HK1155208A - Consensus sequences of chikungunya viral proteins, nucleic acid molecules encoding the same, and compositions and methods for using the same - Google Patents

Description

Chikungunya virus protein consensus sequences, nucleic acid molecules and compositions encoding the chikungunya virus protein consensus sequences, and methods of use thereof

Technical Field

The present invention relates to vaccines and methods for prophylactically and/or therapeutically immunizing individuals against chikungunya virus.

Background

This application claims priority from U.S. provisional application No. 61/042,661, which is incorporated herein by reference.

Immunotherapy refers to the modulation of the immune response of the human body to achieve a desired therapeutic effect. Immunotherapeutics refer to those compositions that, when administered to an individual, modulate the individual's immune system sufficient to ultimately alleviate symptoms associated with an undesired immune response or to ultimately alleviate symptoms by increasing a desired immune response. In some cases, immunotherapy is part of a vaccination regimen in which administration of a vaccine to an individual exposes the individual to an immunogen (in such cases the individual generates an immune response to the immunogen), and an immunotherapeutic increases the immune response and/or selectively enhances a portion (e.g., a cellular immune portion or a humoral immune portion) of the immune response intended to treat or prevent a particular disorder, infection, or disease.

Vaccine regimens can be completed by delivering agents that modulate the immune response in humans to induce an improved immune response. In some vaccination protocols, in which administration of a vaccine to an individual exposes the individual to an immunogen to which the individual generates an immune response, formulations are provided that increase the immune response and/or selectively enhance a portion of the immune response (e.g., a cellular immune portion or a humoral immune portion) that is expected to treat or prevent a particular disorder, infection, or disease.

Vaccines can be used to immunize an individual against a target antigen, such as an allergen, pathogen antigen, or cell-associated antigen associated with a human disease. Antigens associated with cells associated with human diseases include cancer-associated tumor antigens and antigens associated with cells associated with autoimmune diseases.

In designing such vaccines, it has been found that vaccines that produce the target antigen in the cells of the vaccinated individual are effective in eliciting the cellular immune part of the immune system. In particular, live attenuated vaccines, recombinant vaccines that utilize avirulent vectors, and DNA vaccines each allow the production of antigens in the cells of the vaccinated individual, thereby forming the induction of the cellular immune component of the immune system. On the other hand, although killed or inactivated vaccines and subunit vaccines comprising only proteins do induce an effective humoral response, they do not induce a good cellular immune response.

Cellular immune responses are often necessary to provide protection against pathogen infection and to provide effective immune-mediated therapy for treatment of pathogen infection, cancer or autoimmune disease. Thus, vaccines that produce the target antigen in the cells of the vaccinated individual, such as live attenuated vaccines, recombinant vaccines and DNA vaccines using avirulent vectors, are generally preferred.

Chikungunya virus (CHIKV) is an alphavirus native to tropical africa and asia, where it is transmitted to humans by the bite of infected mosquitoes (usually aedes [1 ]). Bucking fever is a disease caused by CHIKV and was first discovered in epidemic form in eastern africa during 1952-1953 [2 ]. Infection of humans by CHIKV may cause syndromes characterized by fever, headache, rash, malaise, nausea, vomiting, myalgia, severe joint pain, and occasional neurological manifestations (e.g. acute limb weakness). It is also associated with fatal hemorrhagic disorders. Other symptoms include muscle pain and pain behind the eye socket. Flexion disease is rarely fatal, but is associated with significant morbidity. Chikungunya has a latency of about 1-2 weeks. The word "flexion" is believed to stem from the description in local dialects of a distorted posture of a patient afflicted with severe joint pain that accompanies the disease [1-3 ].

Buckling is a potential threat to developing countries because it has the epidemic potential to produce a suddenly debilitating disease, and is also a potential threat to developed countries based on its continued spread and considerable military threat due to soldier deployment in emerging endemic conflict areas. CHIKV infection has a significant impact on economy, as the symptoms of the infection incapacitate employees, leading to epidemic downtime in the endemic area, affecting local commerce. This economic impact is greatest for individual family members who are unable to operate for weeks or months. The lack of specific antiviral treatments and the lack of any currently available vaccines to prevent this disease are major obstacles to managing or controlling the new CHIKV outbreak due to debilitating sequelae of infection.

CHIKV is transmitted by infected mosquito bites. Mosquitoes become infected when they ingest CHIKV to infect individuals. Monkeys and possibly other wildlife may also be infected, but their role as CHIKV depots has not been documented. Infected mosquitoes can then transmit the virus to others when they bite. Aedes aegypti (yellow fever mosquito) is bred in domestic containers and aggressively bites humans during the day, being the main carrier of human CHIKV. Aedes albopictus (Asian tiger mosquito) may also play a role in Asian human transmission, where various forest mosquitoes have been found to be infected with viruses [11-17 ]. Because the CHIK pandemic is maintained by human-mosquito-human transmission, the epidemic cycle is similar to that of dengue fever and urban yellow fever. Large-scale outbreaks of CHIK heat have recently been reported for several islands of indian ocean and india.

Since the end of 2004, chikungunya virus has been reproduced in large-scale outbreaks around the world (mainly in the indian ocean islands). In early 2006, the tengwang island suffered an explosive outbreak after the lower propagation period in winter and with the arrival of summer in the southern hemisphere. Reports estimated that 266,000 residents (sharing 770,000 population) were infected with CHIKV, and 248 certificates of death had CHIKV as the likely cause of death [10, 12 ]. Evidence of intrauterine infection and vertical transmission in pregnant women is documented [12, 13, 17 ]. Sequence analysis revealed the presence of geographically clustered virus lineages. Phylogenetic analysis based on part of the E1 sequence revealed the presence of three different CHIKV populations: one is a west african isolate, the other includes an asian isolate, and one is subdivided into east african, middle african, and south african isolates [15, 17 ].

CHIK fever cases have also been reported in 2006 among travelers returning to europe, canada, caribbean (martinnian) and south america (genu yerana) from known endemic areas [5-9 ]. 2005-2006, 12 cases of CHIK fever [10] were serologically and virologically diagnosed in the center for disease control (USA) among travelers to the USA from known areas of CHIK fever epidemics or endemic disease.

These infections have caused public health risks and have attracted the attention of researchers worldwide. Importantly, most chikungunya virus infections resolve completely within weeks or months. However, CHIKV-induced joint pain cases lasting for years have been documented and have further developed into chronic joint problems. The fact that the infection subsides after a longer period of time demonstrates that the immune system can eventually be integrated to control the infection. Moreover, this clearing phenotype demonstrates a role in T cell response clearance. Attempts to early study chikungunya vaccines, such as formalin inactivated vaccines, tween ether inactivated virus vaccines and attenuated live vaccines, have met with some success, however, these attempts have not been continued for various reasons [3 ]. Moreover, all of these vaccines are reported to produce only serological responses, without inducing useful cellular immunity.

Recent epidemics in the indian ocean and tengwang island indicate that a new vector may carry the virus, since aedes aegypti is not found there. In fact, the related Asian tiger mosquito (Aedes albopictus) may be the chief culprit, and has raised possible concerns over the wide spread of the CHIK virus in the world health world.

Therefore, steps should be taken to investigate the method of controlling CHIKV. Unfortunately, there is currently no specific treatment for chikungunya virus and no vaccine is currently available. Recent studies have shown that the membrane E1-a226V mutation directly results in a significant increase in CHIKV infectivity of aedes albopictus, and further demonstrate that single amino acid substitutions can affect vector specificity. This finding provides a reasonable explanation of how this mutant virus causes epidemics in regions lacking typical insect vectors [18 ]. No specific vaccine or specific antiviral treatment for the fever of flexion has been developed. Live attenuated vaccine trials have been conducted in 2000, but the financial source for the project has been interrupted and no vaccine is currently available. However, there are documents detailing several adverse events associated with this previous vaccine and therefore new vaccine strategies must be developed [3, 5 ].

2005-2007 buckling thermal outbreaks highlighted the need for a safe and effective CHIKV vaccine [6 ]. There remains a need for vaccines that can protect individuals from CHIKV infection. There remains a need for a therapeutic method effective in treating individuals having CHIKV infection.

Summary of The Invention

The present invention relates to a composition comprising an isolated nucleic acid molecule encoding a consensus sequence of CHIKV protein E1 or an immunogenic consensus fragment thereof.

The present invention relates to a composition comprising an isolated nucleic acid molecule encoding a consensus sequence of CHIKV protein E2 or an immunogenic fragment thereof.

The present invention relates to a composition comprising an isolated nucleic acid molecule encoding a consensus sequence of the capsid of the CHIKV protein or an immunogenic consensus fragment thereof.

The present invention relates to a composition comprising an isolated nucleic acid molecule encoding a chimeric gene comprising a consensus sequence of CHIKV protein E1, CHIKV protein E2, and CHIKV protein E3, or homologous sequences thereof, or immunogenic consensus fragments thereof, or homologous sequences thereof, which encode immunogenic amino acid sequences that elicit an immune response to each of CHIKV protein E1, CHIKV protein E2, and CHIKV protein E3.

The present invention relates to injectable pharmaceutical compositions comprising an isolated nucleic acid molecule encoding a consensus sequence of CHIKV protein E1 or an immunogenic consensus fragment thereof.

The present invention relates to injectable pharmaceutical compositions comprising an isolated nucleic acid molecule encoding a consensus sequence of CHIKV protein E2 or an immunogenic consensus fragment thereof.

The present invention relates to injectable pharmaceutical compositions comprising isolated nucleic acid molecules encoding a consensus sequence of the capsid of the CHIKV protein or immunogenic consensus fragments thereof.

The present invention relates to injectable pharmaceutical compositions comprising an isolated nucleic acid molecule encoding a chimeric gene comprising the consensus sequences of CHIKV protein E1, CHIKV protein E2 and CHIKV protein E3 or their homologous sequences or their immunogenic consensus fragments or their homologous sequences of immunogenic consensus fragments, which encode immunogenic amino acid sequences that elicit an immune response to each of CHIKV protein E1, CHIKV protein E2 and CHIKV protein E3.

The invention also relates to a method of inducing an immune response to CHIKV in an individual, comprising administering to the individual a composition comprising an isolated nucleic acid molecule encoding a consensus sequence of CHIKV protein E1, or an immunogenic consensus fragment thereof.

