VACCINES AND METHODS TO TREAT LYME DISEASE IN DOGS
The instant invention provides an immunogenic composition comprising an antigenic fragment of OspA protein of Borrelia burgdorferi and a chimeric protein containing antigenic fragments of different phylotypes of OspC protein of Borrelia burgdorferi. Vaccines incorporating the immunogenic composition of the invention, as well as methods of preventing Lyme disease in dogs and/or protecting dogs from Lyme disease using the vaccines are also provided.
This invention is in the field of veterinary medicine. More particularly, this invention is in the field of vaccines treating or preventing Lyme disease in dogs.
BACKGROUNDLyme disease is a bacterial infection caused by pathogenic spirochetes of the genus Borrelia. The infection can occur in humans, dogs, deer, mice and other animals, and is transmitted by arthropod vectors, most notably ticks of the genus Ixodes. Borrelia burgdorferi, the most common cause of Lyme disease in North America, was first cultured in 1982. Borrelia are introduced into the host at the site of the tick bite and this is also the location of the initial characteristic skin lesion, erythema chronicum migrans (ECM). In dogs, Lyme disease manifests with arthritis-induced lameness, anorexia, fever, lethargy, lymphadenopathy, and in some cases, fatal glomerulonephritis. A recent study revealed that the percentage of actively infected dogs in endemic areas can be as high as 11%.
The infection may be treated at any time with antibiotics such as penicillin, erythromycin, tetracycline, and ceftriaxone. Once infection has occurred, however, the drugs may not purge the host of the spirochete, but may only act to control the chronic forms of the disease. Complications such as arthritis and fatigue may continue for several years after diagnosis and treatment.
The canine Lyme disease vaccines were developed to provide protection by primarily inducing OspA borreliacidal antibodies. B. burgdorferi OspC is another potential target for borreliacidal antibody-mediated immunity. This protein appears to have an epitope that is responsible for inducing borreliacidal antibodies, and is not conserved among the pathogenic Borrelia spp. Although the specific function of the OspC protein remains unknown, it has been suggested that OspC expression is required for infection of mammals, but not for infection of ticks. Borrelia express OspC shortly after the tick begins feeding, and must continue to express OspC in order to establish an infection in mammals. Therefore, the “window of effectiveness” of the OspC borreliacidal antibodies is increased significantly, compared to OspA borreliacidal antibodies.
Callister et al., (U.S. Pat. Nos. 6,210,676 and 6,464,985, incorporated by reference herein) have suggested employing an immunogenic polypeptide fragment of OspC, alone or in combination with an OspA polypeptide, to prepare a vaccine to protect humans and other mammals against Lyme disease. Livey et al. (U.S. Pat. No. 6,872,550, incorporated by reference herein) also proposed a vaccine for immunizing against Lyme disease prepared from a combination of recombinant OspA, OspB, and OspC proteins.
However, at least two obstacles need to be overcome before a successful vaccine can be created. First, there are over twenty OspC phylotypes, and it is unclear which ones should be included into a vaccine. Second, suitable epitopes for development of borreliacidal anti-OspC antibodies need to be determined.
Therefore, there remains a longstanding need in the art for an improved vaccine to protect mammals, and especially canines, from Lyme disease.
SUMMARY OF INVENTIONThe instant invention addresses these and other needs by providing, in one aspect, an immunogenic composition comprising: a first protein comprising an amino acid sequence at least 95% identical to SEQ ID NO: 1 (MDPNTVSSFQVDSFLWHVRKRVADQELGDAPFLDRLRRDQKSLRGRGSTLG LDIETATRAGKQIVERILKEESDEALKMTMGKQNVSSLDEKNSVSVDLPGEMNV LVSKEKNKDGKYDLIATVDKLELKGTSDKNNGSGVLEGVKADKSKVKLTISDDL GQTTLEVFKEDGKTLVSKKVTSKDKSSTEEKFNEKGEVSEKIITRADGTRLEYT EIKSDGSGKAKEVLKSYVLEGTLTAEKTTLVVKEGTVTLSKNISKSGEVSVELND TDSSAATKKTAAWNSGTSTLTITVNSKKTKDLVFTKENTITVQQYDSNGTKLEG SAVEITKLDEIKNALK); and a second protein, comprising immunodominant epitopes of OspC phylotypes F and N.
In a set of embodiments, the second protein comprises a plurality of peptides at least 95% identical to immunodominant epitopes from loop 5 (loop peptide) and alpha helix 5 (helix peptide) of one or more OspC phylotypes I, H, C, M, and D, wherein further the loop peptides and the helix peptide from each phylotype are adjacent to each other and wherein the loop peptides and the helix peptides are arranged sequentially; and at least one of: a loop peptide and a helix peptide of OspC phylotype F adjacent to each other, or an amino acid sequence 95% identical to SEQ ID NO 32. In one set of embodiments, if said amino acid sequence 95% identical to SEQ ID NO: 32 is present, it is at the carboxy terminus of said second protein.
In one set of embodiments, the first protein is SEQ ID NO: 1. In another set of embodiments, the loop and helix peptides of phylotypes I, H, N, C, M, D and F are at least 95% identical to SEQ ID NOs: 4-17, respectively.
In another set of embodiments, the immunogenic composition may also comprise additional loop and helix peptides from one or more OspC phylotypes F, T, U, E, A, B, and K, which are, in some embodiments, identical to SEQ ID NOs 16-29, respectively.
In yet another set of embodiments, the immunogenic composition may further comprise at least one additional antigen protective against a microorganism that can cause disease in dogs. The microorganism may be selected from the group comprising canine distemper (CD) virus, canine adenovirus type 2 (CAV-2), canine parainfluenza (CPI) virus, canine parvovirus (CPV), canine coronavirus (CCV), canine herpesvirus, and rabies virus. Antigens from these pathogens for use in the vaccine compositions of the present invention can be in the form of a modified live viral preparation or an inactivated viral preparation. Other pathogens also include Leptospira bratislava, Leptospira canicola, Leptospira grippotyphosa, Leptospira icterohaemorrhagiae, Leptospira pomona, Leptospira hardjobovis, Porphyromonas spp., Bacteriodes spp., Leishmania spp., Ehrlichia spp., Mycoplasma ssp. and Microsporum canis.
In particular embodiments, the immunogenic composition comprises SEQ ID NO: 1 and either SEQ ID NO: 30 or SEQ ID NO: 31.
