disclosure of which is incorporated herein by reference thereto (WO 95/34308). Under particular circumstances adjuvants that induce a Th1 response are desirable.
 

Claim 1 of 3 Claims

1. A method of generating an HIV-specific cytotoxic T-cell (CTL) response in a host, which comprises: administering to the host a T-helper molecule to prime T-helper cells of the immune system of the host, said T-helper molecule being CLP-243 (SEQ ID NO:10) and subsequently administering to the host a mixture of said T-helper molecule and a T-cell inducing HIV-derived molecule to generate an HIV-specific T-cell response in the host, said T-cell inducing HIV-derived molecule being a lipopeptide which is CLP-175 or CLP-176.
 

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