Patent Application
20170202950
The invention relates to immunogenic compositions and methods for immunogen design based on the use of peptides derived from conserved elements within sequences of antigenic proteins and, more in particular, antigenic ancestor p24 sequences from HIV.
HIV-1 vaccine design will have to contend with global HIV-1 sequence diversity. Focusing on highly conserved regions of the HIV-1 genome for vaccine immunogen designs could help to overcome this problem considerably. In addition, mounting immune responses against highly conserved segments of the genome could also prevent the rapid emergence of CTL escape variants as such mutations will likely cause significant reductions in viral replicative fitness. Furthermore, aiming vaccine-induced responses to most conserved targets would avoid inducing responses to highly mutable epitopes that serve as immunodominant decoys that actively defer responses away from protective, invariant epitopes.
Studies with diverse cohorts of clade B and clade C-infected individuals have shown that cytotoxic T-cell responses against HIV-1 Gag are correlated with relative control of HIV-1 in vivo. See Kiepiela P, et al., Nat. Med. 2007; 13:46-53; Masemola A, et al., J. Virol. 2004; 78:3233-3243; Mothe B, et al., Dis. Markers 2009; 27:105-120; and Zuiuga R, et al., J. Virol. 2006; 80:3122-3125. Several hypotheses have been put forward to explain these observations. It has been proposed that the rapid re-presentation of epitopes derived from the Gag proteins contained in the infecting virus particles may render the Gag-specific CTL response more effective than other responses. See Sacha J, et al., J. Immunol. 2007; 178:2746-2754. Alternatively, the high level of conservation of the gag sequence may also reflect the need for a maintained structural integrity of the Gag protein and its low tolerance for escape mutations. See Schneidewind A, et al., J. Virol. 2008; 82:5594-5605. It has also been argued that HIV-1 Gag may contain a high number of CTL epitopes restricted by HLA alleles associated with relative HIV-1 control (such as HLA-B27, B57 and others), although data from large cohort-based studies suggest this is quite unlikely. See Borghans J, et al., PLoS ONE 2007; 2:e920 and Kiepiela, 2007, supra. A specific accumulation of epitopes in Gag presented to these beneficial HLA alleles is also not supported by the described high degree of HLA promiscuity among well-defined CTL epitopes that allow these “good” epitopes to be presented in the context of other HLA alleles. In addition, in all studies reporting the beneficial effects of Gag-specific responses, some HIV-1-infected non-controllers mount detectable responses against Gag as well, raising the question of why these individuals are not able to control viral replication. This has even been reported for Gag-specific responses restricted by the protective HLA-B57 allele in subjects infected by HIV-1 strains with intact wildtype sequences containing the dominant and supposedly protective epitope who nevertheless do not control HIV replication. See Migueles S., et al., J. Virol. 2003; 77:6889-6898. These studies, together with previously published data on the impact of subdominant CTL responses in HIV-1 as well as in SIV infections, suggest that functional characteristics, including functional avidity and variant cross-reactivity may be determinant parameters to mount beneficial, protective CTL responses. See Frahm N., et al., Nat. Immunol. 2006; 7:173-178 and Friedrich T, et al., J. Virol. 2007; 81:3465-3476.
Also, our understanding of the host immune response to any portion of the viral proteome may be incomplete due to technical limitations and the use of antigenic test reagents may also introduce specific bias. Although earlier studies using overlapping peptide (OLP) sets of variable length (15-20 amino acids in length) did not produce significantly different response rates, our own observations in HIV-1 infected individuals using 18mer OLP or optimally defined CTL epitopes, indicated that short 10mer peptides would provide a much more realistic view of the total responses than longer OLP. See Draenert R, et al., J. Immunol. Methods 2003; 275:19-29 and Frahm, 2007, supra. Shorter peptides might require less antigen processing before being effectively presented by HLA class I molecules and may thus identify more responses and responses of lower functional avidity than the corresponding 18mer OLP sets generally used in ex-vivo analyses.
The present invention relates to a prototype vaccine immunogen, experimentally validated, which includes the most conserved regions of HIV-1 p24, referred to as “conserved elements” (CE). The immune reactivity against these regions and against the rest of HIV-1 Gag p24 or of the remainder of the proteome in HIV-1 has been compared in individuals that control HIV well (“HIV-controllers”) and subjects that poorly control their viral replication (“HIV non-controllers”). The design of the CE sequences was based on the identification of regions in the HIV-1 proteome with typically at least 98% sequence conservation across all independent group M sequences at the Los Alamos HIV database. See www.hiv.lanl.gov/content/index, September 2011. For HIV-1 Gag p24, studied as a proof-of-concept protein, this produced 7 segments, ranging from 12 to 24 amino acids in length, corresponding to a total of 124 residues of p24. A side-by-side comparison of 10mer versus 18mer peptides for Gag p24 was conducted, revealing 2-3 times higher response rates when using the shorter peptide test sets.
The results show that when using sensitive peptide test sets, the same overall breadth and magnitude of the CTL responses to Gag p24 can be detected equally by HIV-1 controllers and non-controllers. However, high avidity and cross-reactive responses against Gag p24 are significantly enriched in HIV-1 controllers. These data suggest that HIV-1 non-controllers are unable to induce or maintain responses of desirable functionality to these targets. The results also present functional confirmation of the potential usefulness of this novel CE-based HIV-1 vaccine immunogen approach that can readily be expanded to the rest of the viral genome and which could provide immunity against heterologous viral challenge.
A. Immunogenic Peptides of the Invention
Thus, in a first aspect, the invention relates to a peptide having the capacity to induce HIV-1-specific cytotoxic T lymphocytes (CTL), the sequence of which comprises part or all of a conserved element within a Center-of-Tree (COT) sequence derived from a family of polypeptides encoded by naturally occurring variants of HIV-1.
The term “peptide”, as used herein, refers to a sequence of amino acids, analogues or mimetics having substantially similar or identical functionality. The term “peptide” also includes analogues having synthetic and natural amino acids joined together by peptide bonds.
A cytotoxic T lymphocyte assay can be used to monitor the cellular immune response following subgenomic immunization with a viral sequence against homologous and heterologous HIV strains. See Burke S, et al., J. Inf. Dis. 1994; 170:1110-1119 and Tigges M, et al., J. Immunol, 1996; 156:3901-3910. Conventional assays utilized to detect T cell responses include, for instance, proliferation assays, lymphokine secretion assays, direct cytotoxicity assays and limiting dilution assays. For example, antigen-presenting cells that have been incubated with a peptide can be assayed for their ability to induce CTL responses in responder cell populations. Antigen-presenting cells can be cells such as peripheral blood mononuclear cells (PBMC) or dendritic cells (DC). Alternatively, mutant non-human mammalian cell lines that are deficient in their ability to load MHC class I molecules with internally processed peptides and that have been transfected with the appropriate human MHC class I gene, can be used to test the capacity of a peptide of interest to induce in vitro primary CTL responses.
PBMC can be used as the responder cell source of CTL precursors. The appropriate antigen-presenting cells are incubated with the peptide after which the protein-loaded antigen-presenting cells are incubated with the responder cell population under optimized culture conditions. Positive CTL activation can be determined by assaying the culture for the presence of CTL that kill radiolabeled target cells, both specific peptide-pulsed targets as well as target cells expressing endogenously processed forms of the antigen from which the peptide sequence was derived. For example, the target cells can be radiolabeled with 51Cr and cytotoxic activity can be calculated from radioactivity released from the target cells. Another suitable method allows the direct quantification of antigen-specific T cells by staining with Fluorescein-labeled HLA tetrameric complexes. See Altman J, et al., Proc. Natl. Acad. Sci. USA 1993; 90:10330-10334 and Altman J, et al., Science 1996; 274:94-96. Other relatively recent technical developments include staining for intracellular lymphokines and interferon release assays or ELISpot assays.
The term “conserved element”, as used herein, refers to a region of a sequence which is substantially constant among all related variant sequences when said sequences are compared. For sequence comparison, typically one sequence acts as a reference sequence to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are typically input into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters.
In a preferred embodiment, the sequence is considered as being a conserved element if it shows at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 995 or 100% identity with respect to reference sequence.
Optimal alignment of sequences for comparison can be conducted, for instance, by the Smith-Waterman local homology algorithm, by the Needleman-Wunsch homology alignment algorithm, by the Pearson-Lipman similarity search method, by computerized implementations of these algorithms or by manual alignment and visual inspection. See Smith T, Waterman M, Adv. Appl. Math. 1981; 2:482-489; Needleman S, Wunsch C, J. Mol. Biol. 1970; 48:443-453; Pearson W, Lipman D, Proc. Natl. Acad. Sci. USA 1988; 85:2444-2448; the GAP, BESTFIT, FASTA and TFASTA programs, Wisconsin Genetics Software Package, Genetics Computer Group, Madison, Wis., USA; Ausubel F, et al., Eds., “Short Protocols in Molecular Biology”, 5th Ed. (John Wiley and Sons, Inc., New York, N.Y., USA, 2002).