In another aspect, the instant invention provides a vaccine composition comprising the immunogenic composition as described above. The vaccine can also comprise an adjuvant and a pharmaceutically acceptable carrier. In different embodiments, adjuvants include, without limitations mineral salts, surface-active agents and microparticles, bacterial products, cytokines and hormones, carriers, oil-in-water emulsions and water-in-oil emulsions.
In yet another aspect, the invention also provides a method of preventing Lyme disease in a canine comprising administering to the canine in need thereof an immunologically effective dose of the vaccine composition.
For a better understanding of the instant application, the following non-limiting definitions are provided:
The term “at least 95% identical” includes all percentages of identity including and between 95% and 100%, for example, 96%, 97%, 98%, 99%, etc.
The term “alpha helix 5 region” or “helix 5 region” refers to amino acid sequence located between residues 160 and 200 of OspC phylotype A strain B31, and contains secondary structural elements including a portion of loop 6, alpha helix 5, and the unstructured C-terminal domain (Kumaran et al., 2001).
The term “conservative substitution” denotes the replacement of an amino acid residue by another biologically similar residue, or the replacement of a nucleotide in a nucleic acid sequence such that the encoded amino acid residue does not change or is another biologically similar residue. Examples of conservative substitution include the substitution of one hydrophobic residue such as isoleucine, valine, leucine or methionine for another hydrophobic residue, or the substitution of one polar residue for another polar residue, such as the substitution of arginine for lysine, glutamic for aspartic acid, or glutamine for asparagine, and the like. The term “conservative substitution” also includes the use of a substituted amino acid in place of an unsubstituted parent amino acid, provided that antibodies raised to the substituted polypeptide also immunoreact with the unsubstituted polypeptide.
The term “conservative variation” of a reference protein or a reference nucleic acid refers to a protein or a nucleic acid, respectively, which differs from the reference molecule by only conservative substitution(s).
The term “construct” preceded by a phylotype name (e.g., N-construct or I-construct) refers to an amino acid sequence comprising the loop peptide and the helix peptide.
The term “helix peptide” or “alpha helix peptide” of a certain phylotype of OspC refers to a peptide which is at least 95% identical to an immunodominant epitope from alpha helix 5 region of OspC protein of that phylotype. Thus, for example, helix peptide N refers to a peptide which is at least 95% identical to an immunodominant epitope from alpha helix 5 region of OspC phylotype N.
The term “immunodominant epitope” refers to an epitope on a molecule that induces a dominant, or intense, immune response when compared to other epitopes, including one or both B- and T-cell responses.
The term “linear epitope” refers to an epitope comprising a single, non-interrupted, contiguous chain of amino acids joined together by peptide bonds to form a peptide or polypeptide. Such an epitope can be described by its primary structure, i.e. the linear sequence of amino acids in the chain. Such an epitope, when expressed in a recombinant protein subunit of OspC, retains the ability to bind infection-induced antibodies in a manner similar to the binding of wild-type protein.
The term “loop peptide” of a certain phylotype of OspC refers to a peptide which is at least 95% identical to an immunodominant epitope from loop 5 region of OspC protein of that phylotype. Thus, for example, loop peptide N refers to a peptide which is at least 95% identical to an immunodominant epitope from loop 5 region of OspC phylotype N.
The term “loop 5 region” refers to amino acid sequence generally located between residues 131 and 159 of OspC phylotype A strain B31 and contains secondary structural elements, including a portion of alpha helix 3, loop 5 and alpha helix 4. See Kumaran et al., 2001. The sequence for OspC phylotype A strain B31 is provided in SEQ ID NO: 35 and in
The term “therapeutically effective amount” as used herein means an amount of a microorganism, or a subunit antigen, or polypeptides, or polynucleotide molecules, and combinations thereof, sufficient to elicit an immune response in the subject to which it is administered. The immune response can comprise, without limitation, induction of cellular and/or humoral immunity.
The terms “vaccine” and “vaccine composition,” as used herein, mean a composition which prevents or reduces an infection, or which prevents or reduces one or more signs or symptoms of infection. The protective effects of a vaccine composition against a pathogen are normally achieved by inducing in the subject an immune response, either a cell-mediated or a humoral immune response or a combination of both. Generally speaking, abolished or reduced incidences of infection, amelioration of the signs or symptoms, or accelerated elimination of the microorganism from the infected subjects are indicative of the protective effects of a vaccine composition.
In a broad aspect, the instant invention provides an immunogenic composition capable of inducing antibodies against OspA and OspC proteins of Borrelia burgdorferi. Thus, the composition will include two proteins: the first protein comprising an OspA or a fragment thereof, and a second protein, comprising an OspC protein or a fragment thereof. In some embodiments, the second protein is a chimeric protein comprising multiple fragments of OspC proteins of different phylotypes.
In some embodiments, the first protein comprises a fragment of OspA protein (SEQ ID NO: 2) (MGKQNVSSLDEKNSVSVDLPGEMNVLVSKEKNKDGKYDLIATVDKLELKGTS DKNNGSGVLEGVKADKSKVKLTISDDLGQTTLEVFKEDGKTLVSKKVTSKDKS STEEKFNEKGEVSEKIITRADGTRLEYTEIKSDGSGKAKEVLKSYVLEGTLTAET TLVVKEGTVTLSKNISKSGEVSVELNDTDSSAATKKTAAWNSGTSTLTITVNSK KTKDLVFTKENTITVQQYDSNGTKLEGSAVEITKLDEIKNALK), which is immediately downstream of a viral protein, such as, for example, a fragment of the influenza virus NS1 protein, which is SEQ ID NO: 3 (MDPNTVSSFQVDSFLWHVRKRVADQELGDAPFLDRLRRDQKSLRGRGSTLG LDIETATRAGKQIVERILKEESDEALKMT). An important requirement for the first protein is its ability to generate anti-OspA antibodies in a vaccinated animal. Thus, the full length sequence of the OspA fragment is not necessary, and neither is the 100% identity to SEQ ID NO: 2.
As noted elsewhere in the application, 95% sequence identity is likely to be sufficient to provide suitable level of antibody production. The differing amino acids can be conservative substitutions, and/or are located outside of immunodominant epitope(s) of the OspA fragment.
In other embodiments, shorter OspA fragments can be used. A person of ordinary skill in the art would know how to determine which OspA fragments contain immunodominant epitopes capable of generating borreliacidal antibodies.