The invention also relates to a method of inducing an immune response to CHIKV in an individual comprising administering to the individual a composition comprising an isolated nucleic acid molecule encoding a consensus sequence of CHIKV protein E2, or an immunogenic consensus fragment thereof.

The invention also relates to a method of inducing an immune response to CHIKV in an individual comprising administering to the individual a composition comprising an isolated nucleic acid molecule encoding a consensus sequence of a CHIKV protein capsid, or an immunogenic consensus fragment thereof.

The invention also relates to a method of inducing an immune response to CHIKV in an individual comprising administering to the individual a composition comprising an isolated nucleic acid molecule encoding a consensus sequence of CHIKV protein E1, CHIKV protein E2, and CHIKV protein E3 or a homologous sequence thereof or an immunogenic consensus fragment thereof or a homologous sequence of an immunogenic consensus fragment thereof, which encode immunogenic amino acid sequences that induce an immune response to each of CHIKV protein E1, CHIKV protein E2, and CHIKV protein E3.

The invention also relates to a recombinant vaccine comprising a nucleotide sequence encoding a consensus sequence of CHIKV protein capsid, CHIKV protein E1, CHIKV protein E2 or immunogenic consensus fragments thereof, or an isolated nucleic acid molecule encoding a consensus sequence of CHIKV protein E1, CHIKV protein E2 and CHIKV protein E3 or homologous sequences thereof or immunogenic consensus fragments thereof or homologous sequences of immunogenic consensus fragments thereof, said sequences encoding immunogenic amino acid sequences that elicit an immune response to each of CHIKV protein E1, CHIKV protein E2 and CHIKV protein E3, and to a method of eliciting an immune response to CHIKV in an individual, comprising administering said recombinant vaccine to an individual.

Brief Description of Drawings

FIG. 1: (A) schematic strategy for cloning the IgE-leader CHIKV fusion gene into the pVax1 vector. (B) Agarose gel photograph showing the linear specific bands of the CHIKV plasmids (envelope E1, E2 and capsid) marked by a double cleavage reaction with Kpn1 and Not1 (lane 4) resulting in sizes of 1403bp, 1355bp and 869bp, respectively.

FIG. 2: characterization of CHIKV constructs. (A) Showing S of the synthetic construct³⁵In vitro translation of the tag. The antigens CHIKV-E1, CHIKV-E2 and CHIKV-capsid were translated and immunoprecipitated using specificity E1, E2 and capsid antibodies, respectively, and electrophoresed through a 12% SDS gel, followed by radiographic analysis. Antigen electrophoresis was performed at its predicted molecular weight, confirming expression. (B) Western blot analysis of CHIKV-E1 and CHIKV-capsid constructs in BHK-21 cells (Western blot analysis). Two days after transfection, transfected cell lysates were prepared and immunoblotted with polyclonal CHIKV-E1 antiserum cultured in mice showed expression of 52kDa E1 protein and 36kDa capsid protein.

FIG. 3: antibody ELISA. (A) Mice (B), (C) and (B) C57BL/6 were immunized twice as indicated with 25. mu.g of pVax1 vector or CHIKV plasmid, 2 weeks apart and sacrificed after 1 week. Sera were collected and analyzed for total IgG production by CHIKV-E1, CHIKV-E2, or CHIKV-capsid. The sera were incubated for 1h at 37 ℃ on 96-well plates coated with 2. mu.g/ml of each CHIKV peptide and the antibodies were detected using anti-mouse IgG-HRP. Values represent the mean (± s.d.) of two replicates.

FIG. 4: interferon-gamma response to envelope E1 as measured by enzyme linked immunospot assay (ELISpot). C57BL/6 mice were immunized twice with 25. mu.g of pVax1 vector or pCHIKV-E1, each at 2-week intervals and sacrificed after 1 week. (A) Splenocytes were harvested and cultured overnight in the presence of R10 (negative control) or one of the 10 μ g/ml four peptide pool consisting of 15 peptides overlapping by 9 amino acids across the length of the E1 protein. The response to CHIKV-E1 is shown as a stacked group mean response. (B) Splenocytes were harvested and cultured overnight in the presence of R10 (negative control) or one of the eighteen peptide pools at 10 μ g/ml consisting of 15 peptides overlapping by 9 amino acids across the length of the matrix E1 protein. Spot Formation Units (SFU) were quantified by an automated ELISpot reading analyzer, and raw values were normalized to SFU/million splenocytes. Values represent the average of three replicates.

FIG. 5: interferon-gamma response to CHIKV envelope E2 measured by enzyme linked immunospot (ELISpot). C57BL/6 mice were immunized twice with 25. mu.g of pVax1 vector or pCHIKV-E2, each at 2-week intervals and sacrificed after 1 week. (A) Splenocytes were harvested and cultured overnight in the presence of R10 (negative control) or one of the 10 μ g/ml four peptide pool consisting of 15 peptides overlapping by 9 amino acids across the length of the E2 protein. The response to CHIKV-E2 is shown as a stacked group mean response. (B) Splenocytes were harvested and cultured overnight in the presence of R10 (negative control) or one of the eighteen peptide pools at 10 μ g/ml consisting of 15 peptides overlapping by 9 amino acids across the length of the matrix E2 protein. Spot Formation Units (SFU) were quantified by an automated ELISpot reading analyzer, and raw values were normalized to SFU/million splenocytes. Values represent the average of three replicates.

FIG. 6: interferon-gamma response to CHIKV-capsid as measured by enzyme linked immunospot (ELISpot). C57BL/6 mice were immunized twice with 25. mu.g of pVax1 vector or pCHIKV-capsid, each at 2-week intervals and sacrificed after 1 week. (A) Splenocytes were harvested and cultured overnight in the presence of R10 (negative control) or one of the 10 μ g/ml four peptide pools consisting of 15 peptides overlapping by 9 amino acids across the length of the capsid protein. The response to CHIKV-capsid is shown as the stacked group mean response. (B) Splenocytes were harvested and cultured overnight in the presence of R10 (negative control) or one of the eighteen peptide pools at 10 μ g/ml consisting of 15 peptides overlapping by 9 amino acids across the length of the matrix capsid protein. Spot Formation Units (SFU) were quantified by an automated ELISpot reading analyzer, and raw values were normalized to SFU/million splenocytes. Values represent the average of three replicates.

Figure 7 is a graph showing neutralizing antibody titers to chikungunya virus in sera of patients and healthy individuals (naive) measured using the assay described in example 2.

Preferred embodiments

As used herein, "immunogenic consensus fragment" means a fragment of CHIKV consensus protein that includes sufficient consensus sequence to provide cross-protection to two or more CHIKV strains that does not arise when the corresponding native sequences are used. Fragments are typically 10 or more amino acids in length. Some preferred CHIKV E1 lengths are at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, 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 95, at least 100, at least 105, at least 110, at least 115, at least 120, at least 125, at least 130, at least 135, at least 140, at least 145, at least 150, at least 155, at least 160, at least 165, at least 170, at least 175, at least 180, at least 185, at least 190, at least 195, at least 200, at least 205, at least 210, at least 215, at least 220, at least 225, at least 230, at least 235, at least 240, at least 245, at least 250, at least 255, at least 260, at least 265, at least 270, at least 275, at least 280, at least 285, at least 290, at least 295, at least 300, at least, At least 320, at least 325, at least 330, at least 335, at least 340, at least 345, at least 350, at least 355, at least 360, at least 365, at least 370, at least 375, at least 380, at least 385, at least 390, at least 395, at least 400, at least 405, at least 410, at least 415, at least 420, at least 425, or at least 430. Some preferred CHIKV E1 lengths are 15 or less, 20 or less, 25 or less, 30 or less, 35 or less, 40 or less, 45 or less, 50 or less, 55 or less, 60 or less, 65 or less, 70 or less, 75 or less, 80 or less, 85 or less, 90 or less, 95 or less, 100 or less, 105 or less, 110 or less, 115 or less, 120 or less, 125 or less, 130 or less, 135 or less, 140 or less, 145 or less, 150 or less, 155 or less, 160 or less, 165 or less, 170 or less, 175 or less, 180 or less, 185 or less, 190 or less, 195 or less, 200 or less, 205 or less, 210 or less, 215 or less, 220 or less, 255 or less, 225 or 230 or less, 260 or less, 240 or less, 250 or less, 80 or less, 85 or less, 90 or less, 95 or less, 100 or less, 150 or less, 155 or less, 160 or less, 165 or less, or the like, 265 or less, 270 or less, 275 or less, 280 or less, 285 or less, 290 or less, 295 or less, 300 or less, 305 or less, 310 or less, 315 or less, 320 or less, 325 or less, 330 or less, 335 or less, 340 or less, 345 or less, 350 or less, 355 or less, 360 or less, 365 or less, 370 or less, 375 or less, 380 or less, 385 or less, 390 or less, 395 or less, 400 or less, 415 or less, 420 or less, 425 or less, 430 or less, or 435 or less. Some preferred CHIKV E2 lengths are at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, 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 95, at least 100, at least 105, at least 110, at least 115, at least 120, at least 125, at least 130, at least 135, at least 140, at least 145, at least 150, at least 155, at least 160, at least 165, at least 170, at least 175, at least 180, at least 185, at least 190, at least 195, at least 200, at least 205, at least 210, at least 215, at least 220, at least 225, at least 230, at least 235, at least 240, at least 245, at least 250, at least 255, at least 260, at least 265, at least 270, at least 275, at least 280, at least 285, at least 290, at least 295, at least 300, at least, At least 320, at least 325, at least 330, at least 335, at least 340, at least 345, at least 350, at least 355, at least 360, at least 365, at least 370, at least 375, at least 380, at least 385, at least 390, at least 395, at least 400, at least 405, at least 410, at least 415, or at least 420. Some preferred CHIKV E2 lengths are 15 or less, 20 or less, 25 or less, 30 or less, 35 or less, 40 or less, 45 or less, 50 or less, 55 or less, 60 or less, 65 or less, 70 or less, 75 or less, 80 or less, 85 or less, 90 or less, 95 or less, 100 or less, 105 or less, 110 or less, 115 or less, 120 or less, 125 or less, 130 or less, 135 or less, 140 or less, 145 or less, 150 or less, 155 or less, 160 or less, 165 or less, 170 or less, 175 or less, 180 or less, 185 or less, 190 or less, 195 or less, 200 or less, 205 or less, 210 or less, 215 or less, 220 or less, 255 or less, 225 or 230 or less, 260 or less, 240 or less, 250 or less, 80 or less, 85 or less, 90 or less, 95 or less, 100 or less, 150 or less, 155 or less, 160 or less, 165 or less, or the like, 265 or less, 270 or less, 275 or less, 280 or less, 285 or less, 290 or less, 295 or less, 300 or less, 305 or less, 310 or less, 315 or less, 320 or less, 325 or less, 330 or less, 335 or less, 340 or less, 345 or less, 350 or less, 355 or less, 360 or less, 365 or less, 370 or less, 375 or less, 380 or less, 385 or less, 390 or less, 395 or less, 400 or less, 415 or less, 422 or less. Some preferred CHIKV capsid lengths are at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, 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 95, at least 100, at least 105, at least 110, at least 115, at least 120, at least 125, at least 130, at least 135, at least 140, at least 145, at least 150, at least 155, at least 160, at least 165, at least 170, at least 175, at least 180, at least 185, at least 190, at least 195, at least 200, at least 205, at least 210, at least 215, at least 220, at least 225, at least 230, at least 235, at least 240, at least 245, at least 250, or at least 255. Some preferred CHIKV capsid lengths are 15 or less, 20 or less, 25 or less, 30 or less, 35 or less, 40 or less, 45 or less, 50 or less, 55 or less, 60 or less, 65 or less, 70 or less, 75 or less, 80 or less, 85 or less, 90 or less, 95 or less, 100 or less, 105 or less, 110 or less, 115 or less, 120 or less, 125 or less, 130 or less, 135 or less, 140 or less, 145 or less, 150 or less, 155 or less, 160 or less, 165 or less, 170 or less, 175 or less, 180 or less, 185 or less, 190 or less, 195 or less, 200 or less, 205 or less, 210 or less, 215 or less, 220 or less, 225 or less, 230 or less, 235 or less, 240 or less, 245 or less, 250 or less, 255 or less, or 260 or less. As used in this paragraph, reference to preferred fragment sizes is intended to refer to all permutations of ranges of at least and less than the ranges in between, for example, the ranges can be from any value listed as "at least" size to any value listed as "less than t" size to provide a range of sizes, for example 20-400, 20-30, 40-100, etc.