One example of a useful algorithm is PILEUP. This program creates a multiple sequence alignment from a group of related sequences using progressive, pairwise alignments to show relationship and percent sequence identity. It also plots a tree or dendogram showing the clustering relationships used to create the alignment. See Feng D, Doolittle R, J. Mol. Evol. 1987; 35:351-360. The method is similar to the CLUSTAL algorithm. See Higgins D, Sharp P, Gene 1998; 73:237-244 and CABIOS 1989; 5:151-153. The program can align up to 300 sequences, each of a maximum length of 5,000 nucleotides or amino acids. The multiple alignment procedure begins with the pairwise alignment of the two most similar sequences, producing a cluster of two aligned sequences. This cluster is then aligned to the next most related sequence or cluster of aligned sequences. Two clusters of sequences are aligned by a simple extension of the pairwise alignment of two individual sequences. The final alignment is achieved by a series of progressive, pairwise alignments. The program is run by designating specific sequences and their amino acid or nucleotide coordinates for regions of sequence comparison and by designating the program parameters. For example, a reference sequence can be compared to other test sequences to determine the percent sequence identity relationship using the following parameters: default gap weight (3.00), default gap length weight (0.10), and weighted end gaps.
Other examples of algorithms suitable for determining percent sequence identity and sequence similarity are BLAST and BLAST 2.0 algorithms See Altschul S, et al., Nuc. Acids Res. 1977; 25:3389-3402 and Altschul S, et al., J. Mol. Biol. 1990; 215:403-410. The BLAST and BLAST 2.0 programs are used, with the parameters described herein, to determine percent sequence identity for the nucleic acids and proteins of the invention. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information. See blast.ncbi.nlm nih.gov/blast.cgi, September 2011. This algorithm involves first identifying high scoring sequence pairs (HSPs) through the recognition of short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold. See Altschul, 1997, 1990, supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a word length (W) of 11, an expectation (E) of 10, M=5, N=4 and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a word length of 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix alignments (B) of 50, expectation (E) of 10, M=5, N4, and a comparison of both strands. See Henikoff S, Henikoff J, Proc. Natl. Acad. Sci. USA 1989; 89:10915-10919. The BLAST algorithm also performs a statistical analysis of the similarity between two sequences. See Karlin S, Altschul S, Proc. Natl. Acad. Sci. USA 1993; 90:5873-5787. One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.2, more preferably less than about 0.01. The term “ancestral sequence”, as used herein, refers to a sequence that is at the base node of the maximum likelihood phylogeny of a family of variant sequences. The ancestral sequence is thus approximately equidistant from the different sequences.
The term “Center-of-Tree sequence” or “COT”, as used herein, refers to a sequence from which the average evolutionary distance to each tip of a phylogenetic diagram of related variant sequences has been minimized See Nickle D, et al., Science 2003; 299, 1515-1517.
Typically, the family of variant polypeptides correspond to polypeptides encoded by the genomes of viruses that exist as highly diverse viral populations. However, some highly diverse viruses such as FIV, hepatitis C and endogenous retroviruses like PERV, do not appear to evolve across their host populations through a succession of variants, where one prototypical strain is replaced by successive uniform strains. Instead, an evolutionary tree of viral sequences can form a “star-burst pattern”, with most of the variants approximately equidistant from the center of the star-burst. This star-burst pattern indicates that multiple, diverse circulating strains evolve from a common ancestor. Computational methods can be used to determine ancestral sequences for highly diverse viruses, such as, for example, FIV, HIV-2, hepatitis C and endogenous retroviruses.
Methods for the determination of the COT sequence from a family of sequences are described in the art. See Rolland M, et al., J. Virol. 2007; 81:8507-8514, Mullins J, et al., U.S. Pat. No. 7,655,744 and WO2005/001029. Typically, the determination of the COT or ancestral sequences is carried out by computational methods from the nucleic acid sequences of circulating viruses based on the principle of maximum likelihood. In a preferred embodiment, the sequences are nucleic acid sequences of circulating viruses. The sequences of the viruses in the samples typically share a common feature, such as being from the same viral strain, subtype or group. A phylogeny is constructed by using a model of evolution that specifies the probabilities of nucleotide substitutions in the replicating viral nucleic acids. At positions in the sequences where the nucleotides differ (i.e. at the site of a mutation), the methodology assigns one of the nucleotides to the node (i.e. the branch point of the lineages) such that the probability of obtaining the observed viral sequences is maximized. The assignment of nucleotides to the nodes is based on the predicted phylogeny or phylogenies. For each data set, several sequences from a different viral strain, subtype or group are used as an outgroup to root the sequences of interest. A model of sequence substitutions and then a maximum likelihood phylogeny are determined for each data set (e.g. subtype and outgroup). The maximum likelihood phylogeny is the one that has the highest probability of giving the observed nucleic acid sequences in the samples. The sequence at the base node of the maximum likelihood phylogeny is referred to as the ancestral sequence or most recent common ancestor.
Alternatively, a second method is to choose, for a given nucleotide site and a given node on the tree, the nucleotide that maximizes the probability of obtaining the observed sequences of circulating viruses, allowing for all possible assignments of nucleotides at the other nodes on the tree. This second method maximizes the marginal likelihood of a particular assignment. For these methods, the reconstruction of the ancestral sequence (i.e. ancestral state) need not result in only a single determined sequence, however. It is possible to choose a number of ancestral sequences, ranked in order of their likelihood.
Alternatively, in the case of HIV populations, a second layer of modeling can be added to the maximum likelihood phylogenetic analysis, in particular the layer is added to the model of evolution that is employed in the analysis. This second layer is based on coalescent likelihood analysis. The coalescent is a mathematical description of a genealogy of sequences, taking account of the processes that act on the population. If these processes are known with some certainty, the use of the coalescent can be used to assign prior probabilities to each type of tree. Taken together with the likelihood of the tree, the posterior probability can be determined that a determined phylogenetic tree is correct given the data. Once a tree is chosen, the ancestral states are determined, as described above. Thus, coalescent likelihood analysis can also be applied to determine the sequence of an ancestral viral sequence (e.g. a founder, or Most Recent Common Ancestor (MRCA), sequence).
In a typical embodiment, maximum likelihood phylogeny analysis is applied to determine a COT or an ancestor sequence (e.g. an ancestral viral sequence). Typically, between 20 and 1000 nucleic acid sequence samples are used that have a common feature, such as a viral strain, subtype or group (e.g. samples encompassing a worldwide diversity of the same subtype). Additional sequences from other viruses (e.g. another strain, subtype, or group) are obtained and used as an outgroup to root the viral sequences being analyzed. The samples of viral sequences are determined from presently circulating or endogenous viruses, identified from the database (e.g. GenBank and Los Alamos HIV sequence databases), or from similar sources of sequence information. The sequences are aligned using the CLUSTALW algorithm. See Thompson J., et al., Nucleic Acids Res. 1994, 22:4673-4680. These alignments are refined using GDE. See Smith J, et al., CABIOS 1994; 10:671-675. The amino acid sequences are also translated from the nucleic acid sequences. Gaps are manipulated so that they are inserted between codons.
The sequence of the peptides of the invention comprises part or all of the conserved elements within the COT or, in order to increase sequence diversity coverage, may further include second most frequent variants for each conserved element. The inclusion of such frequent “toggled” amino acids increases coverage at those sites among all group M sequences to >99%.
In a preferred embodiment, the sequence of the peptides of the invention have a length of 10 amino acids of less, including 9 amino acids, 8 amino acids, 7 amino acids, 6 amino acids, 5 amino acids or less.
The term “polypeptide”, as used herein, refers to a polymer in which the monomers are amino acids and are joined together through peptide or disulphide bonds. The term includes post-translational modifications of the polypeptide, for example, glycosylations, acetylations, and phosphorylations.
The acronym “HIV” is used herein to refer to human immunodeficiency viruses generically and includes HIV type 1 (HIV-1), HIV type 2 (HIV-2) or other HIV viruses, including, for example, the HIV-1, HIV-2, emerging HIV and other HIV subtypes and HIV variants, such as widely dispersed or geographically isolated variants. For example, an ancestral viral gene sequence can be determined for the env and gag genes of HIV-1, such as for HIV-1 subtypes A, B, C, D, E, F, G, H, J, and K, and intersubtype recombinants such as AG, AGI, and for groups M, N, O or for HIV-2 viruses or HIV-2 subtypes A or B. In specific embodiments, ancestral viral sequences are determined for the env genes of HIV-1 subtypes B and/or C, or for the gag genes of subtypes B and/or C. In other embodiments, the ancestral viral sequence is determined for other HIV genes or polypeptides, such as pol or the auxiliary genes or polypeptides.