The inventors have surprisingly found that the first protein comprising, from N- to C-terminus, a fragment of the influenza virus NS-1 protein, followed by OspA protein with its signal sequence removed, is particularly suitable for the immunogenic compositions of the instant invention.
Prior art studies are silent as to what phylotypes of OspC are prevalent in invasive Lyme disease in dogs. Most studies have been performed on human samples. Jones et al reports that the most prevalent phylotypes found in joint fluid of the human patients with arthritis are K and A, and typically, and phylotypes A, B, C, D, H, K, N were discovered. Arthritis Rheum 2009 60(7) 2174. Earnhart et al have discovered phylotypes A, B, I, K, C, D, N in blood and/or CSF samples Infect Immun. 2005 73(12): 7869. Other studies typically associated phylotypes A, B, I and K with invasive forms of Lyme disease in humans.
However, it was surprisingly discovered that in dogs, the most prevalent phylotype is OspC F, which was not previously associated with invasive form of Lyme disease, whether in humans or in dogs. Phylotype N, which is associated with invasive Lyme disease in humans, was also associated with invasive Lyme disease in dogs. Additionally, the inventors have discovered that phylotypes T and U, previously not associated with invasive Lyme disease in humans, may cause invasive Lyme disease in dogs.
According to some embodiments, the second protein contains immunodominant epitopes capable of generating immune response against different OspC protein phylotypes. More specifically, the second protein of the immunogenic composition claimed in the instant invention is a chimeric protein that comprises immunodominant epitopes of OspC phylotypes F and N. The immunodominant epitopes may be in the form of loop and/or helix peptides as discussed below, or they may be present within larger fragments of the target OspC protein. A suitable non-limiting example of such fragments is SEQ ID NO: 32 (NNSGKDGNTSANSADESVKGPNLTEISKKITESNAVVLAVKEIETLLSSIDELAT KAIGQKIDANGLGVQANQNGSLLAGAYAISTLITQKLSALNSEDLKEKVAKVKKC SEDFTNKLKNGNAQLGLAAATDDNAKAAILKTNGTNDKGAKELKDLSDSVESLV KAAQVMLTNSVKELTSPVVAESPKKP), which is a fragment of OspC phylotype F protein.
Previous studies demonstrate that the inclusion of conserved region of OspC protein (i.e., conserved among different phylotypes) is important for generation of anti-borrelicidal antibody in mice and humans but not in dogs. See Lovrich et al, Clin. and Vaccine Immunol. May 2007, p. 635-637. Nevertheless, the inventors have surprisingly discovered that the addition of the longer fragment of one of the OspC phylotypes (e.g., phylotype F) is beneficial for the expression level and thus makes the manufacturing of the second protein more efficient.
Buckles et al demonstrated that loop 5 of OspC protein is surface exposed and may be a suitable target for generating borreliacidal antibodies. Clin Vaccine Immunol. 2006 October; 13(10):1162-5. See also WO09135118. However, considering that at least 21 phylotypes of OspC have been described (Seinost et al., Infect Immun. 1999 July; 67(7):3518-24 1999), it remains to be determined what combination provides suitable protection against Lyme disease.
Thus, in some embodiments, the second protein comprises linear epitopes from loop 5 region (loop peptides) and helix 5 regions (helix 5 peptides) of OspC proteins of different phylotypes. Currently considered phylotypes are T, U, E, A, B, K, I, H, N, C, M, D and F. The second protein may thus comprise loop and helix peptides from 2-13 phylotypes of OspC, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 phylotypes. The order of the peptides is not crucial. In some embodiments, the loop peptides are interspaced with the helix peptides, and vice versa. In other words, the loop and helix peptides are arranged sequentially: in such embodiments, no two loop peptides should be present in the second protein without a helix peptide between them, and no two helix peptides should be present without a loop peptide between them.
A person of ordinary skill in the art would be aware how to determine immunodominant epitopes from the loop regions and helix regions of various OspC phylotypes. For example, sera from subjects infected with Borrelia burgdorferi of different phylotypes may be reacted with specific peptides from the loop regions and helix regions of the corresponding phylotypes, and the binding of the antibodies present in the sera to the loop peptides and/or helix peptides can be quantified (e.g., by ELISA, immunoblot, etc), thus providing clues as to which peptides contain immunodominant linear epitopes from a given OspC phylotype.
Similarly, the borreliacidal activity of the antibodies may be determined by methods well known in the art, e.g., generally, by co-incubating cultured Borrelia burgdorferi with the sera from subjects challenged with the immunodominant linear epitopes as described above, and quantification of living and dead Borrelia.
