Novel methods of generating antibodies

Abstract

The invention describes a method of generating antibodies to a mixture of peptidogenic proteins wherein the peptidogenic protein has altered conformational dynamics as compared to a starting protein and wherein the peptidogenic protein has a similar conformation to the starting protein. The peptidogenic proteins can be used to induce an immune response, which can lead to the generation of antibodies and/or can be used to vaccinate a mammal.

1 .- 31 . (canceled) 
     
     
         32 . An isolated polynucleotide or set of polynucleotides encoding a mixture of at least two different peptidogenic proteins derived from a starting protein, wherein each of the peptidogenic proteins has altered conformational dynamics as compared to the starting protein, wherein each of the peptidogenic proteins is similar in conformation to the starting protein, and wherein each of the peptidogenic proteins comprises replacement of at least one non-surface amino acid residue in the starting protein with a different amino acid residue. 
     
     
         33 . The isolated polynucleotide or set of polynucleotides of  claim 32 , wherein the conformational dynamics are altered by:
 a. examining the 3-D structure of the starting protein, identifying non-surface amino acid residues of the starting protein and replacing at least one non-surface amino acid residue in the starting protein to generate the peptidogenic proteins; or   b. examining a model of the 3-D structure of the starting protein, identifying non-surface amino acid residues of the starting protein and replacing at least one non-surface amino acid residue in the starting protein to generate the peptidogenic proteins; or   c. comparing the pattern of conserved amino acid homology across proteins orthologous to the starting protein from different species to identify non-surface amino acid residues of the starting protein and replacing at least one non-surface amino acid residue in the starting protein to generate the peptidogenic proteins; or   d. replacing at least one non-surface amino acid residue of the starting protein to generate the peptidogenic proteins; or   e. replacing at least one non-surface amino acid residue with a smaller amino acid residue; or   f. replacing at least one non-surface amino acid residue with an alanine or glycine; or   g. eliminating at least one disulfide bond in the starting protein.   
     
     
         34 . The isolated polynucleotide or set of polynucleotides of  claim 32 , wherein, in the encoded peptidogenic proteins, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids are replaced in the starting protein. 
     
     
         35 . The isolated polynucleotide or set of polynucleotides of  claim 32 , wherein, in the encoded peptidogenic proteins, the conformational dynamics of the starting protein are altered by replacing:
 a. at least one non-surface threonine with a valine, alanine, glycine or serine; or   b. at least one non-surface cysteine with alanine, valine, glycine, serine or threonine; or   c. at least one non-surface valine with alanine, glycine, leucine or isoleucine; or   d. at least one non-surface leucine with alanine, valine, glycine, or isoleucine; or   e. at least one non-surface isoleucine with alanine, valine, leucine, or glycine; or   f. at least one non-surface proline with methionine, alanine, valine, leucine, isoleucine, or glycine; or   g. at least one non-surface methionine with alanine, valine, leucine, isoleucine, or glycine; or   h. at least one non-surface phenylalanine with tyrosine, methionine, histidine, alanine, valine, leucine, isoleucine, or glycine; or   i. at least one non-surface tyrosine with phenylalanine, methionine, histidine, alanine, valine, leucine, isoleucine, or glycine; or   j. at least one non-surface tryptophan with tyrosine, phenylalanine, methionine, histidine, alanine, valine, leucine, isoleucine, or glycine; or   k. at least one non-surface aspartic acid with glutamic acid, glutamine, asparagine, glycine, serine, threonine, alanine, valine, leucine, or isoleucine; or   l. at least one non-surface asparagine with glycine, serine, threonine, alanine, valine, leucine, isoleucine, glutamine, glutamic acid, or aspartic acid; or   m. at least one non-surface glutamic acid with aspartic acid, asparagine, glutamine, glycine, serine, threonine, alanine, valine, leucine, or isoleucine; or   n. at least one non-surface glutamine with glutamic acid, aspartic acid, asparagine, glutamine, glycine, serine, threonine, alanine, valine, leucine, or isoleucine; or   o. at least one non-surface lysine with arginine, histidine, glycine, serine, threonine, alanine, valine, methionine, leucine, or isoleucine; or   p. at least one non-surface arginine with lysine, histidine, glycine, serine, threonine, alanine, valine, methionine, leucine, or isoleucine; or   q. at least one non-surface histidine with phenylalanine, tyrosine, lysine, arginine, glycine, serine, threonine, alanine, valine, glutamine, asparagine, leucine, methionine or isoleucine; or   r. at least one non-surface alanine with a glycine or proline; or   s. at least one non-surface glycine with an alanine or proline; or   t. at least one non-surface serine with an alanine or glycine; or   u. at least one non-surface residue with a non-natural amino acid; or   v. a combination of any of the above replacements.   
     