In a preferred embodiment, the HIV is a group M HIV. Group M is the predominant circulating HIV-1 group. It has been divided into subtypes, denoted with letters, and sub-subtypes, denoted with numerals. Subtypes A1, A2, A3, A4, B, C, D, E, F1, F2, G, H, J, and K are currently recognized. HIV-1 subtypes, also called clades, are phylogenetically linked strains of HIV-1 that are approximately the same genetic distance from one another; in some cases, subtypes are also linked geographically or epidemiologically. Genetic variation within a subtype can be 15 to 20 percent or more, whereas variation between subtypes or divergent members of the same subtype is usually 25 to 35 percent. Over the past decade, advances in full-genome sequencing of HIV have led to the identification of circulating and unique recombinant forms (CRFs and URFs, respectively). These are the result of recombination between subtypes within a dually infected person, from whom the recombinant forms are then passed to other people. The recombinant progeny are classified as circulating recombinant forms if they are identified in three or more people with no direct epidemiologic linkage; otherwise they are described as unique recombinant forms.
The term “naturally occurring variants”, as used herein, refers to nucleic acid sequences of a selected HIV-1 or HIV-2 gene which can be used for the identification of the ancestral or COT sequences may derive from presently and/or formerly circulating viruses and can be identified from existing databases (e.g. GenBank and Los Alamos sequence databases). The sequence of circulating viruses can also be determined by molecular biology methodologies. See Brown T, “Gene Cloning” (Chapman & Hall, London, England, 1995); Watson R, et al., “Recombinant DNA”, 2nd Ed. (Scientific American Books, New York, N.Y., USA, 1992); Alberts B, et al., “Molecular Biology of the Cell” (Garland Publishing Inc., New York, N.Y., USA, 2008); Innis M, et al., Eds., “PCR Protocols. A Guide to Methods and Applications” (Academic Press Inc., San Diego, Calif., USA, 1990); Erlich H, Ed., “PCR Technology. Principles and Applications for DNA Amplification” (Stockton Press, New York, N.Y., USA, 1989); Sambrook J, et al., “Molecular Cloning. A Laboratory Manual” (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., USA, 1989); Bishop T, et al., “Nucleic Acid and Protein Sequence. A Practical Approach” (IRL Press, Oxford, England, 1987); Reznikoff W, Ed., “Maximizing Gene Expression” (Butterworths Publishers, Stoneham, Mass., USA, 1987); Davis L, et al., “Basic Methods in Molecular Biology” (Elsevier Science Publishing Co., New York, N.Y., USA, 1986), Schleef M, Ed., “Plasmid for Therapy and Vaccination” (Wiley-VCH Verlag GmbH, Weinheim, Germany 2001).
The term “HIV gene” refers to any sequence within the HIV genome that contains information necessary for expression of a polypeptide or protein and may include non-translated sequences. The term is also intended to include any combination of gene(s), gene fragment(s), non-transcribed sequence(s) or non-translated sequence(s) that are present on the same DNA molecule. Thus, the term “gene”, as sometimes used generically, can also include nucleic acid molecules comprising cDNA and cDNA clones.
Non-limiting examples of HIV proteins that may be suitable for use in the embodiments presented herein include the HIV Gag proteins p53, p24, p17, p7, p6, p2 or p1, the HIV Env glycoproteins gp120, gp41 or gp160, HIV enzymes including integrase (p31), reverse transcriptase (p51 or p66), RNase H (p15), protease (p10), the HIV Nef proteins (p25/p27), the HIV Vif protein p23, the HIV Rev protein p19, the HIV Vpr protein (p12/p10), HIV Vpu protein (p16) or HIV Tat proteins (p16/p14).
In a preferred embodiment, the HIV gene is the gag gene. The term “gag gene”, as used herein, refers to a gene that codes for the Gag polyprotein, which is processed during maturation to MA (matrix protein, p17); CA (capsid protein, p24); SP1 (spacer peptide 1, p2); NC (nucleocapsid protein, p7); SP2 (spacer peptide 2, p1) and p6. Sequences of gag genes found in HIV isolates can be readily found (e.g. GenBank and Los Alamos HIV sequence databases).
In a still more preferred embodiment, a polypeptide encoded by the gag gene is p24. As used herein, the term “p24” refers to the gene product of the gag region of HIV, characterized as having an apparent relative molecular weight of about 24,000 daltons designated p24 and which corresponds to the capsid protein of the HIV virion. The term “p24” also refers to modifications and fragments of p24 having the immunological activity of p24.
In a preferred embodiment, the sequences of conserved elements within p24 sequences are selected from the group consisting of the sequences as defined in Table 1.
In a preferred embodiment, the peptide of the invention has a length of 10 amino acids or less. In a still more preferred embodiment, the peptide according to the present invention is selected from the group consisting of the peptides shown in Table 2.
B. Polynucleotides and Vectors
In another aspect, the invention relates to a polynucleotide encoding a peptide according to the invention.
The term “polynucleotide”, as used herein, refers to single-stranded or double-stranded polymers of nucleotide monomers (nucleic acids), including, but not limited to, 2′-deoxyribonucleotides (DNA) and ribonucleotides (RNA) linked by internucleotide phosphodiester bond linkages. It will be understood that the polynucleotides of the invention encode the peptides of the invention without substantially comprising additional regions of the HIV genome. Thus, in the case wherein the peptides of the invention derive from conserved elements within the p24 COT sequence, the polynucleotides of the invention contain those sequences encoded said conserved elements without including additional p24 sequences.
In another aspect, the invention relates to a vector comprising a polynucleotide as defined in the invention.
The term “vector” is used to denote a nucleic acid molecule, linear or circular, that comprises a segment encoding the peptide of interest operably linked to additional segments that provide for its autonomous replication in a host cell of interest. Preferably, the vector is an expression vector, which is defined as a vector, which in addition to the regions of the autonomous replication in a host cell, contains regions operably linked to the polynucleotide of the invention and which are capable of enhancing the expression of the peptide according to the invention.
Thus, suitable vectors according to the present invention include prokaryotic vectors, such as pUC18, pUC19, and Bluescript plasmids and derivatives thereof, like the mp18, mp19, pBR322, pMB9, ColE1, pCR1 and RP4 plasmids; phages and shuttle vectors, such as pSA3 and pAT28 vectors; expression vectors in yeasts, such as 2-micron plasmid type vectors; integration plasmids; YEP vectors; centromeric plasmids and analogues; expression vectors in insect cells, such as the vectors of the pAC series and of the pVL series; expression vectors in plants, such as vectors of the pIBI, pEarleyGate, pAVA, pCAMBIA, pGSA, pGWB, pMDC, pMY, pORE series and analogues; and expression vectors in superior eukaryotic cells either based on viral vectors (e.g. adenoviruses, viruses associated to adenoviruses, retroviruses and lentiviruses) as well as non-viral vectors, such as the pSilencer 4.1-CMV (Ambion), pcDNA3, pcDNA3.1/hyg pHCMV/Zeo, pCR3.1, pEF1/His, pIND/GS, pRc/HCMV2, pSV40/Zeo2, pTRACER-HCMV, pUB6N5-His, pVAX1, pZeoSV2, pCI, pSVL and pKSV-10, pBPV-1, pML2d and pTDT1 vectors.
C. Immunogenic Compositions and Therapeutic Uses Thereof
The results provided in the present invention identify promising immunogenic sequences for a broadly applicable HIV-1 vaccine that covers global HIV-1 diversity. Thus, in another aspect, the invention relates to an immunogenic composition or a vaccine comprising a peptide, a nucleic acid or a vector according to the invention.
The term “immunogenic composition” refers to a composition that elicits an immune response that produces antibodies or cell-mediated immune responses against a specific immunogen. Immunogenic compositions can be prepared, for instance, as injectables such as liquid solutions, suspensions, and emulsions. The term “antigenic composition” refers to a composition that can be recognized by a host immune system. For example, an antigenic composition contains epitopes that can be recognized by humoral and/or cellular components of a host immune system.
The term “vaccine” refers to an immunogenic composition for in vivo administration to a host, which may be a primate, especially a human host, to confer protection against a disease, particularly a viral disease.
In a further aspect, the invention relates to a peptide, a nucleic acid, a vector or an immunogenic composition according to the invention for use in medicine.
In a preferred embodiment, the invention relates to a pharmaceutical composition comprising a peptide, nucleic acid or vector according to the invention and a pharmaceutically acceptable carrier.