In some embodiments, the sequences for the loop peptides and helix peptides are as follows:
Loop peptide I is at least 95% identical to SEQ ID NO: 4 (AKLKGEHTDLGKEGVT);
Helix peptide I is at least 95% identical to SEQ ID NO: 5 (KGADELEKLFESVKNLSKAAKEMLTNSVKE);
Loop peptide H is at least 95% identical to SEQ ID NO: 6 (SEKFAGKLKNEHASLGKKDAT);
Helix peptide H is at least 95% identical to SEQ ID NO: 7 (KGAKELKDLSDSVESLVKA);
Loop peptide N is at least 95% identical to SEQ ID NO: 8 (SDDFTKKLQSSHAQLGVAGGATT);
Helix peptide N is at least 95% identical to SEQ ID NO: 9 (ADELEKLFKSVESLAKAAQDALANSVNELTS);
Loop peptide C is at least 95% identical to SEQ ID NO: 10 (KKLKEKHTDLGKKDAT);
Helix peptide C is at least 95% identical to SEQ ID NO: 11 (AAELEKLFESVENLAKAAKEMLSNS);
Loop peptide M is at least 95% identical to SEQ ID NO: 12 (NKAFTDKLKSSHAELGIANGAAT); Helix peptide M is at least 95% identical to SEQ ID NO: 13 (KGAQELEKLFESVKNLSKAAQETLNNSVKE);
Loop peptide D is at least 95% identical to SEQ ID NO: 14 (SESFTKKLSDNQAELGIENAT);
Helix peptide D is at least 95% identical to SEQ ID NO: 15 (KGAEELVKLSESVAGLLKAAQAILANSVKELTSPVVAESPKKP);
Loop peptide F is at least 95% identical to SEQ ID NO: 16 (SEDFTNKLKNGNAQLGLAAAT);
Helix peptide F is at least 95% identical to SEQ ID NO: 17 (KGAKELKDLSDSVESLVKAAQVMLTNS);
Loop peptide T is at least 95% identical to SEQ ID NO: 18 (STGFTNKLKSGHAELGPVGGNAT);
Helix peptide T is at least 95% identical to SEQ ID NO: 19 (KGAKELKDLSESVEALAKAAQAMLTNS);
Loop peptide U is at least 95% identical to SEQ ID NO: 20 (SEKFTKKLSESHADIGIQAAT);
Helix peptide U is at least 95% identical to SEQ ID NO: 21 (KGAEELDKLFKAVENLSK);
Loop peptide E is at least 95% identical to SEQ ID NO: 22 (STEFTNKLKSEHAVLGLDNLT);
Helix peptide E is at least 95% identical to SEQ ID NO: 23 (KGAAELEKLKAVENLSKAAQDTLKNAVKELTSPIVAESPKKP);
Loop peptide A is at least 95% identical to SEQ ID NO: 24 (SETFTNKLKEKHTDLGKEGVT);
Helix peptide A is at least 95% identical to SEQ ID NO: 25 (KGAEELGKLFESVEVLSKAAKEMLANSVKELTS);
Loop peptide B is at least 95% identical to SEQ ID NO: 26 (SEEFSTKLKDNHAQLGIQGVT);
Helix peptide B is at least 95% identical to SEQ ID NO: 27 (KGVEELEKLSGSLESLS);
Loop peptide K is at least 95% identical to SEQ ID NO: 28 (SEDFTKKLEGEHAQLGIENVT); and
Helix peptide K is at least 95% identical to SEQ ID NO: 29 (AAELEKLFKAVENLAKAAKEM).
In some embodiments, loop and helix peptides from the same phylotype are positioned together, i.e., adjacent to each other. For example, loop peptide from OspC phylotype A and a helix peptide from OspC phylotype A should not be separated by either the loop or the helix peptide from any other OspC phylotype.
Further, while in some embodiments, the loop and helix peptides from the same OspC phylotype are immediately adjacent to each other, in other embodiments, the loop peptide and the helix peptide may be separated by a linker sequence which does not affect the structure of the final protein. The properties of amino acids and their effects on protein structure are well known in the art and persons of ordinary skill in the art would be able to recognize which amino acids are suitable for the linkers.
As will be demonstrated in the Examples, the inventors have surprisingly found that F and N are the most prevalent OspC phylotypes associated with Lyme disease in dogs. The inventors have also found that the presence of loop and helix peptides from phylotypes I, H, N, C, M, D and F provides a very good level of protection against Lyme disease in dogs. While the order of the loop and helix peptides from different phylotypes is not crucial, in some embodiments, the second protein comprises, in N- to C-orientation, an I-construct, a H-construct, a N-construct, a C-construct, a M-construct, a D-construct, followed by an amino acid sequence which is at least 95% identical to a fragment of OspC phylotype F protein (e.g., SEQ ID NO: 32). Thus, in some embodiments, the second protein will comprise an amino acid sequence at least 95% (e.g., 96%, 97%, 98%, 99%, and preferably, 100%) identical to SEQ ID NO: 31 (A12CF).
In other embodiments, the loop and the helix peptides from phylotypes F, T, U, E, A, B, K are included within the second protein. In some embodiments, the second protein, thus, would comprise the following, in N- to C-orientation: a T-construct, a U-construct, a E-construct, an A-construct, a B-construct, a K-construct, the I-construct, the H-construct, the N-construct, the C-construct, the M-construct, and the D-construct. Optionally, the second protein can also comprise an F-construct, which is, in some embodiments, is upstream of the T-construct. Alternatively, or additionally, the second protein can contain the amino acid sequence which is at least 95% (e.g., 96%, 97%, 98%, 99%) identical to the fragment of OspC phylotype F protein (SEQ ID NO: 32).
Other suitable examples of the second protein, as well as methods of making and using same are provided in Application PCT/US2011/056854 (filed on Oct. 19, 2011, inventors R. Marconi and C. Earnhart).
In certain embodiments, the immunogenic composition would comprise SEQ ID NO: 1; and either one of SEQ ID NO: 30 or SEQ ID NO: 31.
The sequences described herein may be manufactured by methods well known in the art. The polypeptides may be produced by direct peptide synthesis using solid-phase techniques (see, e.g., Stewart et al. (1969) Solid-Phase Peptide Synthesis, WH Freeman Co, San Francisco; Merrifield J. (1963) J Am Chem Soc 85:2149-2154). Peptide synthesis may be performed using manual techniques or by automation. Automated synthesis may be achieved, for example, using Applied Biosystems 431A Peptide Synthesizer (Perkin Elmer, Foster City, Calif.), in accordance with the instructions provided by the manufacturer. For example, subsequences may be chemically synthesized separately and combined using chemical methods to provide full-length polypeptides or fragments thereof. Alternatively, such sequences may be ordered from any number of companies which specialize in production of polypeptides. Most commonly, polypeptides may be produced by expressing coding nucleic acids and recovering polypeptides, as described below.
For example, in embodiments where loop peptides and the helix peptides are 100% identical to the fragments of OspC proteins of the target phylotypes, the nucleic acid sequences of such loop and helix peptides are also known or easily accessible from publicly available databases, e.g., Genbank. If the selected loop/helix peptides are somewhat different from the naturally occurring fragments of OspC proteins, the encoding nucleic acid sequences can be easily designed using well known genetic code.
Many organisms display bias for use of particular codons to code for insertion of a particular amino acid in a growing peptide chain. Codon preference or codon bias, differences in codon usage between organisms, is well documented among many organisms. Codon bias often correlates with the efficiency of translation of messenger RNA (mRNA), which is in turn believed to be dependent on, inter alia, the properties of the codons being translated and the availability of particular transfer RNA (tRNA) molecules. The predominance of selected tRNAs in a cell is generally a reflection of the codons used most frequently in peptide synthesis. Accordingly, since the majority of amino acids are encoded by multiple codons (methionine is the exception), the nucleic acid sequences can be tailored for optimal gene expression in a given organism based on codon optimization.