     
         36 . The isolated polynucleotide or set of polynucleotides of  claim 32 , wherein the altered conformational dynamics of the peptidogenic proteins is measured by:
 a. a change in melting temperature as compared to the starting protein; or   b. a change in Gibbs free energy of stabilization or proteolytic sensitivity assay; or   c. a change in Gibbs free energy, wherein the change in Gibbs free energy of stabilization is measured by denaturant modulated equilibrium unfolding, wherein the denaturant is urea or guanidinium hydrochloride.   
     
     
         37 . The isolated polynucleotide or set of polynucleotides of  claim 32 , wherein similarity in conformation to the starting protein is measured by:
 binding of a cross-reacting antibody to both the peptidogenic proteins and the starting protein; optionally   wherein (a) binding of the cross-reactive antibody is measured by an immunoprecipitation assay, surface plasmon resonance, isothermal titration calorimetry, oblique-incidence reflective difference (OI-RD), western blots, radioimmunoassays, ELISA (enzyme linked immunosorbent assay), “sandwich” immunoassays, gel diffusion precipitin reactions, immunodiffusion assays, agglutination assays, complement-fixation assays, immunoradiometric assays, fluorescent immunoassays, and/or protein A immunoassays; and/or   (b) the cross-reacting antibody has a dissociation constant (KD) to the starting protein and the peptidogenic proteins of less than or equal to 10 −9 M; and/or   (c) the cross-reacting antibody has a dissociation constant (KD) to the starting protein and the peptidogenic proteins of less than or equal to 10 −8 M, less than or equal to 10 −7 M, or less than or equal to 10 −6 M.   
     