A “pharmaceutically acceptable carrier,” “pharmaceutically acceptable diluent,” or “pharmaceutically acceptable excipient”, or “pharmaceutically acceptable vehicle,” used interchangeably herein, refer to a non-toxic solid, semisolid or liquid filler, diluent, encapsulating material or formulation auxiliary of any conventional type. A pharmaceutically acceptable carrier is essentially non-toxic to recipients at the employed dosages and concentrations and is compatible with other ingredients of the formulation. For example, the carrier for a formulation containing polypeptides would not normally include oxidizing agents and other compounds that are known to be deleterious to polypeptides. Suitable carriers include, but are not limited to water, dextrose, glycerol, saline, ethanol, and combinations thereof. The carrier can contain additional agents such as wetting or emulsifying agents, pH buffering agents, or adjuvants that enhance the effectiveness of the formulation. Adjuvants could for example be selected from the group consisting of: AlK(SO4)2, AlNa(SO4)2, AlNH4 (SO4), silica, alum, Al(OH)3, Ca3(PO4)2, kaolin, carbon, aluminum hydroxide, muramyl dipeptides, N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-DMP), N-acetyl-nornuramyl-L-alanyl-D-isoglutamine (CGP 11687, also referred to as nor-MDP), N-acetylmuramyul-L-alanyl-D-isoglutaminyl-L-alanine-2-(1 ‘2’-dipalmitoyl-sn-glycero-3-hydroxphosphoryloxy)-ethylamine (CGP 19835A, also referred to as MTP-PE), RIBI (MPL+TDM+CWS) in a 2 percent squalene/TWEEN® 80 emulsion, lipopolysaccharides and its various derivatives, including lipid A, Freund's Complete Adjuvant (FCA), Freund's Incomplete Adjuvants, Merck Adjuvant 65, polynucleotides (e.g. poly IC and poly AU acids), wax D from Mycobacterium tuberculosis, substances found in Corynebacterium parvum, Bordetella pertussis, and members of the genus Brucella, Titermax, ISCOMS, Quil A, ALUN, Lipid A derivatives, choleratoxin derivatives, HSP derivatives, LPS derivatives, synthetic peptide matrixes or GMDP, interleukin 1, interleukin 2, Montanide ISA-51 and QS-21, CpG oligonucleotide, poly I:C, and IL-12, IL-15 and/or GM-CSF. See Hunter R, U.S. Pat. No. 5,554,372 and Jager E, Knuth A, WO1997/28816.
In a further aspect, the invention relates to a peptide, a nucleic acid, a vector, an immunogenic composition or a vaccine according to the invention for use in the treatment or prevention of a disease resulting from HIV-infection. Alternatively, the invention relates to the use of a peptide, a nucleic acid, a vector, an immunogenic composition or a vaccine according to the invention for the manufacture of a medicament for the treatment or prevention of a disease resulting from HIV-infection. Alternatively, the invention relates to a method for the treatment or prevention in a subject of a disease resulting from HIV-infection that comprises the administration to said subject of a peptide, nucleic acid, vector, or immunogenic composition or a vaccine according to the invention.
The term “treat” or “treatment” is used to designate the administration of an immunogenic composition of the invention or of a medicament containing it to control the progression of the disease before or after clinical signs have appeared. Control of the disease progression is understood to mean the beneficial or desired clinical results that include, but are not limited to, reduction of the symptoms, reduction of the duration of the disease, stabilization of pathological states (specifically to avoid additional deterioration), delaying the progression of the disease, improving the pathological state and remission (both partial and total). The control of progression of the disease also involves an extension of survival, compared with the expected survival if treatment was not applied.
The expression “disease associated with a HIV infection” includes a state in which the subject has developed AIDS, but also includes a state in which the subject infected with HIV has not shown any sign or symptom of the disease. Thus, the immunogenic compositions of the invention when administered to a subject that has no clinical signs of the infection can have a preventive activity, since they can prevent the onset of the disease.
The immunogenic compositions are capable of preventing or slowing the infection and destruction of healthy CD4+ T cells in such a subject. It also refers to the prevention and slowing the onset of symptoms of the acquired immunodeficiency disease such as extreme low CD4+ T cell count and repeated infections by opportunistic pathogens such as Mycobacteria spp., Pneumocystis carinii, and Pneumocystis cryptococcus. Beneficial or desired clinical results include, but are not limited to, an increase in absolute naïve CD4+ T-cell count (range 10-3520), an increase in the percentage of CD4+ T-cell over total circulating immune cells (range 1-50 percent), and/or an increase in CD4+ T-cell count as a percentage of normal CD4+ T-cell count in an uninfected subject (range 1-161 percent). “Treatment” can also mean prolonging survival of the infected subject as compared to expected survival if the subject did not receive any HIV targeted treatment.
The beneficial prophylactic or therapeutic effect of an HIV immunogenic composition in relation to HIV infection or AIDS symptoms include, for example, preventing or delaying initial infection of an individual exposed to HIV; reducing viral burden in an individual infected with HIV; prolonging the asymptomatic phase of HIV infection; maintaining low viral loads in HIV infected patients whose virus levels have been lowered via anti-retroviral therapy (ART); increasing levels of CD4 T cells or lessening the decrease in CD4 T cells, both HIV-1 specific and non-specific, in drug naive patients and in patients treated with ART, increasing overall health or quality of life in an individual with AIDS; and prolonging life expectancy of an individual with AIDS. A clinician can compare the effect of immunization with the patient's condition prior to treatment, or with the expected condition of an untreated patient, to determine whether the treatment is effective in inhibiting AIDS.
As used herein, “AIDS” refers to the symptomatic phase of HIV infection, and includes both Acquired Immune Deficiency Syndrome (commonly known as AIDS) and “ARC,” or AIDS-Related Complex, as described by Adler, Brit. Med. J. 294: 1145 (1987). The immunological and clinical manifestations of AIDS are well known in the art and include, for example, opportunistic infections and cancers resulting from immune deficiency.
In a preferred embodiment, the immunogenic compositions of the invention are preventive compositions. “Prevention” is understood to mean the administration of an immunogenic composition of the invention or of a medicament containing it in a initial or early stage of the infection, to avoid the appearance of clinical signs.
The immunogenic compositions of the invention may be useful for the therapy of HIV-1 infection. While all animals that can be afflicted with HIV-1 or their equivalents can be treated in this manner (e.g. chimpanzees, macaques, baboons or humans), the immunogenic compositions of the invention are directed particularly to their therapeutic uses in humans. Often, more than one administration may be required to bring about the desired therapeutic effect; the exact protocol (dosage and frequency) can be established by standard clinical procedures.
The present invention further relates to preventing or reducing symptoms associated with HIV infection. These include symptoms associated with the minor symptomatic phase of HIV infection, including, for example, shingles, skin rash and nail infections, mouth sores, recurrent nose and throat infection and weight loss. In addition, further symptoms associated with the major symptomatic phase of HIV infection, include, for instance, oral and vaginal thrush (Candida), persistent diarrhea, weight loss, persistent cough and reactivated tuberculosis or recurrent herpes infections, such as cold sores (herpes simplex). Other symptoms of full-blown AIDS which can be treated in accordance with the present invention include, for instance, diarrhoea, nausea and vomiting, thrush and mouth sores, persistent, recurrent vaginal infections and cervical cancer, persistent generalized lymphadenopathy (PGL), severe skin infections, warts and ringworm, respiratory infections, pneumonia, especially Pneumocystis carinii pneumonia (PCP), herpes zoster (or shingles), nervous system problems, such as pains, numbness or “pins and needles” in the hands and feet, neurological abnormalities, Kaposi's sarcoma, lymphoma, tuberculosis or other similar opportunistic infections.
Beneficial effects of the peptides, nucleic acids and vectors of the invention include, for example, preventing or delaying initial infection of an individual exposed to HIV, reducing viral burden in an individual infected with HIV, prolonging the asymptomatic phase of HIV infection, maintaining low viral loads in HIV infected patients whose virus levels have been lowered via anti-retroviral therapy (ART), increasing levels of CD4 T cells or lessening the decrease in CD4 T cells, both HIV-1 specific and non-specific, in drug naïve patients and in patients treated with ART, increasing overall health or quality of life in an individual with AIDS and prolonging life expectancy of an individual with AIDS. A clinician can compare the effect of immunization with the patient's condition prior to treatment, or with the expected condition of an untreated patient, or in a clinical trial of individuals treated and untreated with the vaccine to determine whether the treatment is effective in inhibiting AIDS.
D. Antibodies of the Invention
In another aspect, the invention relates to an antibody or a polypeptide comprising an antigen-binding region thereof that binds specifically to a peptide according to the invention.