Methods for producing recombinant polypeptides are also included. One such method comprises introducing into a population of cells any nucleic acid as described above, which is operatively linked to a regulatory sequence effective to produce the encoded polypeptide, culturing the host cells (e.g., yeast, insect, mammalian cells, plant cells, etc) in a culture medium to express the polypeptide, and isolating the polypeptide from the cells or from the culture medium. The nucleic acid is introduced into such cells by any delivery method as is known in the art, including, e.g., transformation, transfection, injection, gene gun, passive uptake, etc. As one skilled in the art will recognize, the nucleic acid may be part of a vector, such as a recombinant expression vector, including a DNA plasmid vector, or any vector as known in the art.
Alternatively, cell-free prokaryotic or eukaryotic-based expression systems may be used.
In some embodiments, the nucleic acid sequence encoding the first and/or second protein, may further comprise a sequence encoding a polypeptide (the “fusion partner”) that is fused to the first and/or second protein, thereby facilitating purification of the fusion protein. In certain embodiments of this aspect of the invention, the fusion partner is a hexa-histidine peptide (SEQ ID NO: 47, HHHHHH), as provided in the pQE vector (Qiagen, Inc.), and described in Gentz et al., Proc Natl Acad Sci USA 86:821-824 (1989), or it may be the HA tag, which corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson, I., et al., Cell 37:767, 1984). The polynucleotide may also contain non-coding 5′ and 3′ sequences, such as transcribed, non-translated sequences, splicing and polyadenylation signals, ribosome binding sites and sequences that stabilize mRNA.
The immunogenic compositions described herein are particularly suitable for preventing or diminishing the severity of symptoms of Lyme disease in dogs. Thus, in another aspect, the instant invention provides a vaccine, comprising the immunogenic composition according to any of the embodiments described above, and a suitable adjuvant.
The first and the second proteins of the immunogenic composition of the instant invention should be present in immunologically effective amount, i.e., in an amount sufficient to trigger the immune response in the dog. In some embodiments, the concentration of the first protein is between 1 and 100 ug/ml (e.g., 5, 10, 20, 30, 40, 50, 60, 70, 80, 90 ug/ml), and the concentration of the second protein is between 1 and 200 ug/ml (e.g., 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190 ug/ml). In some embodiments, the amount of the first protein is between about 10 and 50 ug/ml and the amount of the second protein is between 20 and 100 ug/ml.
Adjuvants suitable for use in accordance with the present invention include, but are not limited to several adjuvant classes such as; mineral salts, e.g., Alum, aluminum hydroxide, aluminum hydroxide gels (e.g., Rehydragel®), aluminum phosphate and calcium phosphate; surface-active agents and microparticles, e.g., nonionic block polymer surfactants, cholesterol, virosomes, saponins (e.g., Quil A, QS-21 and GPI-0100), proteosomes, immune stimulating complexes, cochleates, quarterinary amines (dimethyl diocatadecyl ammonium bromide (DDA)), pyridine, vitamin A, vitamin E; bacterial products such as the RIBI adjuvant system (Ribi Inc.), cell wall skeleton of Mycobacterum phlei (Detox®), muramyl dipeptides (MDP) and tripeptides (MTP), monophosphoryl lipid A, Bacillus Calmete-Guerin, heat labile E. coli enterotoxins, cholera toxin, trehalose dimycolate, CpG oligodeoxnucleotides; cytokines and hormones, e.g., interleukins (IL-1, IL-2, IL-6, IL-12, IL-15, IL-18), granulocyte-macrophage colony stimulating factor, dehydroepiandrosterone, 1,25-dihydroxy vitamin D3; polyanions, e.g., dextran; polyacrylics (e.g., polymethylmethacrylate, Carbopol 934P); carriers e.g., tetanus toxid, diptheria toxoid, cholera toxin B subnuit, mutant heat labile enterotoxin of enterotoxigenic E. coli (rmLT), heat shock proteins; oil-in-water emulsions e.g., AMPHIGEN® (Hydronics, USA); and water-in-oil emulsions such as, e.g., Freund's complete and incomplete adjuvants. In other embodiments, SP oil may also be used. As used herein, the term “SP oil” designates an oil emulsion comprising a polyoxyethylene-polyoxypropylene block copolymer, squalane, polyoxyethylene sorbitan monooleate and a buffered salt solution. In general, the SP oil emulsion will comprise about 1 to 3% vol/vol of block copolymer, about 2 to 6% vol/vol of squalane, more particularly about 3 to 6% of squalane, and about 0.1 to 0.5% vol/vol of polyoxyethylene sorbitan monooleate, with the remainder being a buffered salt solution.
The vaccines described herein may be combination vaccines which include the immunogenic composition described above, in combination with at least one antigen from other canine pathogens, capable of inducing a protective immune response in dogs against disease caused by such other pathogens.
Such other pathogens include, but are not limited to, canine distemper (CD) virus, canine adenovirus type 2 (CAV-2), canine parainfluenza (CPI) virus, canine parvovirus (CPV), canine coronavirus (CCV), canine herpesvirus, and rabies virus. Antigens from these pathogens for use in the vaccine compositions of the present invention can be in the form of a modified live viral preparation, an inactivated viral preparation, or a subunit protein preparation. In other embodiments, a recombinant CDV (Canine Distemper Virus) may also be used. Methods of attenuating virulent strains of these viruses, and methods of making an inactivated viral preparation, are known in the art, and are described in, e.g., U.S. Pat. Nos. 4,567,042 and 4,567,043.
Other pathogens also include Leptospira bratislava, Leptospira canicola, Leptospira grippotyphosa, Leptospira icterohaemorrhagiae, Leptospira pomona, Leptospira hardjobovis, Porphyromonas spp., Bacteriodes spp., Leishmania spp., Ehrlichia spp., Mycoplasma ssp., Anaplasma spp. and Microsporum canis. Antigens from these pathogens for use in the vaccine compositions of the present invention can be in the form of an inactivated whole or partial cell preparation, using methods well-known in the art. For example, methods of making an inactivated whole or partial Leptospira cell preparation are known in the art and are described in, e.g., Yan, K-T, “Aspects of Immunity to Leptospira borgpetersenii serovar hardjo”, PhD Thesis, Appendix 1,1996. Faculty of Agriculture and Food Science, The Queen's University of Belfast; Mackintosh et al., “The use of a hardjo-pomona vaccine to prevent leptospiruria in cattle exposed to natural challenge with Leptospia interrogans serovarhardjo”, New Zealand Vet. J. 28:174-177, 1980; Bolin et. al., “Effect of vaccination with a pentavalent leptopsiral vaccine on Leptospira interrogans serovar hardjo type hardjo-boivs infection of pregnant cattle”, Am. J. Vet. Res. 50:161-165, 1989.