     
         38 . The isolated polynucleotide or set of polynucleotides of  claim 32 , wherein the starting protein is selected from:
 a. an envelope glycoprotein of the human immunodeficiency virus (HIV), HIV gp120, HIV gp41, HIV gp160, an ebola antigen, a viral antigen, a bacterial antigen, a parasite antigen, an allergen, a venom, a toxin, a tumor-associated antigen, a transmembrane domain protein, a G-protein coupled receptor, an ion channel, a hepatitis C virus antigen, a hepatitis B virus antigen, a MERS-CoV antigen, a Zika virus antigen, an influenza virus antigen, a malaria antigen; and/or   b. any one of the malaria antigens selected from MAL13P1.225, PF13_0203, PF11_0164, PFL0375W, PFI1270W, PF10_0104, PF13_0128, PF11_0058, PFA0490W, PF07_0087, PFB0570W, PF13_0180, PF11_0065, PF14_0678, PF13_0125, PF13_0141, PF14_0117, PF11_0098, PF14_0660, PFD0240C, PFE0080C, PFA0660W, PF11_0352, PF11_0055, PFB0475C, PF11_0302, PFA0210C, PF07_0089, PF14_0060, MAL8P1.17, MAL7P1.77, PF11_0099, PF07_0068, PF13_0201, PF07_0094, PF07_0006, PFL0770W, PFI1645C, PF14_0166, PFE0815W, PF11_0174, PFE0475W, PF11_0074, PFL1385C, PF14_0102, PF11_0212, PF07_0129, PFL2570W, PFL1070C, PFB0695C, PF11_0175, MAL13P1.22, PFL0035C, PFI0685W, PFD0425W, PF14_0293, PF14_0344, PFL0560C, PF07_0035, PFL1675C, PFC0435W, PFI1445W, PF13_0354, PFC0110W, PFC0120W, PF08_0078, PF14_0614, PF14_0051, PF11_0076, MAL13P1.262, PFC0640W, PFL2520W, PFC0282W, PF08_0004, PF11_0224, PF13_0272, MAL13P1.171, PFE1340W, PF14_0369, PF14_0178, PFL0870W, PFC0210C, PFI0500W, PF11_0069, PFD0355C, PFI0880C, PF11_0251, PFC0065C, PFC0925W, MAL13P1.121, PF13_0133, PFL2015W, PFD0440W, PFC0330W, PFD0430C, PF14_0462, PF07_0100, MAL7P1.23, PFL1835W, PF07_0047, PF14_0250, PFE0905W, PFA0180W, PFL1210W, PF14_0363, PFB0400W, PFI0920C, PF14_0094, PFA0125C, PF10_0372, PFL2315C, MAL13P1.271, MAL7P1.31, PF10_0317, PF07_0070, MAL7P1.64, PFI0935W, PFA0160C, PFB0465C, PF10_0208, PFB0760W, MAL13P1.206, PF14_0541, PFL0790W, PFL2410W, PF14_0440, PF11_0333, PF10_0242, PFC0590C, PF14_0342, MAL7P1.92, PF14_0593, MAL13P1.49, MAL13P1.172, PF08_0006, PFD1035W, PF11_0052, MAL13P1.79, PF10_0295, PFL1745C, PF14_0677, PFL0600W, PF08_0108, PF08_0081, PF14_0620, PFE0710W, PF11_0270, MAL7P1.149, PFC0810C, PF14_0249, PF13_0116, MAL13P1.60, PF11_0246, PFL2505C, PFB0405W, PF11_0256, PF08_0047, PFC0835C, PFI0605C, PF10_0127, PF11_0344, PF10_0130, PFL2395C, PF08_0008, PF14_0201, PFI1475W, PF13_0182, PF14_0495, or PF13_0277; and/or   c. any one of the Targets selected from A4GCL9, A8T655, B2R7F8, B4DEZ9, B4DTR1, D4HNR6, D5RWT1, F8WCM5, G8EJ09, I6YE93, K9N5Q8, O00141, O00189, O00206, O00220, O00253, O00294, O00300, O00522, O00585, O00754, O14522, O14763, O14773, O14788, O14793, O14807, O14836, O14896, O14931, O14936, O14976, O14994, O15020, O15118, O15230, O15305, O15357, O15372, O15394, O15455, O15520, O43155, O43291, O43323, O43424, O43557, O43570, O43707, O43854, O43897, O43933, O60229, O60494, O60565, O60602, O60706, O60840, O60882, O75197, O75367, O75369, O75436, O75581, O75665, O75695, O75712, O75874, O75888, O75923, O75970, O76041, O76090, O94804, O95150, O95256, O95342, O95407, O95445, O95452, O95633, O95760, O95967, P00167, P00338, P00387, P00403, P00439, P00441, P00451, P00488, P00492, P00519, P00533, P00568, P00734, P00740, P00742, P00746, P00747, P00748, P00751, P00797, P00918, P00966, P00995, P01008, P01009, P01019, P01024, P01031, P01032, P01034, P01111, P01127, P01130, P01137, P01138, P01185, P01241, P01308, P01344, P01374, P01375, P01579, P01583, P01584, P01588, P01589, P01730, P01854, P01889, P01903, P01906, P01909, P01911, P01912, P01920, P02452, P02458, P02461, P02462, P02489, P02647, P02649, P02671, P02675, P02679, P02708, P02730, P02760, P02766, P02768, P02792, P03147, P03372, P03420, P03441, P03446, P03448, P03456, P03457, P03471, P03472