The term “antibody”, as used herein, refers to a protein consisting of one or more proteins substantially encoded by all or part of the recognized immunoglobulin genes, including but not limited to polyclonal antibodies, monoclonal antibodies, and antigen-binding fragments thereof such as, for instance, F(ab′)2 and Fab fragments, and single chain antibodies. The term antibody includes any type of known antibody, such as, for example, polyclonal antibodies, monoclonal antibodies and genetically engineered antibodies, such as chimeric antibodies, humanized antibodies, primatized antibodies, human antibodies and bispecific antibodies.
“Chimeric antibodies” are understood as antibodies constructed with variable regions of an antibody of a species (usually a mammal in which the monoclonal antibody was generated) and constant regions of another species (that species in which the chimeric antibody is going to be used). The objective of said construct is to obtain an antibody with the original monoclonal antibody but which is less immunogenic and better tolerated in the subject who is going to be treated, with an improved serum half-life and which can be recognized by immunological effector mechanisms, i.e., the complement, the Fc receptor of cytotoxic cells or other specific immunoglobulin receptors which show species specificity. In a preferred embodiment, the chimeric antibodies are formed by murine variable regions and human constant regions.
“Humanized antibody” is understood as an antibody from a non-human organism, typically a murine antibody, which conserves the antigen binding properties of the parent antibody, but which is less immunogenic in human beings. This can be achieved by means of different processes, which include (a) grafting the complete nonhuman variable domains into human constant regions to generate chimeric antibodies; (b) grating only the nonhuman complementarity determining regions (CDR) in a human framework and the constant regions, with or without retaining the critical framework residues; and (c) transplanting the complete nonhuman variable domains, but “concealing them” with a section similar to the human variable domain by means of replacing the surface residues.
“Primatized antibody” is understood as a recombinant antibody that has been genetically manipulated to contain the heavy and light variable domains of a monkey antibody (or of another primate), particularly an antibody of a cynomolgus monkey, and containing sequences of a human constant domain, preferably the constant domain of human gamma 1 or 4 immunoglobulin (or a PE variant). The preparation of said antibodies is described in Newman et al., Biotechnology, 10: 1458-1460 (1992); and in patent documents U.S. Pat. No. 5,658,570 and U.S. Pat. No. 6,113,898. It has been described that these antibodies show a high degree of homology with human antibodies, i.e., 85-98%, they have human effector functions, they have lower immunogenicity and can show a high affinity for human antigens. Another very effective means for generating recombinant antibodies is described by Newman, Biotechnology, 10: 1455-1460 (1992).
“Human antibody” is understood as an antibody integrally containing human light and heavy chains as well as constant regions, produced by means of any of the known standard methods. A more extensive definition is found on the “Definitions” section.
The invention also comprises the use of fragments of the different types of antibodies mentioned above which substantially preserve the anti-angiogenic activity of the antibody. The term “antibody fragment” includes antibody fragments such as Fab, F(ab′)2, Fab′, single chain Fv fragments (scFv), diabodies and nanobodies.
Papain digestion of antibodies produces two identical antigen binding fragments referred to as “Fab” fragments, each with a single antigen binding site, and a residual “Fc” fragment, the name of which reflects its capacity for readily crystallizing. Pepsin treatment yields an F(ab′)2 fragment which has two antigen binding sites and which is still capable of cross-linking to the antigen.
“Fv” is the minimal antibody fragment containing a complete antigen binding and antigen recognition site. This region consists of a variable domain of a variable light chain and heavy chain dimer in a strong noncovalent association. In this configuration the three hypervariable regions of each variable domain interact to define an antigen binding site on the surface of the VH-VL dimer. As a whole, the six hypervariable regions confer antigen-antibody specificity to the antibody. However, even a single variable domain (or half an Fv, which comprises only three hypervariable regions specific for an antigen) has antigen recognition and binding capacity, although with less affinity than the complete binding site.
The Fab fragment also contains the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. Fab′ fragments differ from Fab fragments in the addition of a few residues at the carboxy terminus of the domain CH1 of the heavy chain, including one or more cysteines of the antibody hinge region.
The “single chain Fv” or “scFv” antibody fragments comprise the VH and VL domains of an antibody, in which these domains are present in a single polypeptide chain. Preferably, the Fv polypeptide additionally comprises a linker polypeptide between the VH and VL domains which allows the scFv to form the desired structure for antigen binding. For a review of scFv, see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, N.Y., pp. 269-315 (1994).
The term “diabodies” refers to small antibody fragments with two antigen binding sites, those fragments comprising a heavy chain variable domain (VH) connected to a light chain variable domain (VL) in the same polypeptide chain (VH-VL). By means of using a linker which is too short to allow pairing between the two domains in the same chain, the domains are forced to pair with the complementary domains of another chain and create two antigen binding sites. Diabodies are described in further detail in, for example, documents EP 404,097; WO 93/11161; and Hollinger et al., Proc. Natl. Acad. Sci. USA, 90: 6444-6448 (1993).
The term “nanobodies” designates small sized entities (15 kDa) formed solely by the antigen binding region of the heavy chain (VH fragment) of immunoglobulins. Said nanobodies are mainly produced after immunizing animals of the Camelidae family, such as camels, llamas and dromedaries, mainly llamas; and also of the shark family, which have the particularity of having antibodies which naturally lack the light chain and recognize the antigen by the heavy chain variable domain. Nevertheless, the nanobodies derived from these sources require a humanization process for their therapeutic application. Another potential source for obtaining nanobodies is from antibodies derived from different human samples by separating the VH and VL domains of the variable region. Nanobodies present advantages such as a production cost reduction with respect to whole antibodies, stability and the reduction of immunogenicity.
The term “antigen-binding region” of an antibody also includes a synthetic or a genetically engineered polypeptide that binds to a specific antigen, such as polypeptides consisting of the light chain variable region, “Fv” fragments consisting of the variable regions of the heavy and light chains, recombinant single chain polypeptide molecules in which light and heavy variable regions are connected by a peptide linker (“scFv proteins”) and minimal recognition units consisting of the amino acid residues that mimic the hypervariable region.
E. Method for Identifying Controller Patients and for Vaccine Trials
The inventors have demonstrated that the presence of responses to a subset of highly conserved sequence elements (CE) and the maintenance of high avidity responses with broad variant recognition ability are hallmarks of a controlled HIV-1 infection.
Thus, in another aspect, the invention relates to a method for the identification of a HIV controller patient which comprises testing the capability of a sample containing T-cells isolated from said subject to mount a cytotoxic T cell response against at least one 10mer peptide derived from a conserved element within a COT sequence of an HIV protein. If compared to a reference sample the patient is capable of mounting a higher CTL response against at least one 10mer peptide, the subject is considered an HIV controller.
The term “HIV controller”, as used herein, refers to an HIV infected subject who is able to maintain their virus at undetectable or low (<10,000 copies/ml) levels for many years in absence of treatment.
As used herein a “HIV controller” refers to a subject infected with HIV that exhibits a decrease in HIV viral load after the individual is infected with HIV and maintains the decreased HIV viral load over time. A “controller” also refers to an HIV-infected subject who remains asymptomatic with normal CD4 positive T-cell counts and low or undetectable plasma viral loads despite having never been treated with antiretroviral medications. HIV infected individuals that are controllers are capable of maintaining their viral load at a very low levels, for example plasma HIV RNA levels <10,000 copies/ml and, preferably, less than 2000 copies/mL in the absence of antiretroviral therapy, measured three times over a period spanning at least 12 months. See Goudsmit J, et al., AIDS 2002; 16:791-793.
Features of controllers as defined by the HIV Controller Consortium are:
“Subject” means any animal or artificially modified animal Animals include, but are not limited to, humans, non-human primates, cows, horses, sheep, goats, pigs, dogs, cats, rabbits, ferrets, rodents such as mice, rats and guinea pigs, and birds and fowl, such as chickens and turkeys. Artificially modified animals include, but are not limited to, transgenic animals or SCID mice with human immune systems. In the preferred embodiment, the subject is a human.
The terms “center-of-tree”, “cytotoxic T cell response”, “peptide”, “conserved element” and “HIV protein” have been defined in detail above and are used in the same manner in relation to the method for identifying controller HIV patients according to the present invention.
The term “identification an HIV controller”, as used herein refers to the determination of the likelihood that the patient will be able to maintain their virus at undetectable or low (<10,000 copies/ml) levels for many years in the absence of treatment. As will be understood by those skilled in the art, the identification of controller individuals, although preferred to be, need not be correct for 100% of the subjects to be diagnosed or evaluated. The term, however, requires that a statistically significant portion of subjects can be identified as having an increased probability of being a controller. Whether a subject falls within the statistically significant group can be determined without further ado by the person skilled in the art using various well known statistic evaluation tools such as, for example, the determination of confidence intervals, p-value determination, Student's t-test and Mann-Whitney test. See Dowdy S, Wearden S, “Statistics for Research” (John Wiley & Sons, New York, N.Y., USA, 1983). Preferred confidence intervals are at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%. The p-values are, preferably, 0.2, 0.1 or 0.05.