The term "genetic construct" as used herein refers to a DNA or RNA molecule comprising a nucleotide sequence encoding a target protein or an immunomodulatory protein. The coding sequence includes initiation and termination signals operably linked to regulatory elements including a promoter and polyadenylation signals capable of directing expression in the cells of an individual to which the nucleic acid molecule is administered.

The term "expressible form" as used herein refers to a genetic construct containing the necessary regulatory elements operably linked to a coding sequence encoding a target protein or an immunomodulatory protein such that the coding sequence is expressed when present in the cells of an individual.

The phrase "stringent hybridization conditions" or "stringent conditions" as used herein refers to conditions under which a nucleic acid molecule hybridizes to another nucleic acid molecule but not to other sequences. Stringent conditions are sequence dependent and will differ under different circumstances. Longer sequences hybridize specifically at higher temperatures. Generally, stringent conditions are selected to be about 5 ℃ lower than the thermodynamic melting point (Tm) of the particular sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength, pH and nucleic acid concentration) at which 50% of a probe complementary to the target sequence hybridizes to the target sequence at equilibrium. Since the target sequence is generally present in excess, at Tm, 50% of the probe is occupied at equilibrium. Generally, stringent conditions are the following: i.e., wherein the salt concentration is less than about 1.0M sodium ions, typically about 0.01 to 1.0M sodium ions (or other salts), and the temperature is at least about 30 ℃ for short probes, primers or oligonucleotides (e.g., 10 to 50 nucleotides) and at least about 60 ℃ for longer probes, primers or oligonucleotides at pH 7.0 to 8.3. Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide.

"homologous" as used herein refers to sequence homology between two nucleic acid sequences or between two amino acid sequences. Two nucleic acid sequences or two amino acid sequences that are sufficiently homologous to allow immunogenic function are "homologous". Nucleotide and amino acid sequence homology can be found using FASTA, BLAST and gapped BLAST (Altschul et al, Nuc. acids Res., 1997, 25, 3389, which is incorporated herein by reference in its entirety) and PAUP^*4.0b10 software (d.l. swofford, Sinauer Associates, Massachusetts). "percent similarity" with PAUP^*4.0b10 software (d.l. swofford, Sinauer Associates, Massachusetts). The average similarity of the consensus sequences was calculated in comparison to all sequences in the phylogenetic tree. Briefly, the BLAST algorithm, which represents a Basic Local Alignment Search Tool (Basic Local Alignment Tool), is suitable for determining sequence similarity (Altschul et al, J.Mol.biol., 1990, 215, 403-. Software for performing BLAST analysis is publicly available through the national center for Biotechnology information (http:// www.ncbi.nlm.nih.gov /). The algorithm involves first determining high scoring sequence pairs (HSPs) by determining short words of length W in a query sequence that matches or satisfies a positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighbor score threshold (Altschul et al, supra). These initial neighborhood word matches serve as seeds for initiating searches to find HSPs containing them. Word matching extends in both directions along each sequence to maximize cumulative alignmentThe score may be increased. The word matching extension for each direction stops if: 1) decreasing the cumulative alignment score from its maximum realizations by an amount X; 2) (ii) the cumulative score reaches zero or less due to accumulation of one or more negative scoring residue alignments; or 3) reaching the end of either sequence. Blast algorithm parameters W, T and X determine the sensitivity and speed of the alignment. The Blast program uses as defaults the following: word length (W) is 11, BLOSUM62 score matrix (see Henikoff et al proc. natl. acad. sci. usa, 1992, 89, 10915-. The BLAST algorithm (Karlin et al, Proc. Natl. Acad. Sci. USA, 1993, 90, 5873-5787, the entire contents of which are incorporated herein by reference) and gapped BLAST perform a statistical analysis of the similarity between two sequences. One measure of similarity provided by the BLAST algorithm is the minimum sum probability (P (N)), which indicates the probability by which a match between two nucleotide sequences occurs by chance. For example, a nucleic acid is considered similar to another nucleic acid if the smallest sum probability in a test nucleic acid compared to the other nucleic acid is less than about 1, preferably less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001.

In some embodiments, DNA vaccines for CHIKV are provided that can be used to immunize an individual. The vaccine includes both humoral and cellular immunity in vivo.

According to some embodiments, to develop immunogens capable of eliciting cross-reactive immune responses to CHIKV virus (CHIKV), we designed consensus constructs against CHIKV virus envelope E1, E2 and the core capsid. For these constructs, 21 sequences were selected from chikungunya virus isolated from 1952 to 2006 that caused infection and death in humans. The DNA sequence of each gene was selected from the S27 strain (first isolate) and strains of various countries (including the pennismus outbreak isolate) to avoid sampling bias. The DNA sequences were aligned and the most common nucleotide at each position was selected for the synthetic sequence. The deduced amino acid sequences are used to guide the introduction of alignment gaps so that they are introduced between codons that maintain the reading frame. After the consensus sequence was generated, the IgE leader was added N-terminally to enhance expression and secretion, and the construct was optimized by codon optimization and replacement of the existing Kozak sequence with a stronger sequence (GCCGCCACC) (FIG. 1A-SEQ ID NO: 18). For analysis, a polyhistidine tag was added to E1 and the C-terminus of the capsid to confirm expression. These constructs were then prepared in bacterial and purification environments according to the previous studies on analysis, expression and immunogenicity [21 ]. FIG. 1B shows agarose gel electrophoresis of constructs encoding envelope E1, E2 and capsid DNA.

According to another embodiment, we designed chimeric consensus constructs for each of CHIKV virus envelopes E1, E2, and E3. For these constructs, consensus sequences for each of E1, E2, and E3 were prepared and ligated to each other, preferably using sequences encoding protease cleavage sites. In addition, the IgE leader sequence was added to the N-terminus to enhance expression and secretion.

In a preferred embodiment, the construct comprises a CHIKV coding sequence linked to an IgE leader sequence. However, in some embodiments, the CHIKV coding sequence is not linked to the IgE leader sequence, but may optionally be linked to a different leader sequence.

SEQ ID NO: 1 refers to the nucleotide sequence encoding the consensus protein CHIKV-E1 linked to the IgE leader.

SEQ ID NO: 2 refers to the nucleotide sequence encoding the consensus protein CHIKV-E2 linked to the IgE leader.

SEQ ID NO: 3 refers to the nucleotide sequence encoding the CHIKV-capsid consensus protein linked to the IgE leader.

SEQ ID NO: 4 refers to a polypeptide corresponding to SEQ ID NO: 1, but without the IgE leader sequence, of CHIKV-E1 consensus protein.

SEQ ID NO: 5 refers to a polypeptide corresponding to SEQ ID NO: 2, but without the IgE leader sequence, and a nucleotide sequence encoding the coding sequence of CHIKV-E2 consensus protein.

SEQ ID NO: 6 refers to a polypeptide corresponding to SEQ ID NO: 13, but without the IgE leader sequence, encoding the CHIKV-capsid consensus protein.

SEQ ID NO: 7 refers to SEQ ID NO: 1, and is the consensus protein of CHIKV-E1 with the IgE leader sequence.

SEQ ID NO: 8 refers to SEQ ID NO: 2, and the amino acid sequence is CHIKV-E2 consensus protein with an IgE leader sequence.

SEQ ID NO: 9 refers to SEQ ID NO: 3, is CHIKV-capsid consensus protein with IgE leader sequence.

SEQ ID NO: 10 refers to SEQ ID NO: 4, is CHIKV-E1 consensus protein without the IgE leader sequence.

SEQ ID NO: 11 refers to SEQ ID NO: 5, is a CHIKV-E2 consensus protein without the IgE leader sequence.

SEQ ID NO: 12 refers to SEQ ID NO: 6, is CHIKV-capsid consensus protein without IgE leader.

SEQ ID NO: 13 refers to a nucleotide sequence encoding consensus Env, Kozak sequence-IgE leader sequence-CHIKV-E3 coding sequence-cleavage site-CHIKV-E2 coding sequence-cleavage site-CHIKV-E1 coding sequence-termination signal.