, P03474, P03476, P03477, P03478, P03886, P03950, P03951, P04040, P04049, P04062, P04070, P04080, P04155, P04156, P04179, P04180, P04233, P04234, P04275, P04424, P04440, P04578, P04626, P04629, P04661, P04792, P04839, P05019, P05062, P05067, P05089, P05091, P05106, P05107, P05112, P05113, P05121, P05129, P05155, P05156, P05186, P05231, P05386, P05540, P05556, P05961, P05997, P06127, P06213, P06239, P06280, P06340, P06396, P06681, P06684, P06737, P06744, P06804, P06865, P07204, P07225, P07288, P07339, P07477, P07585, P07602, P07686, P07911, P07949, P07954, P08034, P08100, P08118, P08133, P08183, P08235, P08236, P08237, P08246, P08253, P08326, P08473, P08514, P08519, P08567, P08572, P08575, P08581, P08603, P08617, P08708, P08709, P08887, P09417, P09467, P09603, P09619, P09668, P09936, P09960, P0C0L4, P0C0L5, P0DJI8, P0DJI9, P10071, P10321, P10448, P10619, P10721, P10809, P10828, P10912, P11021, P11132, P11166, P11168, P11177, P11215, P11217, P11224, P11230, P11233, P11310, P11362, P11413, P11532, P11586, P11597, P11836, P12023, P12111, P12259, P12318, P12643, P12644, P12821, P12830, P12955, P13103, P13224, P13500, P13569, P13612, P13637, P13716, P13726, P13747, P13765, P13804, P13807, P14138, P14174, P14207, P14210, P14222, P14416, P14672, P14770, P14778, P14780, P14784, P14867, P14921, P14923, P15018, P15259, P15260, P15289, P15291, P15313, P15328, P15391, P15509, P15529, P15692, P15814, P15848, P15907, P15924, P15941, P16109, P16144, P16154, P16278, P16401, P16410, P16422, P16442, P16473, P16871, P16930, P17050, P17174, P17693, P17787, P17858, P17900, P17927, P17948, P18074, P18177, P18206, P18510, P19079, P19235, P19256, P19320, P19438, P19694, P19838, P19876, P20023, P20036, P20138, P20151, P20273, P20701, P20702, P20749, P20807, P20849, P20929, P20933, P21333, P21359, P21439, P21580, P21583, P21709, P21781, P21802, P21817, P21860, P21912, P22105, P22223, P22301, P22362, P22413, P22460, P22735, P22748, P23284, P23415, P23468, P23510, P23560, P23945, P23975, P24723, P24855, P25024, P25025, P25054, P25189, P25445, P26010, P26136, P26927, P27169, P27487, P27909, P27930, P27958, P28062, P28068, P28472, P28482, P28799, P28907, P28908, P29033, P29120, P29216, P29320, P29323, P29400, P29459, P29460, P29590, P29597, P29965, P30044, P30203, P30301, P30460, P30530, P30556, P31371, P31785, P31994, P31995, P31997, P32297, P32754, P33681, P33795, P34059, P34130, P35221, P35222, P35225, P35442, P35462, P35475, P35555, P35579, P35609, P35625, P35670, P35749, P35754, P35968, P35991, P36222, P36871, P36888, P36894, P36896, P36897, P36915, P36941, P36955, P37023, P37173, P38117, P39019, P39023, P39060, P39905, P40225, P40238, P40259, P40394, P41159, P41180, P41181, P41250, P42081, P42261, P42263, P42336, P42568, P42658, P42702, P42768, P43026, P43034, P43235, P43258, P43259, P43260, P43681, P44815, P45381, P45954, P46094, P46379, P46459, P46531, P46783, P46934, P46952, P47756, P48023, P48061, P48167, P48436, P48544, P48643, P49257, P49419, P49588, P49683, P49747, P49768, P49789, P49810, P50440, P50454, P50570, P50897, P50993, P51149, P51159, P51532, P51570, P51608, P51648, P51681, P51688, P51690, P51693, P51787, P51800, P51812, P51813, P52788, P52803, P53004, P53420, P53634, P54098, P54577, P54764, P54802, P54803, P54819, P55008, P55072, P55075, P55268, P55286, P55291, P55789, P56537, P57075, P57735, P58335, P59594, P60033, P60484, P60568, P60900, P60953, P61457, P61626, P61769, P61812, P61916, P62070, P62081, P62736, P62854, P62913, P63092, P63252, P67870, P68032, P68036, P68133, P68871, P69905, P78324, P78363, P78380, P78504, P78509, P78545, P80098, P82279, P84022, P98082, P98160, P98161, Q00653, Q00973, Q01484, Q01955, Q01968, Q02223, Q02297, Q02388, Q02413, Q02487, Q03154, Q03403, Q03426, Q03591, Q04446, Q04609, Q04656, Q04721, Q04771, Q04844, Q05397, Q05495, Q05586, Q06