The method comprises a first step wherein a sample containing T-cells isolated from said patient to mount a cytotoxic T cell response against a peptide derived from a conserved element within a COT sequence of an HIV protein. The term “sample containing T cells” includes, without limitation, peripheral blood mononuclear cells, bone marrow, thymus, tissue biopsy, tumor, lymph node tissue, gut associated lymphoid tissue, mucosal-associated lymph node tissue, spleen tissue, or any other lymphoid tissue. T cells can be isolated as peripheral blood mononuclear cells (PBMC) from a blood sample obtained from the peripheral blood of a subject. PBMCs are prepared from peripheral blood by centrifugation on a gradient of Ficoll and Hypaque (metrizamide). See Walker M, et al., J. Clin. Invest., 2003; 112:1437-1443. Alternatively, T cells may be isolated from leukocytes derived from blood using an apheresis or leukopheresis procedure. Leukocytes derived from leukopheresis filters can be enriched for lymphocytes using density gradient centrifugation, as for example, through a Ficoll-metrizamide gradient.
Then, the samples containing T cells are placed in contact with one or more 10mer peptides derived from a conserved element within a COT region and their ability to mount a cytotoxic cell response is determined. The expression “mounting a cytotoxic T cell response”, as used herein, refers to the ability of the T cell to proliferate as a result of the stimulation of the T cell receptor by one or more of the 10mer peptides.
In a preferred embodiment, the COT sequence is encoded by the gag gene. In a still more preferred embodiment, the protein encoded by the gag gene is p24.
In a preferred embodiment, the conserved element within the p24 COT sequence is selected from the group consisting of ISPRTLNAWVKV (SEQ ID NO:1), LSPRTLNAWVKV (SEQ ID NO:2), VIPMFSALSEGATPDQDLN (SEQ ID NO:3), VIPMFTALSEGATPDQDLN (SEQ ID NO:4), VGGHQAAMQMLKDTINEEAAEWDR (SEQ ID NO:5), VGGHQAAMQMLKETINEEAAEWDR (SEQ ID NO:6), PRGSDIAGTTSTLQEQIGW (SEQ ID NO:7), PRGSDIAGTTSTLQEQIAW (SEQ ID NO:8), KRWIILGLNKIVRMYSPVSI (SEQ ID NO:9), KRWIILGLNKIVRMYSPTSI (SEQ ID NO:10), YVDRFFKTLRAEQA (SEQ ID NO:11), YVDRFYKTLRAEQA (SEQ ID NO:12), LEEMMTACQGVGGPSHK (SEQ ID NO:13) and LEEMMTACQGVGGPGHK (SEQ ID NO:14).
In another preferred embodiment, the plurality of 10mer peptides contain one or more peptides selected from the group consisting of SPRTLNAWV (SEQ ID NO:15), ISPRTLNAW (SEQ ID NO:16), LSPRTLNAW (SEQ ID NO:17), EVIPMFSAL (SEQ ID NO:18), EVIPMFTAL (SEQ ID NO:19), VIPMFTAL (SEQ ID NO:20), SEGATPDQDL (SEQ ID NO:21), GHQAAMQML (SEQ ID NO:22), KDTINEEAA (SEQ ID NO:23), KETINEEAA (SEQ ID NO:24), DTINEEAAEW (SEQ ID NO:25), TSTLQEQIAW (SEQ ID NO:26), KRWIILGLNK (SEQ ID NO:27), GLNKIVRMY (SEQ ID NO:28), VRMYSPVSI (SEQ ID NO:29), VRMYSPTSI (SEQ ID NO:30), RMYSPVSI (SEQ ID NO:31), RMYSPTSI (SEQ ID NO:32), YVDRFFKTL (SEQ ID NO:33), YVDRFYKTL (SEQ ID NO:34), DRFFKTLRA (SEQ ID NO:35), and DRFYKTLRA (SEQ ID NO:36).
Once the cytotoxic T-cell response mounted by the T cell population has been determined, the subject is then classified as controller or not controller depending on whether the response is above the response observed in a reference sample.
The expression “reference sample”, as used herein, refers to a sample which has been either not contacted with a peptide, or to a sample which has been contacted with a peptide which is incapable of triggering a cytotoxic cell response in the sample containing the T cells. Suitable peptides for use in the reference sample include any of the peptides shown above wherein the peptide sequence has been shuffled (i.e. the sequence of amino acids has been randomly altered maintaining the overall composition).
In a preferred embodiment, the determination of the CTL response is measured as functional avidity.
The term “functional avidity”, as used herein, refers to the sum total of the strength of binding of two molecules to one another at multiple sites. Avidity is distinct from affinity, the latter of which refers to the binding strength between an antibody and a simple hapten or antigen determinant. In the case of CTL responses, avidity is typically given as the negative log of the peptide concentration that resulted in 50 percent maximal target lysis and is typically expressed in nM. See Derby M, et al., J. Immunol. 2001; 166:1690-1697.
The functional avidity of responses was determined by performing serial 10-fold limiting peptide dilutions ranging from 100 □g/ml to 10 pg/ml; in duplicates whenever enough PBMCs were available. Half-maximal stimulatory antigen doses (SD50%) were calculated as the peptide concentration needed to achieve a half-maximal number of spots in the ELISpot assay calculated by a sigmoidal dose response curve fit using GraphPad Prism4. Based on cell availability from the different subjects, 219 individual responses in the controllers group and 255 individual responses in the non-controllers group (representing 68% and 80% of all responses in the controllers and non-controllers groups, respectively) were titrated for the functional avidity analyses.
F. Method for the Identification of Immunogenic Peptides
In another aspect, the invention relates to a method for the identification of immunogenic peptides within a family of variant polypeptides which comprises the steps of:
In a first step, the method for the identification of immunogenic peptides according to the invention comprises obtaining the center-of-tree sequence of the family of variant polypeptides.
Typically, the family of variant polypeptides are viral antigens, such as retroviral antigens from the human immunodeficiency virus (HIV) including gene products of the gag, pol, env and nef genes, and other HIV components; hepatitis viral antigens, such as the S, M, and L proteins of hepatitis B virus, the pre-S antigen of hepatitis B virus, and other hepatitis (e.g. hepatitis A, B, and C, viral components such as hepatitis C viral RNA); influenza viral antigens, such as hemagglutinin and neuraminidase and other influenza viral components; measles viral antigens, such as the measles virus fusion protein and other measles virus components; rubella viral antigens, such as proteins E1 and E2 and other rubella virus components; rotaviral antigens, such as VP7sc and other rotaviral components; cytomegaloviral antigens, such as envelope glycoprotein B and other cytomegaloviral antigen components; respiratory syncytial viral antigens, such as the RSV fusion protein, the M2 protein and other respiratory syncytial viral antigen components; herpes simplex viral antigens, such as immediate early proteins, glycoprotein D, and other herpes simplex viral antigen components; varicella zoster viral antigens, such as gpI, gpII, and other varicella zoster viral antigen components; Japanese encephalitis viral antigens, such as proteins E, M-E, M-E-NS1, NS1, NS1-NS2A, 80 percent E, and other Japanese encephalitis viral antigen components; rabies viral antigens, such as rabies glycoprotein, rabies nucleoprotein and other rabies viral antigen components. See Fields B, Knipe D, Eds., “Fundamental Virology”, 2nd Ed. (Raven Press, New York, N.Y., USA, 1991) for additional examples of viral antigens. Antigenic targets that may be delivered using the rAb-DC/DC-antigen vaccines of the present invention include genes encoding antigens such as viral antigens, bacterial antigens, fungal antigens or parasitic antigens. Viruses include picornavirus, coronavirus, togavirus, flavirvirus, rhabdovirus, paramyxovirus, orthomyxovirus, bunyavirus, arenavirus, reovirus, retrovirus, papilomavirus, parvovirus, herpesvirus, poxvirus, hepadnavirus, and spongiform virus. Other viral targets include influenza, herpes simplex virus 1 and 2, measles, dengue, smallpox, polio or HIV. Pathogens include trypanosomes, tapeworms, roundworms, helminthes or malaria. Tumor markers, such as fetal antigen or prostate specific antigen, may be targeted in this manner Other examples include HIV Env proteins and hepatitis B surface antigen.