SEQ ID NO: 14 refers to SEQ ID NO: 13, which is a consensus Env protein sequence, namely an IgE leader sequence-CHIKV-E3-cleavage site-CHIKV-E2-cleavage site-CHIKV-E1 coding sequence.

SEQ ID NO: 15 refers to a polypeptide corresponding to SEQ ID NO: 13, i.e. the coding sequence of CHIKV-E3-cleavage site-CHIKV-E2-cleavage site-CHIKV-E1-coding sequence-termination signal.

SEQ ID NO: 16 refers to SEQ ID NO: 15, which is a consensus Env protein sequence without the IgE leader sequence, i.e., the sequence encoding CHIKV-E3-cleavage site-CHIKV-E2-cleavage site-CHIKV-E1.

SEQ ID NO: 17 refers to the amino acid sequence of the IgE leader sequence.

SEQ ID NO: 18 denotes the preferred Kozak sequence.

When the nucleic acid molecule encoding the consensus protein is taken up by the cells of the individual, the nucleotide sequence encoding the consensus protein is expressed in the cells, thereby delivering the protein to the individual. Aspects of the invention provide methods of delivering a consensus protein coding sequence on a plasmid, or as part of a recombinant vaccine and as part of an attenuated vaccine.

According to some aspects of the invention, compositions and methods are provided for prophylactically and/or therapeutically immunizing an individual against a pathogen or abnormal disease-associated cell. The vaccine may be any type of vaccine, such as an attenuated live vaccine, a recombinant vaccine or a nucleic acid vaccine or a DNA vaccine.

In some embodiments, the vaccine comprises one, two, or all three consensus proteins. In some embodiments, the vaccine comprises coding sequences for two common proteins on the same nucleic acid molecule. In some embodiments, the vaccine comprises coding sequences for two proteins shared in common on two different nucleic acid molecules. In some embodiments, the vaccine comprises coding sequences for three common proteins on the same nucleic acid molecule. In some embodiments, the vaccine comprises coding sequences for three consensus proteins, wherein the coding sequences for two consensus proteins are on the same nucleic acid molecule and the coding sequence for a third consensus protein is on a second nucleic acid molecule. For example, one nucleic acid molecule comprises the coding sequences for E1 and E2 and comprises the coding sequence for a capsid; either one nucleic acid molecule comprises the coding sequence for E1 and the capsid and comprises the coding sequence for E2, or one nucleic acid molecule comprises the coding sequence for E2 and the capsid and comprises the coding sequence for E1. In some embodiments, the vaccine comprises coding sequences for three proteins in common, wherein there are three different nucleic acid molecules, and each nucleic acid molecule comprises a different coding sequence.

In some embodiments, the coding sequence of consensus E1, including the IgE leader sequence, is SEQ ID NO: 1. in some embodiments, the coding sequence of consensus E1 without the IgE leader sequence is SEQ ID NO: 4. in some embodiments, the consensus E1 protein comprising the IgE leader sequence is SEQ ID NO: 7 or a fragment thereof. In some embodiments, the consensus E1 protein is SEQ ID NO: 10 or a fragment thereof. In some embodiments, the consensus E1 protein containing the IgE leader sequence is 80%, 90%, 95%, 98%, or 99% homologous to SEQ ID NO: 7. in some embodiments, the consensus E1 protein without the IgE leader sequence is 80%, 90%, 95%, 98%, or 99% homologous to SEQ ID NO: 10. in some embodiments, the coding sequence of consensus E2, including the IgE leader sequence, is SEQ ID NO: 2. in some embodiments, the coding sequence of consensus E2 without the IgE leader sequence is SEQ ID NO: 5. in some embodiments, the consensus E2 protein comprising the IgE leader sequence is SEQ ID NO: 8 or a fragment thereof. In some embodiments, the consensus E2 protein is SEQ ID NO: 11 or a fragment thereof. In some embodiments, the consensus E2 protein containing the IgE leader sequence is 80%, 90%, 95%, 98%, or 99% homologous to SEQ ID NO: 7. in some embodiments, the consensus E1 protein without the IgE leader sequence is 80%, 90%, 95%, 98%, or 99% homologous to SEQ ID NO: 11. in some embodiments, the coding sequence of the consensus capsid including the IgE leader sequence is SEQ ID NO: 3. in some embodiments, the coding sequence of consensus E1 without the IgE leader sequence is SEQ ID NO: 6. in some embodiments, the consensus E1 protein comprising the IgE leader sequence is SEQ ID NO: 9 or a fragment thereof. In some embodiments, the consensus E1 protein is SEQ ID NO: 12 or a fragment thereof. In some embodiments, the consensus E1 protein containing the IgE leader sequence is 80%, 90%, 95%, 98%, or 99% homologous to SEQ ID NO: 9. in some embodiments, the consensus E1 protein without the IgE leader sequence is 80%, 90%, 95%, 98%, or 99% homologous to SEQ ID NO: 12.

the plurality of genes may be on a single nucleic acid molecule or on a plurality of nucleic acid molecules. For example, one nucleic acid molecule comprises the coding sequences for E1 and E2 and comprises the coding sequence for a capsid; either one nucleic acid molecule comprises the coding sequence for E1 and the capsid and comprises the coding sequence for E2, or one nucleic acid molecule comprises the coding sequence for E2 and the capsid and comprises the coding sequence for E1. In some embodiments, the vaccine comprises coding sequences for three consensus proteins, wherein there are three different nucleic acid molecules and each nucleic acid molecule comprises a different coding sequence. The vaccine may comprise a combination of two or more consensus sequences of E1, E2, and the capsid.

In some embodiments, the vaccine comprises consensus CHIKV Env comprising E1, E2, and E3 linked together as a single chimeric gene. In some embodiments, the individual consensus sequences are linked to each other by a sequence encoding a protease cleavage site. In some embodiments, the chimeric gene comprises a fragment of each of E1, E2, and E3, such that expression of the chimeric gene in an individual results in an immune response to each of E1, E2, and E3. In some embodiments, the chimeric gene comprises SEQ ID NO: 13 or a fragment thereof comprising a sequence sufficient for expression of the protein in an individual to mount an immune response to each of E1, E2, and E3. In some embodiments, the chimeric gene comprises at least 85% homologous to SEQ ID NO: 13 or a fragment thereof comprising a sequence sufficient for expression of the protein in an individual to mount an immune response to each of E1, E2, and E3. In some embodiments, the chimeric gene comprises at least 90% homologous to SEQ ID NO: 13 or a fragment thereof comprising a sequence sufficient for expression of the protein in an individual to mount an immune response to each of E1, E2, and E3. In some embodiments, the chimeric gene comprises at least 95% homologous to SEQ ID NO: 13 or a fragment thereof comprising a sequence sufficient for expression of the protein in an individual to mount an immune response to each of E1, E2, and E3. In some embodiments, the chimeric gene comprises at least 98% homologous to SEQ ID NO: 13 or a fragment thereof comprising a sequence sufficient for expression of the protein in an individual to mount an immune response to each of E1, E2, and E3. In some embodiments, the chimeric gene comprises at least 99% homologous to SEQ ID NO: 13 or a fragment thereof comprising a sequence sufficient for expression of the protein in an individual to mount an immune response to each of E1, E2, and E3. In some embodiments, the chimeric gene comprises SEQ ID NO: 15 or a fragment thereof comprising a sequence sufficient for expression of the protein in an individual to mount an immune response to each of E1, E2, and E3. In some embodiments, the chimeric gene comprises at least 85% homologous to SEQ ID NO: 15 or a fragment thereof comprising a sequence sufficient for expression of the protein in an individual to mount an immune response to each of E1, E2, and E3. In some embodiments, the chimeric gene comprises at least 90% homologous to SEQ ID NO: 15 or a fragment thereof comprising a sequence sufficient for expression of the protein in an individual to mount an immune response to each of E1, E2, and E3. In some embodiments, the chimeric gene comprises at least 95% homologous to SEQ ID NO: 15 or a fragment thereof comprising a sequence sufficient for expression of the protein in an individual to mount an immune response to each of E1, E2, and E3. In some embodiments, the chimeric gene comprises at least 98% homologous to SEQ ID NO: 15 or a fragment thereof comprising a sequence sufficient for expression of the protein in an individual to mount an immune response to each of E1, E2, and E3. In some embodiments, the chimeric gene comprises at least 99% homologous to SEQ ID NO: 15 or a fragment thereof comprising a sequence sufficient for expression of the protein in an individual to mount an immune response to each of E1, E2, and E3.

In some embodiments, the vaccine comprises consensus CHIKV Env encoding the consensus amino acid sequences of E1, E2, and E3 linked together. In some embodiments, the individual consensus sequences are linked to each other by a sequence encoding a protease cleavage site. In some embodiments, the chimeric gene encodes a fragment of each of E1, E2, and E3, wherein the amino acid sequence encoded thereby is immunogenic and elicits an immune response to each of E1, E2, and E3. In some embodiments, the consensus protein CHIKV Env comprises SEQ ID NO: 14 or a fragment thereof comprising sufficient sequence wherein the amino acid sequence encoded thereby is immunogenic and elicits an immune response to each of E1, E2, and E3. In some embodiments, the CHIKV Env consensus protein comprises at least 85% homologous to SEQ ID NO: 14 or a fragment thereof, wherein the amino acid sequence encoded thereby is immunogenic and elicits an immune response to each of E1, E2, and E3. In some embodiments, the CHIKV Env consensus protein comprises at least 90% homologous to SEQ ID NO: 14 or a fragment thereof, wherein the amino acid sequence encoded thereby is immunogenic and elicits an immune response to each of E1, E2, and E3. In some embodiments, the CHIKV Env consensus protein comprises at least 95% homologous to SEQ ID NO: 14 or a fragment thereof, wherein the amino acid sequence encoded thereby is immunogenic and elicits an immune response to each of E1, E2, and E3. In some embodiments, the CHIKV Env consensus protein comprises at least 98% homologous to SEQ ID NO: 14 or a fragment thereof, wherein the amino acid sequence encoded thereby is immunogenic and elicits an immune response to each of E1, E2, and E3. In some embodiments, the CHIKV Env consensus protein comprises at least 99% homologous to SEQ ID NO: 14 or a fragment thereof, wherein the amino acid sequence encoded thereby is immunogenic and elicits an immune response to each of E1, E2, and E3. In some embodiments, the CHIKV Env consensus protein comprises at least 85% homologous to SEQ ID NO: 16 or a fragment thereof, wherein the amino acid sequence encoded thereby is immunogenic and elicits an immune response to each of E1, E2, and E3. In some embodiments, the CHIKV Env consensus protein comprises at least 90% homologous to SEQ ID NO: 16 or a fragment thereof, wherein the amino acid sequence encoded thereby is immunogenic and elicits an immune response to each of E1, E2, and E3. In some embodiments, the CHIKV Env consensus protein comprises at least 95% homologous to SEQ ID NO: 16 or a fragment thereof, wherein the amino acid sequence encoded thereby is immunogenic and elicits an immune response to each of E1, E2, and E3. In some embodiments, the CHIKV Env consensus protein comprises at least 98% homologous to SEQ ID NO: 16 or a fragment thereof, wherein the amino acid sequence encoded thereby is immunogenic and elicits an immune response to each of E1, E2, and E3. In some embodiments, the CHIKV Env consensus protein comprises at least 99% homologous to SEQ ID NO: 14 or a fragment thereof, wherein the amino acid sequence encoded thereby is immunogenic and elicits an immune response against each of E1, E2, and E3.