124, Q06187, Q07001, Q07108, Q07599, Q07954, Q07FI5, Q08048, Q08334, Q08345, Q08881, Q09428, Q09470, Q0A2R1, Q0A448, Q0ED31, Q0Z7S6, Q0ZME7, Q0ZNA6, Q12791, Q12809, Q12879, Q12965, Q13002, Q13009, Q13018, Q13093, Q13126, Q13224, Q13231, Q13253, Q13410, Q13421, Q13424, Q13478, Q13496, Q13501, Q13563, Q13614, Q13620, Q13651, Q13698, Q13753, Q13822, Q13835, Q13873, Q13936, Q14019, Q14103, Q14108, Q14114, Q14117, Q14126, Q14204, Q14315, Q14376, Q14563, Q14571, Q14573, Q14654, Q14833, Q14956, Q14974, Q15027, Q15046, Q15109, Q15116, Q15149, Q15223, Q15262, Q15303, Q15465, Q15582, Q15746, Q15822, Q15831, Q15833, Q15848, Q16288, Q16348, Q16515, Q16552, Q16566, Q16787, Q16827, Q189K3, Q189K5, Q18PE1, Q1HLC5, Q1HP67, Q1PHM6, Q28090, Q289M7, Q29974, Q29980, Q29983, Q2M1P5, Q2N0S5, Q2N0S6, Q30154, Q30201, Q32ZE1, Q3T906, Q3UV74, Q3ZC95, Q3ZLH8, Q46342, Q49DS8, Q4KMG0, Q53SF7, Q59FL8, Q59H50, Q5D862, Q5JWF2, Q5T2T1, Q5VV43, Q5VWQ8, Q5VYX0, Q67333, Q69994, Q6DQ33, Q6IQ55, Q6KF10, Q6PHW0, Q6Q1S2, Q6TXC0, Q6VMK1, Q6XV28, Q71U36, Q75760, Q7L0Q8, Q7LG56, Q7Z6G8, Q7Z6Z7, Q82559, Q84850, Q86UR5, Q86UX7, Q8IU80, Q8IWT1, Q8IZI9, Q8IZJ0, Q8IZP0, Q8IZU9, Q8J581, Q8JDI3, Q8N0W4, Q8N157, Q8N1C3, Q8N5C8, Q8N608, Q8NBP7, Q8NCM8, Q8NFY4, Q8NG31, Q8Q7Z9, Q8TDA6, Q8TDX5, Q8TEW8, Q8TF72, Q8WWA0, Q8WX93, Q8WXD0, Q8WXH0, Q8WXI7, Q91MA7, Q92185, Q92542, Q92597, Q92692, Q92736, Q92743, Q92765, Q92766, Q92796, Q92834, Q92838, Q92887, Q92956, Q93052, Q93099, Q969J5, Q96B97, Q96F46, Q96FE5, Q96HC4, Q96J02, Q96J92, Q96MS0, Q96P20, Q96PD4, Q96PX8, Q96QZ7, Q993A8, Q99466, Q99497, Q99877, Q99972, Q99985, Q9BQB4, Q9BUL8, Q9BWV1, Q9BXJ0, Q9BXM0, Q9BXR6, Q9BZ11, Q9C000, Q9D1H1, Q9DBR4, Q9EQF4, Q9GZV9, Q9H0F7, Q9H0U3, Q9H221, Q9H222, Q9H251, Q9H3Z4, Q9H4F8, Q9H6X2, Q9HB21, Q9HBA0, Q9HBE4, Q9HCE7, Q9HCK4, Q9HD26, Q9HDB5, Q9IGQ6, Q9J3E7, Q9NPF7, Q9NQ25, Q9NQ38, Q9NQC7, Q9NS40, Q9NVA2, Q9NVD7, Q9NY72, Q9NZ08, Q9NZN1, Q9NZQ7, Q9NZV8, Q9P1W8, Q9UBP0, Q9UBQ7, Q9UBV7, Q9UBX5, Q9UGJ0, Q9UGM3, Q9UHF4, Q9UHG0, Q9UHN1, Q9UHP7, Q9UIR5, Q9UKL4, Q9UKM7, Q9UKQ2, Q9ULV1, Q9ULV5, Q9UM47, Q9UM54, Q9UNA1, Q9UNG2, Q9UPW8, Q9W7Y7, Q9WFX3, Q9WMX2, Q9Y275, Q9Y281, Q9Y2Q5, Q9Y496, Q9Y4CO, Q9Y4J8, Q9Y5K2, Q9Y5K6, Q9Y6D5, Q9Y6N8, or Q9Y6Q6; and/or   d. a tumor associated antigen selected from MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9, MAGE-A10, MAGE-A11, MAGE-A12, GAGE-I, GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7, GAGE-8, BAGE-I, RAGE-1, LB33/MUM-1, PRAME, NAG, MAGE-Xp2 (MAGE-B2), MAGE-Xp3 (MAGE-B3), MAGE-Xp4 (MAGE-B4), MAGE-C1/CT7, MAGE-C2, NY-ESO-I, LAGE-I, SSX-I, SSX-2(HOM-MEL-40), SSX-3, SSX-4, SSX-5, SCP-I and XAGE, melanocyte differentiation antigens, p53, ras, CEA, MUC1, PMSA, PSA, tyrosinase, Melan-A, MART-1, gp100, gp75, alpha-actinin-4, Bcr-Abl fusion protein, Casp-8, beta-catenin, cdc27, cdk4, cdkn2a, coa-1, dek-can fusion protein, EF2, ETV6-AML1 fusion protein, LDLR-fucosyltransferaseAS fusion protein, HLA-A2, HLA-A11, hsp70-2, KIAA0205, Mart2, Mum-2, and 3, neo-PAP, myosin class I, OS-9, pml-RAR alpha fusion protein, PTPRK, K-ras, N-ras, Triosephosphate isomerase, GnTV, Herv-K-mel, NA-88, SP17, and TRP2-Int2, (MART-I), E2A-PRL, H4-RET, IGH-IGK, MYL-RAR, Epstein Barr virus antigens, EBNA, human papillomavirus (HPV) antigens E6 and E7, TSP-180, MAGE-4, MAGE-5, MAGE-6, p185erbB2, p180erbB-3, c-met, nm-23H1, PSA, TAG-72-4, CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras, alpha-fetoprotein, 13HCG, BCA225, BTAA, CA 125, CA 15-3 (CA 27.29\BCAA), CA 195, CA 242, CA-50, CAM43, CD68\KP1, CO-029, FGF-5, G250, Ga733 (EpCAM), HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB\170K, NY-CO-1, RCAS1, SDCCAG16, TA-90 (Mac-2 binding protein\cyclophilin C-associated protein), TAAL6, TAG72, TLP, TPS, tyrosinase related proteins, TRP-1, TRP-2, or cytomegalovirus phosphoprotein 65 (pp65).   
     