In accordance with the present invention, vaccines can be administered to a dog of at least 6 weeks old, or at least 7 weeks old, or at least 8 or 9 weeks old. The administration can be done by any known routes, including the oral, intranasal, mucosal topical, transdermal, and parenteral (e.g., intravenous, intraperitoneal, intradermal, subcutaneous or intramuscular). Administration can also be achieved using needle-free delivery devices. Administration can also be achieved using a combination of routes, e.g., first administration using a parental route, and subsequent administration using a mucosal route. In some embodiments, routes of administration include subcutaneous and intramuscular administrations.
All publications cited in the specification, both patent publications and non-patent publications, are indicative of the level of skill of those skilled in the art to which this invention pertains. All these publications are herein fully incorporated by reference, to the same extent as if each individual publication were specifically and individually indicated as being incorporated by reference.
For a clearer understanding of the invention, the following examples are set forth below. These examples are merely illustrative, and are not understood to limit the scope or underlying principles of the invention in any way. Indeed, various modifications of the invention, in addition to those shown and described herein, will become apparent to those skilled in the art from the examples set forth hereinbelow and the foregoing description. Such modifications are also intended to fall within the scope of the appended claims.
EXAMPLES Example 1 Determination of B. Burgdorferi OspC Phylotypes Associated with Lyme Disease in DogsAdult Ixodes scapularis ticks were collected in southern Rhode Island by flagging. The percentage of ticks infected with B. burgdorferi was determined through direct fluorescent microscopy using standard methods and labeled anti-B. burgdorferi antibody.
All procedures were conducted in compliance with regulations of the Animal Welfare Act and the dogs were maintained in accordance with Farm Canine Husbandry Standard Operating Procedures. Fifteen purpose-bred dogs of both sexes (7 males, 8 females; 9 to 10 weeks of age; Marshall Bioresources) were assigned identification numbers and divided into four study groups designated as T01 (n=4), T02 (n=4), T03 (n=4) and T04 (n=3). The dogs were fitted with Elizabethan collars and housed in one-over-one condo style cages. One day prior to tick infestation serum was collected from each dog. Dogs in study groups T01, T02, T03 and T04 were infested with 0, 25, 50 or 75 adult Ixodes scapularis ticks, respectively, using secured infestation chambers placed on each side of the midthorax. The ticks were fed to repletion, removed and serum samples and skin biopsies were collected at 49 and 90 days (relative to the start of infestation). Seroconversion was assessed with the SNAP 4DX test (IDEXX). To cultivate spirochetes, a portion of each skin biopsy was placed in BSK-H media (6% rabbit serum; 37EC, 5% CO2). Clonal populations were obtained from the cultures by sub-surface plating as previously described. Colonies were excised from the plates and placed in BSK-H media for cultivation.
DNA was extracted from skin biopsies using the Qiagen DNeasy Kit as instructed by the supplier. In addition, DNA was extracted from cultures of the clonal populations of B. burgdorferi as previously described. The ospC gene was PCR amplified using DNA extracted from tissues (100 ng) and from DNA obtained from boiled B. burgdorferi cell lysates (1:1 supernatant; GoTaq polymerase). All PCR was performed using standard conditions. A portion of each reaction was assessed by agarose gel electrophoresis and ethidium bromide staining. The remaining PCR products were excised from the gels (Qiagen Gel Extraction Kit; QIAGEN) and annealed with the pET46Kk/LIC vector (Novagen). The plasmids were propagated in E. coli NovaBlue cells (Novagen). Colonies were screened for the ospC gene by PCR. The templates for PCR were generated by boiling a portion of each ospC positive, E. coli colony. Portions of the colonies were also inoculated into LB media (2 ml), grown overnight, harvested by centrifugation and plasmid extracted using the Qiagen MiniPrep kit (QIAGEN). The primers used for PCR are as follows (5′ to 3′):
DNA sequencing was performed by Eurofins MWG Operon. Neighbor joining trees were generated using ClustalX 2.0.10 software in the multiple alignment mode with the default settings and a Gonnet matrix and visualized using N-J Plot version 2.2.
ResultsAnalysis of the Prevalence of B. Burgdorferi in Ticks Collected from Rhode Island.
Using direct fluorescent microscopy it was determined that ˜50% of the Ixodes scapularis ticks field-collected in southern Rhode Island were infected with B. burgdorferi. This is consistent with previously reported tick infection rates in this area.
Infection of Dogs with Borrelia burgdorferi Through Tick Infestation.
At the start of this study, all dogs were confirmed to be sero-negative for through immunoblot analyses and through the use of the B. burgdorferi using the SNAP 4DX assay (IDEXX). To infect dogs with B. burgdorferi via the natural transmission route, field collected ticks were fed on dogs. Since the infection rate in the ticks was ˜50% increasing numbers of ticks (0, 25, 50 or 75) were placed on the dogs. Serum samples were collected 49 days after tick infestation and immunological status evaluated. Of the dogs infested with ticks, 10 of 11 were sero-positive for B. burgdorferi. All negative controls dogs (not infested with ticks) were seronegative. Total DNA was extracted from skin biopsies collected from each dog and tested for B. burgdorferi by PCR with ospC and flaB primer sets. All seropositive dogs yielded ospC and flaB amplicons of the predicted size. All seronegative dogs were PCR negative for both genes.
Analysis of OspC Diversity in Strains Found in Infected Dog Tissues.
