WO2016059517A1

WO2016059517A1 - Peptide for anti-ebola vaccine

Info

Publication number: WO2016059517A1
Application number: PCT/IB2015/057709
Authority: WO
Inventors: Jean-Pierre Spinosa; Darja Kanduc
Original assignee: STRELNIKOV, Evgeny
Priority date: 2014-10-14
Filing date: 2015-10-08
Publication date: 2016-04-21

Abstract

Based on the concept according to which protein immunogenicity more likely resides in unique/rare peptide sequences and aiming at defining epitopic peptides that might serve for therapeutic anti-Ebolavirus (EV) vaccines, a set of 45 EV proteomes was searched for peptide motifs unique to EVs and absent in human proteins. The search highlighted one peptide, LWQQWDR, that represent the molecular signature of the 45 EVs and is absent in the human proteome at the pentapeptide level. Synthetic oligopeptide constructs based on such unique viral fragment might be used for constructing synthetic oligopeptides for vaccine formulations, thus offering the possibility of hitting 1) multiple EV species/strains, 2) with high specificity and 3) without crossreactivity-associated adverse events. A rationale for constructing LWQQWDR-based synthetic oligopeptides is also described.

Description

Field of invention

The present invention relates to immunogenic peptides that are useful for evoking specific and effective immune responses in immunotherapies and immunodiagnostics.

State of the art

EV infection causes haemorrhagic fever, a severe illness characterized by an abrupt onset and a case fatality rate of up to 90%, Currently, no treatments are available for EV, in spite of recurrent EV outbreaks [1-3].

Recent attempts to define immune tools against EBOLA have been unsuccessful. For example, human adenovirus type 5 vectors (rAdS) encoding ebolavirus glycoprotein has been shown to generate protective immunity against acute lethal Zaire ebolavirus challenge in macaques, but fail to protect animals immune to Ad5, suggesting natural Ad5 exposure may limit vaccine efficacy in humans, and, in addition, it was seen that immune protection can wane over several months [4].

Moreover, EBOLA rapidly accumulates interhost and intrahost genetic and, consequently, phenetic variations [5]. Hence, potentially effective immunotherapeutic tools and vaccines can rapidly lose any efficacy.

in addition, since exposure to EBOLA may be lethal, special containment scientific-clinical units and biosafety level 4 facilities are mandatory, this way severely limiting the research to realize efficacious diagnostics, prevention, and vaccines for contrasting EBOLA,

As a consequence of the lack of immune tools to diagnose, prevent and fight Ebola, outbreaks of EV disease regularly occur and threaten Africa and the eniire world [6]. The human, ecological and economical damages are unquantifiable [7].

Description of the invention

The problems mentioned in the previous chapter have been solved with the use of a specific sequence as defined in the claims.

Based on the concept that peptide uniqueness modulates self-nonself discrimination, so that peptide sequences not present in the host may be highly immunogenic towards the host [8-12], EV proteins were searched for peptide motifs unique to EVs and absent in the human proteome. The purpose consisted in defining epitopic peptides that might immediately serve for therapeutic anti-Ebola vaccines and, in the long-term, for prevention and diagnostics of EV worldwide. The invention describes a pathway that starts from the five known EV species (Sudan, Zaire, Tai Forest, Bundibugyo, and Reston) and derived strains (for a total of 45 EV proteomes equal to 240,836 amino acids) and has one short fragments (LWQQWDR, 7 amino acids) as an arrival point for designing anti-EV vaccines able to specifically hit multiple EV species/strains, exempt from possible collateral adverse events deriving from cross-reactions with the host proteins, immediately realizable since the method does not imply manipulation of infected sera or viral particles, usable for prevention as well as diagnostics, and the usage of which can be extended to non- human primates too.

The proteome from Sudan EV, strain Gulu, (GenBank accession: AY729654.1 ; 5190 amino acid (aa) long) was analysed for pentapeptide matching to the human proteome. The EV proteome consists of eight proteins: NP, nucleoprotein; vp35, polymerase cofactor vp35; vp40, matrix protein vp40; sGP, secreted glycoprotein; GP, Envelope glycoprotein; vp30, minor nucleoprotein vp30; vp24, membrane-associated protein vp24; L, RNA-directed RNA polymerase. The aa sequences 1 -295 of secreted glycoprotein (sGP) and giycoprotein (GP) are identical. EV protein sequences and further details are reported at ht^://www .ncbi.nlm.nih.gov/nuccore/AY729654.