     
         39 . The isolated polynucleotide or set of polynucleotides of  claim 32 , wherein the peptidogenic proteins are derived:
 a. from the same starting protein; or   b. from multiple starting proteins; or   
       from multiple related starting proteins. 
     
     
         40 . The isolated polynucleotide or set of polynucleotides of  claim 32 , wherein the polynucleotides:
 c. are synthesized in vitro; or   d. comprise DNA; or   e. comprise in vitro transcribed (IVT) mRNA; or   f. comprise IVT mRNA comprising a poly(A) tail; or   g. comprise IVT mRNA comprising a 5′ Cap,   
       optionally wherein the polynucleotides are:
 1) not associated with any targeting components; or 
 2) are associated with a targeting component capable of targeting the polynucleotides to a cell or an organ; or 
 3) are associated with a targeting component capable of targeting the polynucleotides to a cell or an organ, wherein the targeting component is a vector. 
 
     
     
         41 . The isolated polynucleotide or set of polynucleotides of  claim 32 , wherein the mixture of peptidogenic proteins further comprises the starting protein. 
     
     
         42 . The isolated polynucleotide or set of polynucleotides of  claim 32 , wherein each of the peptidogenic proteins has increased conformational dynamics as compared to the starting protein. 
     
     
         43 . An isolated mixture of polypeptides encoded by the isolated polynucleotide or set of polynucleotides of  claim 32 . 
     
     
         44 . The isolated mixture of polypeptides of  claim 43 , wherein the conformational dynamics are altered by:
 h. examining the 3-D structure of the starting protein, identifying non-surface amino acid residues of the starting protein and replacing at least one non-surface amino acid residue in the starting protein to generate the peptidogenic proteins; or   i. examining a model of the 3-D structure of the starting protein, identifying non-surface amino acid residues of the starting protein and replacing at least one non-surface amino acid residue in the starting protein to generate the peptidogenic proteins; or   j. comparing the pattern of conserved amino acid homology across proteins orthologous to the starting protein from different species to identify non-surface amino acid residues of the starting protein and replacing at least one non-surface amino acid residue in the starting protein to generate the peptidogenic proteins; or   k. replacing at least one non-surface amino acid residue of the starting protein to generate the peptidogenic proteins; or   l. replacing at least one non-surface amino acid residue with a smaller amino acid residue; or   m. replacing at least one non-surface amino acid residue with an alanine or glycine; or   n. eliminating at least one disulfide bond in the starting protein.   
     
     
         45 . The isolated mixture of polypeptides of  claim 43 , wherein at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids are replaced in the starting protein. 
     