Nucleic acid molecules can be delivered using any of several known techniques, including DNA injection (also known as DNA vaccination), recombinant vectors (e.g., recombinant adenoviruses, recombinant adeno-associated viruses, and recombinant vaccinia viruses).

DNA vaccines are described in U.S. patent nos. 5,593,972, 5,739,118, 5,817,637, 5,830,876, 5,962,428, 5,981,505, 5,580,859, 5,703,055, 5,676,594 and the priority applications cited therein, which are all incorporated herein by reference. In addition to the delivery protocols described in those applications, alternative methods of delivering DNA are described in U.S. patent nos. 4,945,050 and 5,036,006, both of which are incorporated herein by reference.

Routes of administration include, but are not limited to, intramuscular, intranasal, intraperitoneal, intradermal, subcutaneous, intravenous, intraarterial, intraocular and oral as well as topical, transdermal, by inhalation or suppository or to mucosal tissues (e.g., by lavage to vaginal, rectal, urethral, buccal and sublingual tissues). Preferred routes of administration include injection into mucosal tissue, intramuscular, intraperitoneal, intradermal and subcutaneous. The genetic construct may be administered by means including, but not limited to: conventional syringes, needle-less injection devices or "microprojectile bombardment gene guns".

Another route of administration involves the use of electroporation to deliver gene constructs, described in U.S. Pat. nos. 5,273,525, 5,439,440, 5,702,359, 5,810,762, 5,993,434, 6,014,584, 6,055,453, 6,068,650, 6,110,161, 6,120,493, 6,135,990, 6,181,964, 6,216,034, 6,233,482, 6,241,701, 6,347,247, 6,418,341, 6,451,002, 6,516,223, 6,567,694, 6,569,149, 6,610,044, 6,654,636, 6,678,556, 6,697,669, 6,763,264, 6,778,853, 6,939,416, 6,939,862, and 6,958,060, which are hereby incorporated by reference.

Examples of electroporation devices and electroporation methods that are preferred for facilitating DNA vaccine delivery include those described in: U.S. Pat. No. 7,245,963 to Draghia-Akli et al, U.S. patent publication 2005/0052630 to Smith et al, the contents of both of which are hereby incorporated by reference in their entirety. Also preferred are electroporation devices and methods for facilitating DNA vaccine delivery provided in co-pending and commonly owned U.S. patent application serial No. 11/874072 filed on day 10/17 of 2007, which is in accordance with the claims of 35 USC 119(e) to U.S. provisional application serial No. 60/852,149 filed on day 10/17 of 2006 and U.S. provisional application serial No. 60/978,982 filed on day 10/10 of 2007, all of which are hereby incorporated by reference in their entirety.

The following is an example of an embodiment using electroporation techniques, and is discussed in more detail in the above-mentioned patent references: the electroporation device may be configured to deliver a pulse of energy to a desired mammalian tissue that produces a constant current similar to a user-preset current input. The electroporation device includes an electroporation component and an electrode member or a handle member. The electroporation component may include and incorporate one or more of the various elements of the electroporation device, including: the device comprises a controller, a current waveform generator, an impedance tester, a waveform recorder, an input element, a state reporting element, a communication port, a memory component, a power supply and a power switch. The electroporation component can function as one element of the electroporation device, and the other element is a separate element (or component) in communication with the electroporation component. In some embodiments, the electroporation component can function as more than one element of the electroporation device, which can also be in communication with other elements of the electroporation device that are separate from the electroporation component. Delivery of vaccines using electroporation is not limited by the elements of the electroporation device being present as part of an electromechanical or mechanical device, as the elements can function as one device or in communication with each other with separate elements. The electroporation component is capable of delivering an energy pulse that produces a constant current in the desired tissue and includes a feedback mechanism. The electrode assembly includes an electrode array having a plurality of electrodes arranged in a space, wherein the electrode assembly receives the energy pulse from the electroporation component and delivers it to the desired tissue via the electrodes. At least one of the plurality of electrodes is neutral in energy pulse delivery and measures impedance in the desired tissue and communicates the impedance to the electroporation component. A feedback mechanism may receive the measured impedance and may adjust the energy pulse delivered by the electroporation component to maintain a constant current.

In some embodiments, the plurality of electrodes may deliver the pulse of energy in a dispersed pattern. In some embodiments, the plurality of electrodes may deliver the energy pulses in a decentralized pattern by controlling the electrodes in a programmed sequence that is input to the electroporation component by a user. In some embodiments, the programming sequence includes a plurality of pulses delivered sequentially, wherein each pulse of the plurality of pulses is delivered by at least two active electrodes (neutral electrodes with one measured impedance), and wherein a next pulse of the plurality of pulses is delivered by a different one of the at least two active electrodes (neutral electrodes with one measured impedance).

In some embodiments, the feedback mechanism is performed by hardware or software. Preferably, the feedback mechanism is performed by an analog closed loop circuit. Preferably, this feedback occurs every 50, 20, 10 or 1 mus, but is preferably real-time or immediate (i.e., determined to be substantially immediate by measuring response time using prior art techniques). In some embodiments, the neutral electrode measures impedance in the desired tissue and communicates the impedance to a feedback mechanism, and the feedback mechanism responds to the impedance and adjusts the energy pulse to maintain the constant current at a value similar to the preset current. In some embodiments, the feedback mechanism continuously and instantaneously maintains a constant current during the delivery of the energy pulse.

When taken up by a cell, the gene construct may remain present in the cell as a functional extrachromosomal molecule. The DNA may be introduced into the cells in the form of a plasmid, where it is transiently present. Alternatively, the RNA may be administered to the cell. It is also contemplated to provide the gene construct as a linear minichromosome, including a centromere, a telomere, and an origin of replication. The genetic construct may constitute part of the genetic material in a vector of a live attenuated microorganism or recombinant microorganism that is administered to a subject. The genetic construct may be part of the genome of a recombinant viral vaccine, wherein the genetic material is maintained extrachromosomally. The gene construct includes regulatory elements necessary for gene expression of the nucleic acid molecule. The element comprises: a promoter, a start codon, a stop codon, and a polyadenylation signal. In addition, enhancers are often required for gene expression of sequences encoding target proteins or immunomodulatory proteins. It is essential that these elements be operably linked to the sequence encoding the desired protein and that the regulatory elements be operable in the individual to whom they are administered.

The start codon and stop codon are generally considered to be part of the nucleotide sequence encoding the desired protein. However, it is essential that these elements are functional in the individual to whom the gene construct is administered. The initiation codon and the stop codon must be in frame with the coding sequence.

The promoter and polyadenylation signal used must be functional within the cells of the individual.

Examples of promoters useful in the practice of the present invention, particularly in the production of human gene vaccines, include, but are not limited to, promoters from simian virus 40(SV40), Mouse Mammary Tumor Virus (MMTV), human immunodeficiency virus (MV) (e.g., BIV Long Terminal Repeat (LTR) promoter), moloney virus, ALV (avian leukemia virus), Cytomegalovirus (CMV) (e.g., CMV immediate early promoter), Epstein Barr Virus (EBV), Rous Sarcoma Virus (RSV), and promoters from human genes such as human actin, human myosin, human heme, human muscle creatine, and human metallothionein.

Examples of polyadenylation signals that may be used in the practice of the present invention, particularly in the production of human gene vaccines, include, but are not limited to, the SV40 polyadenylation signal, the calf growth hormone polyadenylation (bgh-PolyA) signal, and the LTR polyadenylation signal. In particular, the SV40 polyadenylation signal, known as the SV40 polyadenylation signal in the pCEP4 plasmid (Invitrogen, San Diego Calif.), was used.

In addition to the regulatory elements required for DNA expression, other elements may be included in the DNA molecule. Such other elements include enhancers. Enhancers may be selected from the group including, but not limited to: human actin, human myosin, human heme, human muscle creatine and viral enhancers (e.g. those from CMV, RSV and EBV).

The genetic construct may have a mammalian origin of replication to maintain the construct extrachromosomally and to produce multiple copies of the construct within the cell. Plasmids pVAX1, pCEP4 and pREP4 from Invitrogen (San Diego, Calif.) contain the EB virus origin of replication and the nuclear antigen EBNA-1 coding region, which produces high copies of non-integrating episomal replication.

In some preferred embodiments for immunological applications, the delivered nucleic acid molecule comprises a nucleotide sequence encoding a consensus protein and additionally comprises a protein gene that further enhances the immune response to such target protein. Examples of such genes are those encoding other cytokines and lymphokines such as alpha-interferon, gamma-interferon, Platelet Derived Growth Factor (PDGF), TNF, GM-CSF, Epidermal Growth Factor (EGF), IL-1, IL-2, II-4, IL-6, IL-10, IL-12, IL-15, IL-28, including a deletion of the signal sequence and optionally IL-15 from the IgE signal peptide.