Sequence similarity analyses of EV proteins vs the human proteome was conducted using EV 5-roers sequentially overlapped by four residues (i.e., MMATQ, MATQH, ATQHT, TQHTQ, and so forth), and PIR peptide match program (pir.georgetown.edu/pirwww/search/peptide.shtml) (13). Each viral pentapeptide was used to search the human proteome for instances of the same pentapeptide. Any such occurrence was termed a match.

Comparative peptide matching analyses were conducted on 45 EV proteomes that were selected from NCBI nucleotide database using as keywords "Ebola virus" and "complete genome". The name and the NCBI GenBank accession numbers of the analyzed EV are listed in Table 2.

The 45 EV proteomes were searched for common aa fragments by sequence alignment analyses that used ClustalW algorithm (http^/vvww^uniprot^rg/progranVVquei-y^clustahv&sort^score)^^) Molecular modeling was obtained using Spartan software (https://www .wavefun.com/products/spartan.html)

The primary aa sequence of the proteins from Sudan ebolavirus strain Gulu, Accession: AY729654.1 , was analysed for similarity to the human proteome using pentapeptides as scanning probes and PIR exact matching program (13). In fact, a grouping of five aa residues represents a minimal functional unit in immunobiology (15, and further references therein), thus constituting an appropriate measurement unit for fiinctional-stractural-immunological analyses (16). The pentapeptide similarity profile of the EV proteins versus the human proteome is illustrated in Fig I . In agreement with results obtained from the analyses of numerous viral and microbial organisms (14-24), EV protein sequences present pentapeptides rarely (or not at all) represented in the human proteome and pentapeptides frequently recurring in human proteins, in a few cases, the pentapeptide frequency is highest, i.e. a pentapeptide repeatedly recurs in the host proteome. On the whole, 67,286 exact matches (including multiple occurrences) are common to EV and human proteomes.

The immunological implications of such extensive viral-versus-human peptide matching are of clinical importance. In fact, given the fact that 5 amino acids form a minimal unit able to evoke humoral and/or cellular immune responses as well as to act in immune recognition (12, 13), the pentapeptide matches illustrated in Figure 1 are a measure of the potential snimunological cross-reactivity existing between EV and the human host, and implies that utilization of entire viral antigens in vaccine formulations to fight EV might carry a high crossreactivity risk, thus potentially leading to thousand of undesired autoimmune responses against the human proteins sharing matches with EV.

Figure 1 represents similarity profile of the proteins from Sudan ebolavirus strain Gulu, Accession: AY729654.1, versus the human proteome at the pentapeptide level. Pentapeptide aa sequences in one-letter code. The analysed 8 EV proteins are shown in the upper part of the figure. EV protein abbreviations are detailed under Methods.

The pentapeptide profiling portrayed in Figure 1 indicates an immunologic crossreactivity risk that is directly proportional to the number of viral-versus-human pentapeptide matches. On the other hand, the data shown in Figure 1 also suggest that aa regions unique to the virus and with no counterpart in the human proteome might represent a possible approach for specifically hitting EV without damaging host proteins, cells, and tissues. Accordingly, pentapeptides uniquely present in EV were singled out. The results are reported in Table 1 that reports the pentapeptides exclusively owned by the Sudan EV strain Gulu, Accession: AY729654.1. and, therefore, representing the peptide viral signatures. Seventeen out of the 301 unique pentapeptides listed in Table 1 are present twice, by being part of the NH2 domain common to sGP and GP (see under Methods).

Tab!e 1. The peptide signatures of Sudan EV strain Guiu, GenBank accession: AY729654.1

Of relevance, many pentapeptides listed in Table 1 consecutively overlap thus forming long peptide sequences e, eg, GWVCIFQ in vp35; GNHMQIS, GLMHNQN, EPHDWTK, NDDNWWTG in GP; LWQQWDR in VP30; STWFVTN, IKYCNQCY, VFDWMHF, DTRFRNTC, DGMCWTN, STWHHEL, VCHRFNH in L. The presence of such long peptide fragments appeared of interest for possible anti-EV vaccine formulations so that next step consisted in investigating whether they were exclusively associated with Sudan ebolavirus strain Guiu, Accession: AY729654.1. To this aim, the primary aa sequences of the proteins from Sudan ebolavirus strain Guiu, Accession: AY729654.1, were aligned with the aa sequences of the proteins from 44 EVs, retrieved as described under Methods and listed in Table 2. On the whole, the set of EVs listed in Table 2 includes fi ve species, four of which ca use disease in humans (Sudan, Zaire, Tai Forest, and Bundibugyo) whereas the fifih (Resion) causes disease in non-human primates, but not in humans.