The determination of the centre-of-tree sequences from a family of variant sequences is usually carried out as described above. See Nickle D, et al., Science 2002; 296(5577):2354-2360; Rolland, 2007, supra; and Mullins, 2005, supra.
In a preferred embodiment, the sequences used for obtaining the COT sequence are of viral origin. In a still more preferred embodiment, the related peptides of viral origin are derived from HIV. In yet another embodiment, the sequences derived from HIV derive from a polypeptide encoded by the gag gene. In another preferred embodiment, the polypeptide encoded by the gag gene is p24.
In a second step, the method for the identification of immunogenic peptides within a family of variant polypeptides involves the identification of conserved sequence elements within the center-of-tree sequence. Conserved elements are those regions of the sequence that are substantially constant among all related variants when the variant sequences are compared. The identification of the conserved elements can be carried out as described above using any preferred pairwise or multiple sequence alignment algorithm.
In a third step, the method for the identification of immunogenic peptides within a family of variant polypeptides involves producing peptides, the sequence of which is comprised within said conserved sequence elements. Methods for obtaining and producing the peptides of the present invention are not particularly limited. Chemically synthesized peptides or recombinant peptides produced by gene recombination techniques are available. In a preferred embodiment, the peptides have a length of 10 amino acids or less.
In a fourth step, the method for the identification of immunogenic peptides within a family of variant polypeptide comprises testing the peptides obtained in the previous step for their immunogenicity. The step can be carried out essentially as described above based on the ability of the peptides to induce a biological response in a sample containing T cells such as proliferation assays, lymphokine secretion assays, direct cytotoxicity assays, and limiting dilution assays.
The invention is described hereinafter by way of the following examples that are merely illustrative and not limitative of the scope of the invention.
Materials and Methods
A. Study Subjects
Chronically HIV-1 infected individuals were recruited from the HIV-1 Unit in Hospital Germans Trias i Pujol, Badalona, Spain. They fulfilled the following inclusion criteria: sustained viral load <10,000 RNA copies and CD4 cell counts >350 cells/mm3 for the controllers group (n=25) and viral load >50,000 RNA copies/ml and CD4 cell counts <350 cells/mm3 for the non-controllers (NC) group (n=25). Individuals with primary HIV-1 infection and subjects on antiretroviral treatment were excluded from the study. Furthermore, all patients were HLA typed at high resolution using SSP-PCR at the immunology department of the Hospital Germans Trias i Pujol. Individuals expressing any of the previously described protective alleles (HLA-B27, -B57 or -B58) were excluded from the analysis. See Mothe B, et al., Dis. Markers 2009; 27:105-120. Informed consent was obtained from all participants and the study was approved by the institutional review board of the Hospital Germans Trias i Pujol. Individuals in the NC group were slightly older than in the C group (p=0.04) but did not differ significantly with regards to time since diagnosis of HIV-1 infection. Likely route of HIV-1 acquisition and gender distribution was not significantly different in both groups either. The median HIV-1-1 RNA levels were 200,000 copies/ml (52,000-1,200,000) in NC. The median CD4 cell count was 642 cells/mm3 (434-1114) and 98 cells/mm3 (11-361) for the C and NC, respectively. These and additional demographic details are included in Table 3.
aData are expressed as median (min-max range),
bn, (%),
CUD: undetectable viremia (<49 copies/ml)
B. Synthetic Peptides Set
An overlapping peptide set of 223 peptides of 10 amino acids in length (overlapping by 9 residues) spanning the entire group M Center-of-Tree (COT-M) Gag p24 sequence was synthesized at the peptide synthesis facility of the Massachusetts General Hospital using 9-Fluorenylmethyloxycarbonyl (Fmoc)-chemistry. An additional eighty-eight 10-mer peptides were generated to cover the most frequently occurring variants in the 7 CE regions (1 variant per CE). In order to determine the relative immunodominance of Gag specific responses, the analysis also included a previously described comprehensive overlapping peptide (410 18mer OLP) set spanning the entire viral proteome. See Frahm N, et al., Aids 2008; 22:447-456. The OLP sequence was based on the consensus-B sequence of 2001 available at the HIV immunology database (www.hiv.lanl.gov/content/sequence/HIV/mainpage.html, September 2011). Peptides were generally 18mers varying from 15-20 amino acids in length and overlapping by 10 amino acids, designed using the PeptGen algorithm at the Los Alamos HIV database (www.hiv.lanl.gov/content/sequence/PEPTGEN/peptgen.html, September 2011).
C. ELISpot Assay
PBMCs were separated from whole blood within 4 h of venopuncture and used directly for the IFN-γ ELISpot screening. Each COT-M Gag p24 overlapping peptide and the 88 variant peptides were added at a final concentration of 14 □g/ml. For all assays, between 75,000-100,000 PBMC per well were added in 140 ul of R10 96-well polyvinylidene plates (Millipore, Bedford, Mass., USA). The IFN-γ Mabtech kit was used for detection of IFN-γ secretion following manufacturer instructions. In parallel, CTL responses to the clade B full proteome were assessed using the 18mer peptide set in a previously described matrix outline, followed by deconvolution of reactive pools and reconfirmation of each response at a single peptide level on the following day. See Frahm N, et al., J. Virol. 2004; 78:2187-2200. The number of spots was counted using a “CTL ELISpot Reader Unit” and the magnitude of responses was expressed as spot forming cells (SFC) per million input cells. The threshold for positive responses was defined as at least 5 spots per well and responses exceeding “mean number of spots in negative control wells plus 3 standard deviations of the negative control wells” and “three times the mean of negative control wells”, whichever was higher. As a conservative approach and not to overestimate the breadth of responses, positive responses to 3 consecutive 10mers in the COT-M Gag p24 peptide set were counted as 1 response. Similarly, reactivity to 2 consecutive 18mer peptides was counted as 1 response when testing with the full proteome 18mer peptide set. The highest magnitude of the sequential responses was taken as the magnitude for each response. Responses reacting with flanking 10mer peptides that only partly overlapped with the CE regions were considered as targeting the actual CE when the 10mer overlapped with at least 8 amino acids of the CE.
D. Functional Avidity
The functional avidity of responses was determined by performing serial 10-fold limiting peptide dilutions ranging from 100 □g/ml to 10 pg/ml; in duplicates whenever enough PBMCs were available. Half-maximal stimulatory antigen doses (SD50%) were calculated as the peptide concentration needed to achieve a half-maximal number of spots in the ELISpot assay calculated by a sigmoidal dose response curve fit using GraphPad Prism4 (GraphPad Software Inc., San Diego, Calif., USA). Based on cell availability, 219 individual responses in the controllers group and 255 individual responses in the non-controllers group—representing 68% and 80% of all responses in the controllers and non-controllers groups, respectively, were titrated for the functional avidity analyses.
E. Gag p24 Sequencing
Viral RNA was extracted from 1 millilitre of plasma spun at 9000 g for 1.5 hour (QIAamp Viral RNA Kit™, QIAGEN, Valencia, Calif., USA). From the pelleted RNA, the whole gag region was reverse-transcribed and amplified in a One-Step reaction (SuperScript® III One-Step RT-PCR System with Platinum® Taq High Fidelity, Invitrogen, Carlsbad, Calif., USA) under the following conditions: 30 min at 52° C. for the reverse transcription step; 2 min at 94° C.; followed by 35 cycles at 94° C. during 30 sec, 58° C. during 30 sec and 68° C. during 2 min; followed by a final extension step at 68° C. during 5 min. Primers used for the RT-PCR were: Gag U761 (HXB2: 761→778) 5′-TTT GAC TAG CGG AGG CTA G-3′ (SEQ ID NO:37) and Gag D2397 (HXB2: 2397→2376) 5′-CCC CTA TCA TTT TTG GTT TCC A-3′ (SEQ ID NO:38). One microliter of the RT-PCR product was subsequently used as a template for a second, nested round of PCR (Platinum® Taq DNA Polymerase High Fidelity, Invitrogen, Carlsbad, Calif., USA), using primers p24 U1070 (HXB2: 1070→1088) 5′-TAA AAG ACA CCA AGG AAG CT-3′ (SEQ ID NO:39) and p24 D2063 (HXB2: 2063→2044) 5′-TCT TTC ATT TGG TGT CCT TC-3′ (SEQ ID NO:40). PCR cycling conditions were: 2 min at 94° C.; followed by 35 cycles at 94° C. during 30 sec, 54° C. during 30 sec and 68° C. during 2 min; followed by a final extension step at 68° C. during 5 min. The final PCR products were column-purified (QIAquick PCR Purification Kit, QIAGEN, Valencia, Calif., USA) and sequenced bidirectionally. Sequences were assembled using Sequencher® 4.10.1 (Gene Codes Corp., Ann Arbor, Mich., USA). Assembled sequences were codon-aligned using the Hidden Markov Model implemented in the tool HIValign (www.hiv.lanl.gov/content/index, September 2011). Autologous gag p24 bulk sequences were obtained for 21 of the 25 HIV-1 non-controllers included in our study.