The compositions used in the present methods may further comprise one or more of the following proteins and/or nucleic acid molecules encoding such proteins, as set forth in U.S. serial No. 10/139,423, corresponding to U.S. publication No. 20030176378 (which is incorporated herein by reference): major histocompatibility complex antigens including major histocompatibility complex class I antigens or major histocompatibility complex class II antigens; death domain receptors including, but not limited to, Apo-1, Fas, TNFR-1, p55, WSL-1, DR3, TRAMP, Apo-3, AIR, LARD, NGRF, DR4, DR5, KILLER, TRAIL-R2, TRICK2, and DR 6; death signals, i.e., proteins that interact with death domain receptors, including but not limited to FADD, FAP-1, TRADD, RIP, FLICE, and RAIDD; or death signals including ligands that bind to death domain receptors and initiate apoptosis, including but not limited to FAS-L and TNF; and mediators of interaction with death domain receptors including, but not limited to, FADD, MORT1, and MyD 88; toxins, including cell killing proteins, include, but are not limited to, insect and snake venom, bacterial exotoxins (e.g., pseudomonas exotoxin), double-stranded ribosome inactivating proteins (e.g., ricin), including single-chain toxins and gelonin.

The compositions used in the present methods may further comprise one or more of the following proteins and/or nucleic acid molecules encoding such proteins, as set forth in U.S. serial No. 10/560,650, corresponding to U.S. publication No. 20070041941 (which is incorporated herein by reference): IL-15, including fusion proteins comprising a non-IL-15 signal peptide linked to an IL-15 protein sequence, e.g., a fusion protein comprising an IgE signal peptide linked to an IL-15 protein sequence, CD40L, TRAIL; RAILreeDRC5, TRAIL-R5, RANK, 5, Ox40LIGAND, NKG 25, F461811 or MICA, MICB, NKG 25, CD153(CD30 5), Fos, c-jun, Sp-1, Ap 5, Ap-2, p5, p65Rel, MyD 5, IRAK, TRAF 5, IkB, NIK, SAP 5, JNK1B 5, JNK2B 5, JNK1A κ 5, JNK2A 5, JNK3A 5, K3A 3B 5, TNF-NF-P-3, TNF-NF-P-3, human-NF-P-3, TNF-P-3, human-P-3, TNF-3-P-3, human-3-NF-3, human-3-NF-3, human-3, human, MadCAM-1, NGF IL-7, VEGF, TNF-R, Fas, CD40L, IL-4, CSF, G-CSF, GM-CSF, M-CSF, LFA-3, ICAM-2, ICAM-1, PECAM, P150.95, Mac-1, LFA-1, CD34, RANTES, IL-8, MIP-1 α, E-selector, CD2, MCP-1, L-selector, P-selector, FLT, Apo-1, Fas, TNFR-1, P55, WSL-1, DR3, TRAMP, Apo-3, AIR, LARD, NGRF, DR4(TRAIL), DR5, KIER, TRAIL-R2, TRICK2, DR6, ICE, VLA-1 and CD86 (B7.2).

The compositions used in the present methods may further comprise one or more of the following proteins and/or nucleic acid molecules encoding such proteins, as set forth in U.S. serial No. 10/560,653, corresponding to U.S. publication No. 20070104686 (which is incorporated herein by reference): fos, c-jun, Sp-1, Ap-2, p38, p65Rel, MyD88, IRAK, TRAF6, IkB, inactivated NIK, SAP K, SAP-1, JNK, interferon responsive genes, NFkB, Bax, TRAIL, TRAILrec 5, TRAIL-R3, TRAIL-R4, RANK, RANK LIGAND, Ox40, Ox40LIGAND, NKG2D, MICA, MICB, NKG2A, NKG2B, NKG2C, NKG2E, NKG2F, TAP1 and TAP 2.

If it is desired for any reason to eliminate the cells receiving the gene construct, additional elements serving as targets for cell destruction may be added. The herpes virus thymidine kinase (tk) gene may be included in the genetic construct in an expressible form. The drug ganciclovir can be administered to an individual and causes selective killing of any cells that form tk, thereby providing a means to selectively destroy cells with the genetic construct.

To maximize protein production, regulatory sequences well suited for gene expression in the cell to which the construct is administered may be selected. Furthermore, codons may be selected which are most efficiently transcribed in the cell. One of ordinary skill in the art can prepare DNA constructs that are functional in a cell.

In some embodiments, a genetic construct may be provided to produce a coding sequence for an immunomodulatory protein described herein linked to an IgE signal peptide.

One method of the invention comprises the step of administering the nucleic acid molecule intramuscularly, intranasally, intraperitoneally, subcutaneously, intradermally, or topically or by lavage to mucosal tissue selected from the group consisting of inhalation, vaginal, rectal, urethral, buccal, and sublingual.

In some embodiments, the nucleic acid molecule is delivered to the cell in conjunction with administration of a polynucleotide function enhancer or gene vaccine facilitator. Polynucleotide function enhancers are described in U.S. patent nos. 5,593,972 and 5,962,428, both of which are incorporated herein by reference. Genetic vaccine promoters are described in U.S. patent No. 5,739,118, which is incorporated herein by reference. The adjuncts administered in conjunction with the nucleic acid molecules may be administered as a mixture with the nucleic acid molecules or separately at the same time, before or after administration of the nucleic acid molecules. In addition, other agents that may be used as transfection and/or replication agents and/or inflammatory agents and that may be co-administered with polynucleotide function enhancers include growth factors, cytokines and lymphokines, such as alpha-interferon, gamma-interferon, GM-CSF, Platelet Derived Growth Factor (PDGF), TNF, Epidermal Growth Factor (EGF), IL-1, IL-2, IL-4, IL-6, IL-10, IL-12, and IL-15, as well as fibroblast growth factors, surfactants (e.g., Immune Stimulation Complexes (ISCOMS)), LPS analogs (including monophosphoryl lipid A (WL)), muramyl peptides, quinone analogs, and small vesicles such as squalene, hyaluronic acid may also be used in conjunction with the administration of the gene construct. In some embodiments, an immunomodulatory protein can be used as an enhancer of polynucleotide function. In some embodiments, the nucleic acid molecule is provided in combination with a (lactide-glycolide) copolymer (PLG) to enhance delivery/uptake.

The pharmaceutical composition according to the invention comprises about 1 nanogram to about 2000 micrograms of DNA. In some preferred embodiments, the pharmaceutical composition according to the invention comprises from about 5 nanograms to about 1000 micrograms of DNA. In some preferred embodiments, the pharmaceutical composition contains from about 10 nanograms to about 800 micrograms of DNA. In some preferred embodiments, the pharmaceutical composition contains about 0.1 to about 500 micrograms of DNA. In some preferred embodiments, the pharmaceutical composition contains about 1 to about 350 micrograms of DNA. In some preferred embodiments, the pharmaceutical composition contains about 25 to about 250 micrograms of DNA. In some preferred embodiments, the pharmaceutical composition contains about 100 to about 200 micrograms of DNA.

The pharmaceutical composition according to the invention is formulated according to the mode of administration to be used. Where the pharmaceutical compositions are injectable pharmaceutical compositions, they are sterile, pyrogen-free and particulate-free. Preferably, isotonic formulations are used. In general, isotonic additives may include sodium chloride, glucose, mannitol, sorbitol, and lactose. In some cases, isotonic solutions such as phosphate buffered saline are preferred. Stabilizers include gelatin and albumin. In some embodiments, a vasoconstrictor is added to the formulation.

According to some embodiments of the invention, there is provided a method of inducing an immune response to chikungunya virus. The vaccine may be a live attenuated vaccine, a recombinant vaccine or a nucleic acid or DNA vaccine.

The nucleic acid molecule may be provided as a plasmid DNA, a nucleic acid molecule of a recombinant vector or as part of the genetic material provided in an attenuated vaccine. Alternatively, in some embodiments, the consensus proteins may be delivered as proteins accompanying or replacing the nucleic acid molecules encoding them.

The genetic construct may comprise a nucleotide sequence encoding a target protein or an immunomodulatory protein operably linked to regulatory elements required for gene expression. According to the present invention, there is provided a combination of genetic constructs, including one construct comprising an expressible form of a nucleotide sequence encoding a target protein and one construct comprising an expressible form of a nucleotide sequence encoding an immunomodulatory protein. Delivery of a DNA or RNA molecule comprising a combination of gene constructs into a living cell results in expression of the DNA or RNA and production of the target protein and one or more immunomodulatory proteins. Resulting in an enhanced immune response to the target protein.

In addition to the use of expressible forms of immunomodulatory protein coding sequences to improve genetic vaccines, the invention also relates to improved live attenuated vaccines and improved vaccines that use recombinant vectors to deliver foreign genes encoding antigens. Live attenuated vaccines and vaccines that use recombinant vectors to deliver foreign antigens are described in U.S. patent nos.: 4,722,848; 5,017,487, respectively; 5,077,044, respectively; 5,110,587; 5,112,749, respectively; 5,174,993; 5,223,424, respectively; 5,225,336, respectively; 5,240,703, respectively; 5,242,829, respectively; 5,294,441, respectively; 5,294,548, respectively; 5,310,668, respectively; 5,387,744, respectively; 5,389,368, respectively; 5,424,065, respectively; 5,451,499, respectively; 5,453,364, respectively; 5,462,734, respectively; 5,470,734, respectively; and 5,482,713, all of which are incorporated herein by reference. Gene constructs comprising nucleotide sequences encoding consensus proteins, or immunogenic consensus fragments thereof, operably linked to regulatory sequences that can function in vaccines to achieve expression are provided. The gene constructs are incorporated into attenuated live vaccines and recombinant vaccines to produce improved vaccines according to the invention.

The present invention provides improved methods of immunizing individuals comprising the step of delivering gene constructs to the cells of individuals as part of vaccine compositions, including DNA vaccines, attenuated live vaccines and recombinant vaccines. The genetic construct comprises a nucleotide sequence encoding a consensus protein, or immunogenic consensus fragment thereof, operably linked to regulatory sequences that can function in a vaccine to achieve expression. The vaccine forms a cross-protection against different strains.