Table 2. Names and NCBI GenBank accessions of EV species/strains analysed for unique peptide fragments

Then, EV sequences were searched for the peptide fragments formed by consecutively overlapping pentapeptiaes unique to Sudan eboiavirus strain Gulu, Accession: AY729654.1. Table 3 illustrates that only 1 peptide fragment, LWQQWDR, barcodes the 45 EVs, while the other peptide fragments are absent or present aa substitutions thus resulting useless for usage in developing specific immune tools. Indeed, one single amino acid change can alter immunorecognition and immunoreactivity (17).

Table 3. LWQQWDR sequence is 100% conserved among 45 EV strains and variants.

Numbers indicate EV strains/variants as reported in Table 2. Not conserved sequences in bold Conserved sequences with a grey background. GNHMQIS sequence was not considered since alignment presenied gaps.

Constructing a LWQQWDR-based oligopeptide as an immunogen against EV

LWQQWDR may be the basis for constructing synthetic oligopeptides to be used directly as antigens in vaccine formulations. LWQQWDR sequences may be assembled into LWQQWDR multimers. Since the tail-head junction point may generate pentapeptides present in the human proteome, LWQQWDR sequences can be joined using as linker one amino acid or a stretch of amino acids chosen in such a way that none of the newly generated pentapeptides at the junction point is present in the human proteome.

LWQQWDRhmccLWQQWDRhmccLWQQWDRhmccLWQQWDR (with aa linker given in small letters) is an example of such a synthetic oligopeptide construct. Of special relevance, the presence of Cys residues in the linkers adds the additional advantage of catalyzing the construct polymerization given the tendency of Cys to form S-S bridges.

In sum, usage of the construct LWQQWDRhrnccLWQQWDRhmccLWQQWDRhmccLWQQWDR as immunogen offers numerous advantages since:

- The construct is formed by overlapping pentapeptides "never seen" in the human proteome. As "nonself ' to the human host they have an immunogenic potential;

- Foreign epitopic peptides concentrated along a muitimer induce better antibody responses [18];

- Will have a highest specificity

- Will not produce crossreactivity-associated adverse events.

- Is formed by a sequence, LWQQWDR, with a highest level of conservativeness among 45 EV species/strains. That is, it lias the potential to evoke immune responses capable of hitting 45 EV species/strains, thus representing a solution to the problem presented by EV tendency to rapidly mutate and accumulate genetic and, consequently, phenetic variations [5].

- Its synthesis does not imply the use of viral particles or risky procedures. Its synthesis and use are innocuous, thus eliminating the need of special containment units and biosafety level 4 facilities

- Can consequently allow fast procedures, fast protocols and fast trials.

- Because of the presence of Cys residues in the linkers and the consequent tendency to polymerize, the construct can acquire the dimension of a medium-weight antigen. Indeed, already as a dimer, the construct has a molecular weight of 1 IkDa. This provides, as a further advantage, the possibility of eliminating carrier proteins in the immunization procedure. In general, molecules must be 10-12 kDa to elicit an immune response, so that it is necessary to conjugate the peptide to a carrier protein such as keyhole limpet hemocyanin prior to immunization. In the present case, the peptide uniqueness rationale that led to identify the EV heptapeptide LWQQWDR prevents from conjugating LWQQWDR to a carrier protein because of the includible crossreactivity risk. Just as an example, the hemocyanin pentapeptide LTAEE is present hundred of times in the human proteome.

Conclusion EV outbreaks need an urgeni, rationale, effective solution. A svntheiic oligopeptide construct formed by the highly conserved viral LWQQWDR peptide, wiih the junction points "obscured" to the human host by ad hoc chosen aa linkers, appears to be such a solution.