To determine the ospC genotype of strains that persisted in the skin of dogs exposed to ticks, ospC was PCR amplified from DNA extracted from skin biopsies. The resulting amplicons were cloned into pET46 Ek/LIC and the plasmids propagated in E. coli. Plasmid was then isolated from no less than 5 separate E. coli colonies and the ospC sequences determined. Sequence alignment and dendogram construction (
To further define the range of ospC genotypes present in the infected dogs, cultures from the skin biopsies were plated to yield clonal populations. By this approach, strains expressing ospC types that were not detected by PCR of biospy samples can be identified. Individual B. burgdorferi colonies were then tested for ospC by PCR (
In this study the inventors determined the ospC genotype of Lyme disease spirochete strains that successfully established infection and persisted in dogs. Field collected Ixodes scapularis ticks from Rhode Island were fed on laboratory dogs and the ospC genotype of strains present in skin after 49 days was determined. A total of 11 different OspC types were identified. OspC type F, which has not been previously detected in humans, was the most frequently detected OspC type (50% of infected dogs). Types B, N and U, which occur with very low frequency in humans were also detected. Two ospC types that have not been previously defined (DRI85a and DRI85e) were also recovered. The diversity observed in this study is consistent with earlier studies that demonstrated the maintenance of several ospC phyletic types within a local B. burgdorferi population. In that the ticks used in this study were collected from a single geographic region, it is possible that strains expressing other OspC types that are not well represented in Rhode Island are also competent to infect dogs. In spite of this caveat, this study is the first to demonstrate that OspC types not previously associated with human infection can efficiently infect dogs, thus facilitating the rational design of a new generation canine Lyme disease vaccine.
Example 2 Efficacy of Recombinant Chimeric Borrelia burgdoferi OspC/OspA Vaccines in DogsThirty dogs, all in good general health, were chosen for the study. Blood samples were collected prior to the initial vaccination. Dogs received one of the following vaccines, as described in Table 2: T01: PBS (control product); T02: ug/ml OspA+30 ug/ml A12CF (SEQ ID NO: 31); T03: 20 ug/ml OspA+30 ug/ml A10CF (SEQ ID NO: 30). (A12CF consists of epitopes from multiple OspC phylotypes, linked together to form a single polypeptide. A10CF also consists of epitopes from multiple OspC phylotypes; its design is similar to that of A12CF.) Dogs were vaccinated twice, at 8 and 11 weeks of age, and then challenged at 14 weeks of age. Following vaccination, dogs were observed for 20 minutes for reactions or abnormalities. Injection sites were observed on Days 1, 2, 3 and 22, 23, 24 for swelling, pain, heat, abscess, drainage, etc. Each dog was fitted with an Elizabethan [E] collar one day prior to placing the ticks, and the dogs were monitored for their ability to move, eat and drink with E-collars in place. Twenty to forty pairs (male, female) of Ixodes scapularis adult ticks, collected from the northeast USA, were placed along the dorsal midline of each dog, and allowed to feed until repletion for a period of 7 to 10 days. Serum samples and skin biopsies were collected at prescribed intervals, and assayed to monitor infection. Replete or unattached, non-viable ticks were collected and stored at 4° C. At the end of the challenge, remaining ticks were removed and stored, and dogs were treated with a topical acaricide according to label, followed by a second application 30 days later. Dogs were observed daily for overall physical appearance and behavior. Clinical observations were performed (lameness and ataxia); if either was observed, that dog's body temperature (tympanic) was measured/recorded daily, until the clinical signs subsided. Blood collections and skin biopsies were performed per protocol, other than deviations in scheduling/timing, based on receipt of ticks and subsequent infestation. Punch skin biopsies were taken near the general site of tick attachments on the dorsal cervical region, and timed to coincide with blood collections. Final biopsies were taken immediately after euthanasia, and prior to necropsy.
No reactions or abnormalities were observed in any dogs following vaccination, nor were any abnormalities at the injection site (swelling, pain, heat, abscess, drainage, etc.). Body temperatures, measured using a tympanic probe, did not show an appreciable or sustained elevation. Intermittent lameness occurred in two dogs vaccinated with A12CF+OspA (T02), and one dog in the non-vaccinated group (T01) was lame on Days 192-193. Ataxia was not observed in any dogs during the study. Abnormal health events, including pyoderma, bite wounds, abrasions, loose stools, otitis externa, etc., were observed in some dogs during the study, but none were attributed to the vaccines or vaccinations.
A serological response, indicative of active Borrelia burgdoferi infection, was observed on Day 146 in 8 of 10 control dogs (T01), and in one dog in T02. Nine control dogs (T01) were serologically positive on Day 177, and all dogs in T01 were positive at the study conclusion. In contrast, there was only one dog in each vaccinated group that was positive from Day 177 to the conclusion of the study.
The ticks used in the study were dual infected with B. burgdoferi and Anaplasma. The results of the serological assay indicate that ticks successfully transmitted Anaplasma to the dogs. This supports the specificity of the vaccine constructs (T02, T03) against B. burgdoferi only.
ELISA values, expressed as geometric mean titers to each of OspA and OspC, were significantly different when comparing T01 versus T02, and T01 versus T03, on all days, with the exception of the comparison of T01 vs. T02 for OspC on Day 146 (Table 3).
Serum samples collected during the post-challenge phase from control dogs (T01) and dogs in T02 (A12CF+OspA) were assayed in an ELISA which is specific for live B. burgdoferi organisms. The geometric mean titers for T01 vs T02 were: at Day 146, 90 vs 6; Day 177, 116 vs 7; and Day 198, 87 vs 7. Thus, these results support the vaccine's protective effect against B. burgdoferi.
Skin punch biopsy samples were cultured for viable spirochetes. In group T01, 4 dogs on Day 146, and 5 dogs on Day 177, were culture positive. One dog in each of T02 and T03 had a spirochete-positive skin culture on Day 177. No positive culture was obtained from any group at the conclusion of the study.
Skin punch biopsies were also assessed by PCR, using flab- and ospC-specific primers, for the presence of B. burgdoferi on Day 146. Five dogs in T01 were positive for flab, while 3 were positive for ospC. No dogs in either T02 or T03 were positive for either PCR reaction.
Examinations of joints and skin sections microscopically demonstrated that vaccination with either the T02 or T03 vaccine protected against infection (data not shown). Vaccinated dogs had fewer changes in their joints and skin as is characteristic of Lyme disease. If such changes were present, they were less severe in vaccinated dogs when compared to tissues from non-vaccinated control dogs. There was a slight difference between the two vaccines (T02; T03), based on the number of dogs with lesions in their joints (6 for T02; 7 for T03). However, a definitive conclusion cannot be drawn as to which construct provided better protection.
In conclusion, both A12CF+OspA (T02) and A10CF+OspA (T03) were efficacious in protecting dogs against Borrelia burgdoferi infection as transmitted by Ixodes scapularis ticks.
Claims
1. An immunogenic composition comprising:
- A) a first protein comprising an amino acid sequence at least 95% identical to SEQ ID NO: 1; and
- B) a second protein, comprising immunodominant epitopes of OspC phylotypes F and N.