Examples

Example 1

Here we provide data for a novel consensus-based method for CHIKV vaccine design using DNA vaccine strategy. Vaccine cassettes were designed based on the consensus sequence specific for the CHIKV capsid and the envelope with several modifications. Expression of capsid, envelope E1 and E1 was evaluated using T7-coupled transcription/translation and immunoblot analysis. C57BL/6 mice were immunized by intramuscular injection of plasmids encoding CHIK-capsid, E1 and E1 using adaptive constant current electroporation technique. Analysis of cellular immune responses (including epitope mapping) showed that electroporation of these constructs elicited robust and broad cellular immunity. In addition, antibody ELISA demonstrated that these synthetic immunogens were able to elicit high titers of antibodies that recognize the native antigen. Taken together, these data support further studies using CHIK consensus antigens in potential vaccine mixtures.

In this study, we designed a vaccine cassette based on a consensus sequence and a substituted immunoglobulin E leader sequence with several modifications including codon optimization, RNA optimization, addition of Kozak sequences, specific for capsid (Cap) and envelope (E1) and envelope (E2). The vaccine cassette was introduced into the DNA vaccine vector pVax1 at a specific site. Inserts of vaccine constructs were checked using specific restriction enzyme and primer sequencing using the T7 promoter. The final construct was expressed efficiently based on in vitro expression and using in vivo western blot analysis. This confirms that the viral construct was correctly expressed and processed for further immunogenicity studies.

Recently, the use of EP to deliver DNA vaccines has attracted considerable attention. Recent studies of IM immunization + EP in small animal models and non-human primates consistently report increases in cellular responses, particularly antibody responses [20-22 ].

Materials and methods

Cells and animals:

the BHK-21 cell line obtained from ATCC was cultured and maintained in DMEM medium supplemented with 10% fetal bovine serum. Mammalian plasmid expression vector pVax1 was purchased from Invitrogen ((Carlsbad, CA). 3-4 week old female C57BL/6 mice (Jackson laboratories, Indianapolis, IN) for these experiments and divided into three experimental groups (n4) all animals were housed IN a temperature controlled light-cycle facility according to the guidelines of the Institutional Animal Care and Use Committee (IACUC) of the national institute of health (Bethesda, MD, USA) and the university of pennsylvania (Philadelphia, PA, USA).

CHIKV DNA construct and synthesis:

the CHIKV core and envelope genes were designed by synthesis of synthetic primers followed by DNA-PCR amplification using consensus strain prediction sequences gleaned from the NCBI database of all CHIKV viruses. The consensus sequence was optimized for expression, which included codon and RNA optimization (GeneArt, Regensburg, Germany), followed by insertion into the pVax1 expression vector (Invitrogen).

In vitro and in vivo expression:

construct expression was confirmed using the T7 promoter in the pVax1 backbone and a coupled T7-based transcription/translation system (Promega, Madison, WI) containing the S35-methionine CHIKV gene. The synthesized proteins were immunoprecipitated using anti-E1 antibody, anti-E2 antibody, or anti-capsid antibody. Immunoprecipitated proteins were electrophoresed on 12% NuPage SDS-PAGE gels (Invitrogen, CA) and subsequently fixed and dried. Autoradiography was performed to detect the incorporated S35-labeled gene product. In vivo expression, BHK-21 cells (1X 10)⁶) Transfection with CHIKV construct was performed using Fugene transfection method (Roche, NJ). 72 hours after transfection, proteins (50. mu.g) were fractionated on SDS-PAGE (12%) and transferred to PVDF membrane (Bio-Rad, Hercules, Calif.). Immunoblot analysis was performed using specific antisera cultured in mice, and the expressed proteins were visualized with horseradish peroxidase-conjugated goat anti-mouse IgG using an ECL detection system (Amersham Pharmacia Biotech, Piscataway, N.J.) [19]。

Immunization and electroporation

Animals were primed with plasmid DNA using standard protocols [20 ]. Groups of four mice were immunized twice, 2-3 times, with the pCHIKV gene (25. mu.g), spaced 2 weeks apart, and sacrificed 1 week after the last immunization. All immunizations were delivered to the quadriceps muscle by in vivo Electroporation (EP) (VGX Pharmaceuticals Inc, Blue Bell, PA) in a total volume of 100 μ Ι. Animals were sacrificed 7 days after the last immunization, after which serum and spleen were collected for immunological analysis. Blood was obtained from control and immunized mice 1 week after the second and third immunizations, respectively. Square wave pulses were used in all experiments and delivered using a constant current EKD designed and tested in our laboratory [20-22 ]. A three-electrode array (3-EA) was used in the mouse experiments. The 3-EA is composed of three 26-gauge solid stainless steel electrodes, forms an isosceles triangle, has two long sides of 0.5mm and a short side of 0.3mm, and is fixed together by non-conductive plastic. The specific EP conditions for the mouse experiments were 0.1 amp, three pulses, 52 ms/pulse with 4 seconds pulse intervals using a constant current. The delay time between plasmid injection and EP was about 20 seconds. The sequence of events for plasmid administration/EP was as follows: the disposable electrode assembly was placed in the handle container, the start button on the handle was pressed and the animal experimental group number was entered, 50 μ l of DNA construct (25 μ g total DNA) plasmid was injected using an insulin syringe, the needle was immediately placed in the area around the injection site, the start button on the handle was pressed, and after 4 seconds of countdown, the pulse was delivered. After 5 seconds of electroporation, the array was gently removed from the muscle. All electrodes were fully inserted into the muscle during all treatments [21, 22 ]. All DNA was prepared using endotoxin free Qiagen columns. All animals were housed in a temperature controlled light cycle facility at pennsylvania university and were managed according to guidelines of the american national institute of health and pennsylvania university.

Cellular response: ELISPOT assay

ELISPOT assay was performed as previously described [23]. Briefly, ELISpot 96-well plates (microwells) were coated with anti-mouse IFN-. gamma.capture antibody and incubated at 4 ℃ for 24h (R)&D Systems). The next day, plates were washed and blocked with 1% BSA for 2 h. 20 ten thousand splenocytes from immunized mice were added to each well and 5% CO at 37 deg.C₂In the presence of RPMI 1640 (negative control), ConA (positive control) or a specific peptide antigen (10. mu.g/ml; Invitrogen) overnight. The peptide library consisted of 15 peptides overlapping by 11 amino acids. After 24h of stimulation, the cells were washed and then incubated with biotinylated anti-mouse IFN-. gamma.antibody (R) at 4 ℃&D Systems) were incubated together for 24 h. Plates were washed and streptavidin-alkaline phosphatase (R) was added&D Systems) were added to each well and incubated at room temperature for 2 h. The plate was washed and 5-bromo-4-chloro-3' -indolylphosphonate p-toluidine salt and tetrazolium chloride blue (chromogen developer; R) were added to each well&D Systems). The plates were then rinsed with distilled water and dried at room temperature. Spots were counted by an automated ELISPOT reader (CTL Limited) [21-23 ]]。

Humoral immune response: antibody ELISA

Antibody levels and humoral immune responses to vaccination were determined for each CHIKV DNA construct after the first injection of each DNA. Briefly, 96-well high-binding polystyrene plates (Corning, NY) were coated overnight at 4 ℃ with synthetic specific peptide (2. mu.g/ml) diluted in PBS. The next day, plates were washed with PBST (PBS, 0.05% Tween 20), blocked with 3% BSA in PBST for 1h, and incubated with 1: 100 dilutions of sera from immunized and naive mice for 1h at 37 ℃. Bound IgG was detected using a 1: 5,000 dilution of goat anti-mouse IgG-HRP (Research Diagnostics, NJ). Bound enzyme was detected by addition of chromogen base solution TMB (R & D Systems) and read at 450nm on a Biotek EL312e Bio-Kinetics reader. All serum samples were tested in duplicate [22 ].

Results

Consensus construct expression:

expression of the three CHIKV consensus constructs was verified using a variety of techniques. To visualize the proteins produced in vitro, S is performed³⁵Labeled in vitro T7 coupled with transcriptional and translational analysis. The translation product was immunoprecipitated using a histidine-tag antibody and subjected to gel analysis. SDS-PAGE and radiographic analysis showed that electrophoresis of each construct (envelope E1, E2 and capsid) was performed at its theoretically predicted molecular weight (FIG. 2A). We next attempted to examine the expression of these constructs in vivo in mammalian cells. After transfection into BHK-21 cells, the protein was extracted 3 days later and expression was detected by western blot analysis using specific polyclonal antibodies (fig. 2B). Upon immunoblotting with specific antibodies, a 52kDa protein was observed in cells transfected with the envelope E1 construct and a 36kDa protein was observed in cells transfected with the capsid construct.

Humoral immunogenicity

We hypothesize that the strength of our common immunogen to defend against the virulent CHIK virus depends on the cellular immune part of the immune system. Moreover, cross-reactive but non-neutralizing antibodies may provide some degree of protection against disease severity. To determine whether our constructs elicit an antibody response, we performed an antibody ELISA on CHIK-immunized mouse sera to determine the antibody titer of sera obtained after DNA immunization, and tested the antibody response by ELISA. anti-E1 specific IgG antibodies in the sera of mice immunized with envelope E1 were significantly higher than in the sera of mice immunized with vehicle control (fig. 3A). Similarly, anti-E2-specific IgG antibodies and capsid-specific IgG antibodies in the sera of mice immunized with envelope E2 and capsid construct, respectively, were significantly higher than in the sera of mice immunized with vector control (fig. 3B & C). These results further support: alternative means of plasmid delivery, particularly electroporation, increase antibody production in response to DNA vaccine immunogens.

Cellular immunogenicity

The ability of E1, E2, and the capsid construct to induce CD8+ CTL responses was determined by IFN- γ ELISpot analysis. Consensus envelope constructs E1, E2 and capsid vaccine were able to induce strong IFN- γ responses in C57BL/6 mice after three immunizations (fig. 4A, 5A and 6A). For molecular characterization of the cellular immune response induced by membrane E1, ELISpot analysis was performed against a peptide library spanning the entire membrane E1. 74 15 peptides spanning residues 1-435 of the E1 protein (9 amino acid overlaps between them) and peptides spanning residues 1-423 of the E2 protein were used. The envelope induces the dominant epitope HSMTNAVTI in the E1 protein (fig. 4B) and IILYYYELY in the E2 protein (fig. 5B). Similarly, for capsid proteins, ELIspot analysis was performed on peptide libraries spanning the entire capsid protein. 45 15 peptides spanning capsid protein residues 1-261 were used, with a 9 amino acid overlap between them. Construct capsids induced dominant epitope ACLVGDKVM (fig. 5B). Interestingly, the dominant epitope induced by construct CHIKV-E1 carries the 226A-V mutation, indicating that the construct can also efficiently induce an immune response to the newly emerging mutant virus. This finding may indicate that the immune response can drive virus evolution during cell selection.