References

[ 1 ] http://www.cdc.gov/vhfyebola/

[2] ht^://www.who.int/csr/disease/ebola/en/

[3] http://ww^v.cdc.gov/vht7eboia/outbreaks/giii!iea/

[4] Stanley DA, Honko AN, Asiedu C, Trefiy JC, Lau-Kilby AW, Johnson JC, Hensley L, Ammendoia V, Abbate A, Grazioli F, Foulds KE, Cheng C, Wang L, Donaldson MM, Colloca S, Folgori A, Roederer M, Nabei GJ, Mascola J, Nicosia A, Cortese R, Koup RA, Sullivan NJ.Chimpanzee adenovirus vaccine generates acute and durable protective immunity against ebolavirus challenge. Nat Med. 2014;20( 10): 1 126-1129.

[5] Gire SK, Goba A, Andersen KG, Sealfon RS, Park DJ, Kanneh L, Jalloh S, Momoh M, Fullah M, Dudas G, Wohl S, Moses LM, Yozwiak NL, Winnicki S, Matranga CB, Malboeuf CM, Qu J, Gladden ADQ, Schaffner SF, Yang X, Jiang PP, Nekoui M, Colubri A(l 1), Coomber MR, Fonnie MQ, Moigboi A, Gbakie M, Kamara FK, Tucker V, Konuwa E, Saffa S, Sellu J, Jalloh AA, Kovoma A, Koninga J, Mustapha I, Kargbo K, Foday M, Yillah M, Kanneh F, Robert W, Massally JL, Chapman SB, Bochicchio J, Murphy C, Nusbaum C, Young S, Birren BW, Grant DS, Scheiffelin JS, Lander ES, Happi C, Gevao SM, Gnirke A, Rambaut A, Garry RF, Khan SH, Sabeti PC. Genomic surveillance elucidates Ebola virus origin and transmission during the 2014 outbreak. Science. 2014;345(6202): 1369-72.

[6] Bausch, D.G.; Schwarz, L. Outbreak of ebola virus disease in Guinea: where ecology meets economy. PLoS Negl. Trop. Dis., 2014, 8, e3056

[7] Hampton T. Largest-ever outbreak of Ebola virus disease thrusts experimental therapies, vaccines into spotlight, JAMA. 2014 Sep 10;312(10):987-9.

[8] Kanduc, D. Immunogenicity in peptide-immunotherapy: from self/nonself to similar/dissimilar sequences. In: Sigalov AB, editor, Multichain Immune Recognition Receptor Signaling: From

Spatiotemporal Organization to Human Disease, Landes Biosciences, Austin, TX, 2008; 198-207.

[9] Kanduc, D. "Self-nonself" peptides in the design of vaccines. Curr. Pharm. Des., 2009, 15, 3283-

3289.

[10] Kanduc, D. Protein information content resides in rare peptide segments. Peptides, 2010, 31 , 983- 988.

[11] Kanduc, D. The self/nonself issue: A confrontation between proteomes. Self Nonself, 2010, 1, 255- 258.

[ 12] Kanduc, D. Peptide cross-reactivity: the original sin of vaccines. Front. Biosci., 2012, 4, 1393-1401 .

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[ 14] Thompson, J.D., Higgins, D.G., Gibson, T,J. Clustal W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 1994, 22, 4673-4680. Kanduc, D. Peniapeptides as minimal functional units in cell biology and immunology. Curr. Protein Pept. Sci., 2013, 14, 111-120.

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Claims

1. Use of the sequence LWQQWDR as a basis for constructing synthetic oligopeptides to be used direcily as antigens in vaccine formulations.

2. Synthetic oligopeptide construct comprising the LWQQWDR peptide to be used as immunogen in an active and/or passive vaccine against infectious diseases.

3. Construct according to claim 2 comprising the sequence

LWQQWDRhmccLWQQWDRhmccLWQQWDRhmccLWQQWDR.

4. Construct according to claim 2 or 3 to be used in an active and/or passive vaccine against Ebola Virus (EV) in humans and non-human primates

5. Construct according to claim 4 io be used against any or all of the 45 EV strains and variants disclosed in the preseni document.

6. Pharmaceutical preparation comprising the construct of claim 3, 4 or 5.

7. Pharmaceutical preparation according to claim 6 to be used as a vaccine for prevention, therapeutics and diagnostics of Ebola infection.