2. The immunogenic composition of claim 1, wherein the first protein comprises SEQ ID NO: 1.
3. The immunogenic composition of claim 1 or 2, wherein the second protein comprises:
- i) a plurality of peptides at least 95% identical to immunodominant epitopes from loop 5 region (loop peptide) and alpha helix 5 region (helix peptide) of OspC phylotype I, H, C, M, and D; and
- ii) at least one of: a) a loop peptide of OspC phylotype F and a helix peptide of OspC phylotype F adjacent to each other, or b) an amino acid sequence 95% identical to SEQ ID NO 32.
4. The immunogenic composition of any one of claims 1 and 2, wherein:
- Loop peptide I is SEQ ID NO: 4;
- Helix peptide I is SEQ ID NO: 5;
- Loop peptide H is SEQ ID NO: 6;
- Helix peptide H is SEQ ID NO: 7;
- Loop peptide N is SEQ ID NO: 8;
- Helix peptide N is SEQ ID NO: 9;
- Loop peptide C is SEQ ID NO: 10;
- Helix peptide C is SEQ ID NO: 11;
- Loop peptide M is SEQ ID NO: 12;
- Helix peptide M is SEQ ID NO: 13;
- Loop peptide D is SEQ ID NO: 14;
- Helix peptide D is SEQ ID NO: 15.
5. The immunogenic composition of any one of claims 1-3, wherein the second protein comprises an amino acid sequence 95% identical to SEQ ID NO 32 at the carboxy terminus of said second protein.
6. The immunogenic composition of any one of claims 1-5, wherein the second protein comprises, in N- to C-orientation, an I-construct, a H-construct, a N-construct, a C-construct, a M-construct, a D-construct.
7. The immunogenic composition of any one of claims 1-6, wherein the second protein further comprises a plurality of peptides 95% identical to immunodominant epitopes from loop 5 region (loop peptide) and alpha helix 5 region (helix peptide) of OspC phylotypes T, U, E.
8. The immunogenic composition of claim 7, wherein the second protein comprises, in N- to C-orientation, a T-construct, a U-construct, an E-construct.
9. The immunogenic composition of claim 7 or 8, wherein
- Loop peptide T is SEQ ID NO: 18;
- Helix peptide T is SEQ ID NO: 19;
- Loop peptide U is SEQ ID NO: 20;
- Helix peptide U is SEQ ID NO: 21;
- Loop peptide E is SEQ ID NO: 22;
- Helix peptide E is SEQ ID NO: 23;
10. The immunogenic composition of any one of claims 1-9, wherein the second protein further comprises a plurality of peptides 95% identical to immunodominant epitopes from loop 5 region (loop peptide) and alpha helix 5 region (helix peptide) of OspC phylotypes A, B, K.
11. The immunogenic composition of claim 10, wherein the second protein comprises, in N- to C-orientation, an A-construct, a B-construct, a K-construct.
12. The immunogenic composition of claim 11, wherein
- Loop peptide A is SEQ ID NO: 24;
- Helix peptide A is SEQ ID NO: 25;
- Loop peptide B is SEQ ID NO: 26;
- Helix peptide B is SEQ ID NO: 27;
- Loop peptide K is SEQ ID NO: 28;
- Helix peptide K is SEQ ID NO: 29.
13. The immunogenic composition of any one of claims 10-12, wherein the second protein comprises, in N- to C-orientation, the T-construct, the U-construct, the E-construct, the A-construct, the B-construct, the K-construct.
14. The immunogenic composition of claim 13, wherein the second protein comprises, in N- to C-orientation, the T-construct, the U-construct, the E-construct, the A-construct, the B-construct, the K-construct, the I-construct, the H-construct, the N-construct, the C-construct, the M-construct, the D-construct.
15. The immunogenic composition of claim 12, wherein the F-construct is upstream of the T-construct, and wherein the amino acid sequence 95% identical to SEQ ID NO 32 is downstream of the D-construct.
16. The immunogenic composition of claim 15, wherein
- Loop peptide F is SEQ ID NO: 16;
- Helix peptide F is SEQ ID NO: 17.
17. The immunogenic composition according to any one of claims 1-16, wherein the loop peptides and the helix peptide from each phylotype are adjacent to each other.
18. The immunogenic composition according to any one of claims 1-17, wherein the loop peptides and the helix peptides for each phylotype are arranged sequentially.
19. The immunogenic composition of any one of claims 1-18, wherein the loop peptide for each phylotype is upstream of the corresponding helix peptide for that phylotype.
20. The immunogenic composition according to any one of claims 1-19, wherein
- a. the first protein is SEQ ID NO: 1; and
- b. the second protein is SEQ ID NO: 30.
21. The immunogenic composition according to any one claims 1-19, wherein
- a. the first protein is SEQ ID NO: 1; and
- b. the second protein is SEQ ID NO: 31.
22. The immunogenic composition of any one of claims 1-21 further comprising at least one additional antigen preventing or reducing the pathological effects of infection with a microorganism that can cause disease in dogs.
23. The immunogenic composition of claim 22, wherein said microorganism is selected from the group consisting of canine distemper (CD) virus, canine adenovirus type 2 (CAV-2), canine parainfluenza (CPI) virus, canine parvovirus (CPV), canine coronavirus (CCV), canine herpesvirus, and rabies virus.
24. A vaccine composition comprising the immunogenic composition according to any one of claims 1-23 and an adjuvant.
25. The vaccine composition of claim 24, wherein the adjuvant is selected from the group consisting of mineral salts, surface-active agents and microparticles, bacterial products, cytokines and hormones, carriers, oil-in-water emulsions and water-in-oil emulsions.
26. A method of preventing or reducing the pathological effects of infection with Borrelia burgdorferi in a canine comprising administering the canine in need thereof an immunologically effective dose of the vaccine composition according to any one of claims 24 and 25.
Type: Application
Filed: Apr 17, 2013
Publication Date: Mar 24, 2016
Patent Grant number: 9562079
Inventors: Robert M. Lohse (Kalamazoo, MI), Patrick F. M. Meeus (Richland, MI), Jason J. Millership (Portage, MI), Zhichang Xu (Kalamazoo, MI), Richard Thomas Marconi (Midlothian, VA), Christopher G. Earnhart (Spotsylvania, VA)
Application Number: 13/864,658