F. Statistical Analyses
All values are presented as median values unless otherwise stated. GraphPad Prism version 4.00 for Windows (GraphPad Software Inc., San Diego, Calif., USA) was used to compare response rates in both groups and subgroup analyses. Mann-Whitney test and Wilcoxon matched paired test were used for unpaired and paired comparisons, respectively. Spearman rank correlation was used to assess association between CD4 cell counts and the fraction of detectable responses targeting individual viral proteins.
A number of cohort-based analyses, some including several hundred individuals, have analyzed the differential contribution of protein-specific T cell responses to the overall cellular immunity against HIV-1. The most consistent observation is that HIV-1 infected individuals with relative control of in vivo viral replication target predominantly epitopes located in HIV-1 Gag. On the other hand, subjects with elevated viral loads were consistently found to mount their broadest and strongest responses towards HIV-1 Nef. Using a 18mer overlapping peptide set, we assessed the distribution of HIV-1-specific T cell responses in 25 HIV-1 controller and in 25 non-controllers by IFN-γ ELISpot assays. See Frahm, 2004, supra. To limit the potential bias by the presence of highly immunodominant epitopes restricted by HLA-B27 and -B57/58, whose responses are known to be associated with virologic control, the 50 tested subjects included only individuals without these HLA alleles. Overall, the majority of responses were detected against OLP located in the HIV-1 Gag, Pol and Nef proteins, as seen in earlier studies See Frahm, 2004, supra. HIV-1 controllers showed an overall broader breadth (median of 19 responses, range 7-53) than non-controllers (12 responses, range 2-55, p=0.025), translating also into a greater total magnitude of the response (18,881 SFC/106 input PBMC, range 2,690-44,720) compared to non-controllers (9,000 SFC/106 input PBMC, range 850-114,630, p=0.0353). The magnitude of individual responses in controllers and non-controllers was, however, not significantly different (p=0.2). As expected from earlier studies in another clade B infected cohort of comparable size in Peru, the HIV-1 controller subjects directed 29% of their total HIV-1-specific responses towards OLP located in Gag (18% in non-controllers, p=0.0336) whereas non-controllers targeted Nef more frequently (17% of their total responses) than controllers (12%, p=0.0076, data not shown). See Zuniga R, et al., J. Virol. 2006; 80:3122-3125. We also found a statistically significant direct association between the relative breadth of Gag-specific CTL responses and the CD4 T-cell counts of the 50 individuals analyzed (p=0.03, Spearman's rank test, data not shown). Also, we detected an inverse correlation between relative immunodominance of Gag and the viral loads in the 50 individuals tested (p=0.0039, data not shown). These data confirm the associations seen between immunodominant Gag responses and lower viral loads, both in clade B and C infections, even in a small cohort of individuals without any protective HLA alleles, and suggest that an increased breath of the Gag response may provide increasing additional benefit for viral control.
Among Gag-specific T cell responses, those targeting the p24 subunit have been particularly implicated in mediating viral control. See Zuñiga, 2006, supra. Indeed, more controllers showed a response to at least one Gag p24 OLP (24/25 individuals, 96%) compared to HIV-1 non-controllers (20/25, 80%). In addition, breadth of the Gag p24 response was higher in the controllers group as well, with a median of 3 responses to p24 in controllers (range 0-12) compared to 1 response in non-controllers (range 0-9, p=0.04). These differences were also significant when focusing on the 18 OLP (18mers) that span the 7 CE in p24, with a median of 2 responses in controllers (range 0-6) compared to 1 response in non-controllers (range 0-6, p=0.03). This data, when considered together, confirm earlier reports on the relative benefits of Gag specific responses. However, it also demonstrates clearly that Gag-specific responses can be detected in HIV-1 non-controllers. Why these responses do not contribute more effectively to viral control remains unclear. It may be possible that HIV-1 non-controllers mount responses to Gag at the same level as HIV-1 controllers, but with functional properties that do not allow their detection with the same ease as in controllers. Among different factors, the functional avidity of these responses and thereby the need for sensitive peptide test sets, may differ between the two groups.
In order to increase sensitivity of detection of responses, all individuals were tested against a set of 223 peptides of 10 amino acids in length (overlapping by 9 residues) spanning the group M Center-of-Tree (COT-M) Gag p24 sequence. Eighty-nine of the 223 10mer peptides covered the 7 CE regions that were identified when designing the CE-based p24 immunogen sequence (40%). This sequence represents a cross-clade, HIV-1 group M DNA immunogen designed to focus immune responses on conserved protein elements (CE) that are considered essential to the function of the virus while precluding responses against immunodominant decoys. The inclusion criteria for the CE was high sequence conservation within the HIV-1 group M (>98%). See Roland, 2007, supra. Sequence diversity coverage was further increased by including the single second most frequent variants at one amino acid for each CE. Allowing for the inclusion of such frequent “toggled” amino acids increased the CE coverage of all group M sequences to >99%. See Llano, 2009, supra. The 7 CE segments, as well as the optimal epitopes, have been described. See
When the COT-M Gag p24 10mers set of peptides was used in parallel to the 18mer OLP set (30 peptides, overlapping by 10 residues), significantly more responses were identified with the 10mers both in the controllers (p=0.0002) and non-controllers group (p=0.0006). Of note, the increase in response rates was more pronounced in the non-controller group (3-fold) than in the controllers (2-fold). This indicates that the non-controllers had proportionally greater enhancement of response detection towards the tested peptide in comparison to the controller group. See
As HIV-1 controllers and non-controllers showed comparable total response rates to the 7 CE, we next asked whether any of the individual CE performed significantly differently between the two groups. Indeed, three CE were targeted by at least 40% more controllers than non-controllers (CE #4, #5 and #6,
Although the CE-immunogen approach is based on the identification of the most conserved segments of the HIV-1 genome, the inclusion of single second most frequent variants can further increase coverage of global group M sequence diversity. To incorporate these variants in the ex-vivo immune analyses, the 223 10mer peptides included in the above screenings were complemented by 88 additional overlapping 10mer variant peptides; 42 of which were covering sequence variants located in CE regions. As for the COT-M sequence peptides, the median number of responses to these p24 variants was greater in controllers (median of 4 responses; range 2-9) than in HIV-1 non-controllers (median 2 responses, range 0-16) and reached statistical significance despite the smaller number of tested peptides (p=0.02). See
The variant recognition data was also analyzed in comparison to the reactivity to the “wild type” COT-M sequence. This allowed for the identification of cases in which responses were only detected by using the variant peptide and the assessment of the level of cross-reactivity of responses (i.e. responses that reacted with both the COT-M and the variant peptides). Of the 50 subjects tested, 23 controllers and 17 non-controllers had detectable responses to COT-M peptides for which a variant peptide was tested. Among controllers, a median of 50% of variant peptides were reactive when the COT-M sequence elicited a response. In HIV-1 non-controllers, individuals reacted with a median of 31% to the tested variants of reactive COT-M peptides (p=0.2). See
To rule out that the overall reduced variant response rates in the non-controllers was due to mismatches between the tested peptide sequences and the autologous viral sequence in the CE region, autologous Gag p24 bulk sequences were obtained for 21 of the 25 HIV-1 non-controllers. The analysis showed a 99% identity between the test set and the autologous viral sequences as only 24 amino acid polymorphisms were identified across all 7 CE segments for the 21 individuals analyzed. See
Based on cell availability, a total of 474 individual responses (219 responses in HIV-1 controllers and 255 responses in non-controllers) were titrated to define their functional avidity. As shown in
In order to test directly whether functional avidity was related to the ability to recognize peptide variants, titrated responses were grouped into high, intermediate and low avidity responses and their variant recognition potentials were compared. Indeed, responses with SD50% in the first quartile of all titrated responses (<1,401 ng/ml) showed cross-reactivity with their variants in 67% of all cases, whereas fewer (48% and 33%) of responses of intermediate or low functional avidity were cross-reactive with their 10mer variants. See
This application claims the priority of U.S. application Ser. No. 13/884,634, filed Jul. 25, 2013; International Patent Application No. PCT/EP2011/069870 filed on Nov. 10, 2011, and of U.S. Provisional Patent Application No. 61/412,060 filed on Nov. 10, 2010. The disclosures of the foregoing international patent application and U.S. provisional patent application are hereby incorporated by reference herein in their respective entireties.
| Number | Date | Country | |
|---|---|---|---|
| Parent | 13884634 | Jul 2013 | US |
| Child | 15463176 | US |