Discussion of the related Art

Evaluation of the immune response elicited in C57BL/6 mice showed that the constructs were highly immunogenic and elicited T cell immune responses in terms of IFN- γ response and proliferation. ELISpot data from this study indicate that the intensity of IFN- γ response is fundamental in terms of the number of spots obtained. Although envelope proteins and capsids in other related alphaviruses are known to be immunogenic, little is known about the immunogenicity of the envelope and capsid proteins. Matrix peptide pools from different regions of the envelope E1 and capsid elicit IFN- γ production by splenocytes, identifying the dominant T cell epitopes HSMTNAVTI and ACLVGDKVM in E1 and capsid proteins, respectively. The total IgG levels of vaccinated mice were found to be increased compared to the total IgG levels of unvaccinated controls, indicating the induction of a strong humoral immune response. In addition to the ability of these vaccines to boost protection against multiple chikungunya virus challenge, follow-up studies to further analyze the type of antibody response elicited are currently in progress.

This study shows that these constructs can be further studied as vaccine candidates. However, since this study is limited to demonstrating high expression and immunogenicity of vaccine constructs following intramuscular injection followed by electroporation in mice, detailed assessment of immunogenicity of the vaccine constructs in more models, including non-human primate models, is important. The described synthetic cassette constructs appear to be a convenient tool for further investigation of chikungunya virus immunobiology.

Example 2

Assays to measure neutralizing antibody titers were designed that provide a viable rapid diagnosis of clinical human CHIKV infection and applied to preclinical serum monitoring of susceptible vertebrate hosts.

CHIKV was identified by RT-PCR. RNA was extracted from patient sera using QIAamp viral RNA minikit. A Quiagen one-step RT-PCR kit was used for one-step RT-PCR testing. In the gene encoding the viral envelope protein E2, the amplification product was 305 bp. When cells were observed, CHIKV developed a foamy cytopathic effect (CPE), in which cell rounding was seen after 24-48 hours post infection (pi).

The microneutralization assay was designed as follows. The neutralization assay is based on the reaction of an antigen with an antibody. The presence of homologous antibodies in the patient sera that inhibit the known virus titer against CHIKV virus was observed. The serum is serially diluted (typically, e.g., 1: 10 to 1: 640) and incubated with known titers of CHIKV under conditions and for an amount of time sufficient for the antibodies in the serum to inhibit the virus. After incubation, if the virus has not been neutralized by antibodies in serum, the mixture is added to the recipient cells under conditions that would result in infection of the cells with the virus. The highest dilution of serum that inhibited virus propagation was recorded as antibody titer. CPE (cytopathic effect) can be used as a measure of viral propagation in cells.

CHIKV neutralization tests were performed using patient samples. FIG. 7 is a graph showing neutralizing antibody titers of sera of pre-immunized and DNA-immunized mice. Mice were immunized with constructs encoding E1, E2, capsid, E1+ E2, or vector pVax. Sera were serially diluted (1: 10 to 1: 640) and incubated with CHIKV (100TCID50) for 90 min at 37 ℃. After incubation, the mixture was added to Vero cells (15,000 cells/well) in a 96-well flat bottom plate and incubated at 5% CO₂The culture was carried out at 37 ℃ for 5 days in an atmosphere. The highest titer at which no CPE (cytopathic effect) was observed was recorded as the neutralizing antibody titer.

Reference to the literature

The following references are incorporated herein by reference.

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Claims (26)

1. A nucleic acid molecule encoding one or more proteins selected from the group consisting of:

consensus CHIKV Env, including immunogenic consensus fragments of CHIKV E1 consensus protein or CHIKV E1 consensus protein, CHIKV E2 consensus protein or CHIKV E2 consensus protein, and immunogenic consensus fragments of CHIKV E3 consensus protein or CHIKV E3 consensus protein;

CHIKV E1 consensus protein;

an immunogenic consensus fragment of CHIKV E1 consensus protein;

CHIKV E2 consensus protein;

an immunogenic consensus fragment of CHIKV E2 consensus protein;

CHIKV capsid consensus protein;

an immunogenic consensus fragment of the CHIKV capsid consensus protein;

and homologues thereof.

2. A composition comprising the nucleic acid molecule of claim 1.

3. A composition, comprising: one or more nucleic acid molecules of claim 1, wherein the composition comprises two or more different nucleic acid molecules.

4. A composition according to claim 3, comprising:

nucleic acid molecules encoding the proteins

An immunogenic consensus fragment of CHIKV E1 consensus protein or CHIKV E1 consensus protein, and an immunogenic consensus fragment of CHIKV E2 consensus protein or CHIKV E2 consensus protein;

an immunogenic consensus fragment of CHIKV E1 consensus protein or CHIKV E1 consensus protein, and an immunogenic consensus fragment of CHIKV capsid consensus protein or CHIKV capsid consensus protein;

an immunogenic consensus fragment of CHIKV E2 consensus protein or CHIKV E2 consensus protein, and an immunogenic consensus fragment of CHIKV E capsid consensus protein or CHIKV capsid consensus protein; and

an immunogenic consensus fragment of CHIKV E1 consensus protein or CHIKV E1 consensus protein, and an immunogenic consensus fragment of CHIKV E2 consensus protein or CHIKV E2 consensus protein, and an immunogenic consensus fragment of CHIKV capsid consensus protein or CHIKV capsid consensus protein.

5. The composition of any one of claims 1-4, wherein the isolated nucleic acid molecule encodes CHIKV E1 consensus protein.

6. The composition of any one of claims 1-4, wherein the isolated nucleic acid molecule comprises SEQ ID NO: 1 or SEQ ID NO: 3.

7. the composition of any one of claims 1-4, wherein the isolated nucleic acid molecule encodes the polypeptide of SEQ ID NO: 7 or a homologous protein thereof, or SEQ ID NO: 10 or a homologous protein thereof.

8. The composition of any one of claims 1-4, wherein the isolated nucleic acid molecule encodes CHIKV E2 consensus protein.

9. The composition of any one of claims 1-4, wherein the isolated nucleic acid molecule comprises SEQ ID NO: 2 or SEQ ID NO: 5.

10. the composition of any one of claims 1-4, wherein the isolated nucleic acid molecule encodes the polypeptide of SEQ ID NO: 8 or a homologous protein thereof, or SEQ ID NO: 11 or a homologous protein thereof.

11. The composition of any one of claims 1-4, wherein the isolated nucleic acid molecule encodes CHIKV capsid consensus protein.

12. The composition of any one of claims 1-4, wherein the isolated nucleic acid molecule comprises SEQ ID NO: 3 or SEQ ID NO: 6.

13. the composition of any one of claims 1-4, wherein the isolated nucleic acid molecule encodes the polypeptide of SEQ ID NO: 9 or a homologous protein thereof, or SEQ ID NO: 12 or a homologous protein thereof.

14. The composition of claim 1, wherein the isolated nucleic acid molecule encodes CHIKV Env consensus protein.

15. The composition of claim 14, wherein the isolated nucleic acid molecule encodes the polypeptide of SEQ ID NO: 14 or SEQ ID NO: 16.

16. the composition of claim 1, wherein the isolated nucleic acid molecule comprises SEQ ID NO: 13 or SEQ ID NO: 15.

17. the composition of any one of claims 1-16, wherein the isolated nucleic acid molecule encodes an IgE leader sequence.

18. The composition of any one of claims 1-17, wherein the nucleic acid molecule is a plasmid.

19. An injectable pharmaceutical composition comprising the composition of any one of claims 1-18.

20. A method of inducing an immune response to CHIKV in an individual comprising administering to the individual a composition of any one of claims 1-19.

21. The method of claim 20, wherein the composition is administered using electroporation.

22. A recombinant vaccine comprising a nucleotide sequence encoding one or more proteins selected from the group consisting of:

a CHIKV Env consensus protein comprising an immunogenic consensus fragment of CHIKV E1 consensus protein or CHIKV E1 consensus protein, an immunogenic consensus fragment of CHIKV E2 consensus protein or CHIKV E2 consensus protein, and an immunogenic consensus fragment of CHIKV E3 consensus protein or CHIKV E3 consensus protein;

CHIKV E1 consensus protein;

an immunogenic consensus fragment of CHIKV E1 consensus protein;

CHIKV E2 consensus protein;

an immunogenic consensus fragment of CHIKV E2 consensus protein;

CHIKV capsid consensus protein;

an immunogenic consensus fragment of the CHIKV capsid consensus protein; and

their homologues.

23. The recombinant vaccine of claim 24, wherein the recombinant vaccine is a recombinant vaccinia vaccine.

24. A method of inducing an immune response to CHIKV in an individual comprising administering to the individual the recombinant vaccine of claim 22.

24. A composition comprising one or more proteins selected from the group consisting of:

a CHIKV Env consensus protein comprising an immunogenic consensus fragment of CHIKV E1 consensus protein or CHIKV E1 consensus protein, an immunogenic consensus fragment of CHIKV E2 consensus protein or CHIKV E2 consensus protein, and an immunogenic consensus fragment of CHIKV E3 consensus protein or CHIKV E3 consensus protein;

proteins homologous to a CHIKV Env consensus protein comprising an immunogenic consensus fragment of CHIKV E1 consensus protein or CHIKV E1 consensus protein, an immunogenic consensus fragment of CHIKV E2 consensus protein or CHIKV E2 consensus protein, and an immunogenic consensus fragment of CHIKV E3 consensus protein or CHIKV E3 consensus protein;

CHIKV E1 consensus protein;

an immunogenic consensus fragment of CHIKV E1 consensus protein;

CHIKV E2 consensus protein;

an immunogenic consensus fragment of CHIKV E2 consensus protein;

CHIKV capsid consensus protein;

an immunogenic consensus fragment of the CHIKV capsid consensus protein; and

their homologues.

25. An injectable pharmaceutical composition comprising the composition of claim 24.

26. A method of inducing an immune response to CHIKV in an individual comprising administering to the individual the composition of claim 24 or 25.