     
         46 . The isolated mixture of polypeptides of  claim 43 , wherein the conformational dynamics of the starting protein are altered by replacing:
 w. at least one non-surface threonine with a valine, alanine, glycine or serine; or   x. at least one non-surface cysteine with alanine, valine, glycine, serine or threonine; or   y. at least one non-surface valine with alanine, glycine, leucine or isoleucine; or   z. at least one non-surface leucine with alanine, valine, glycine, or isoleucine; or   aa. at least one non-surface isoleucine with alanine, valine, leucine, or glycine; or   bb. at least one non-surface proline with methionine, alanine, valine, leucine, isoleucine, or glycine; or   cc. at least one non-surface methionine with alanine, valine, leucine, isoleucine, or glycine; or   dd. at least one non-surface phenylalanine with tyrosine, methionine, histidine, alanine, valine, leucine, isoleucine, or glycine; or   ee. at least one non-surface tyrosine with phenylalanine, methionine, histidine, alanine, valine, leucine, isoleucine, or glycine; or   ff. at least one non-surface tryptophan with tyrosine, phenylalanine, methionine, histidine, alanine, valine, leucine, isoleucine, or glycine; or   gg. at least one non-surface aspartic acid with glutamic acid, glutamine, asparagine, glycine, serine, threonine, alanine, valine, leucine, or isoleucine; or   hh. at least one non-surface asparagine with glycine, serine, threonine, alanine, valine, leucine, isoleucine, glutamine, glutamic acid, or aspartic acid; or   ii. at least one non-surface glutamic acid with aspartic acid, asparagine, glutamine, glycine, serine, threonine, alanine, valine, leucine, or isoleucine; or   jj. at least one non-surface glutamine with glutamic acid, aspartic acid, asparagine, glutamine, glycine, serine, threonine, alanine, valine, leucine, or isoleucine; or   kk. at least one non-surface lysine with arginine, histidine, glycine, serine, threonine, alanine, valine, methionine, leucine, or isoleucine; or   ll. at least one non-surface arginine with lysine, histidine, glycine, serine, threonine, alanine, valine, methionine, leucine, or isoleucine; or   mm. at least one non-surface histidine with phenylalanine, tyrosine, lysine, arginine, glycine, serine, threonine, alanine, valine, glutamine, asparagine, leucine, methionine or isoleucine; or   nn. at least one non-surface alanine with a glycine or proline; or   oo. at least one non-surface glycine with an alanine or proline; or   pp. at least one non-surface serine with an alanine or glycine; or   qq. at least one non-surface residue with a non-natural amino acid; or   rr. a combination of any of the above replacements.   
     
     
         47 . The isolated mixture of polypeptides of  claim 43 , wherein the altered conformational dynamics of the peptidogenic proteins is measured by:
 d. a change in melting temperature as compared to the starting protein; or   e. a change in Gibbs free energy of stabilization or proteolytic sensitivity assay; or   f. a change in Gibbs free energy, wherein the change in Gibbs free energy of stabilization is measured by denaturant modulated equilibrium unfolding, wherein the denaturant is urea or guanidinium hydrochloride.   
     
     
         48 . The isolated mixture of polypeptides of  claim 43 , wherein similarity in conformation to the starting protein is measured by:
 binding of a cross-reacting antibody to both the peptidogenic proteins and the starting protein; optionally   wherein (a) binding of the cross-reactive antibody is measured by an immunoprecipitation assay, surface plasmon resonance, isothermal titration calorimetry, oblique-incidence reflective difference (OI-RD), western blots, radioimmunoassays, ELISA (enzyme linked immunosorbent assay), “sandwich” immunoassays, gel diffusion precipitin reactions, immunodiffusion assays, agglutination assays, complement-fixation assays, immunoradiometric assays, fluorescent immunoassays, and/or protein A immunoassays; and/or   (b) the cross-reacting antibody has a dissociation constant (KD) to the starting protein and the peptidogenic proteins of less than or equal to 10 −9 M; and/or   (c) the cross-reacting antibody has a dissociation constant (KD) to the starting protein and the peptidogenic proteins of less than or equal to 10 −8 M, less than or equal to 10 −7 M, or less than or equal to 10 −6 M.   
     
     
         49 . The isolated mixture of polypeptides of  claim 43 , wherein the starting protein is selected from:
 e. an envelope glycoprotein of the human immunodeficiency virus (HIV), HIV gp120, HIV gp41, HIV gp160, an ebola antigen, a viral antigen, a bacterial antigen, a parasite antigen, an allergen, a venom, a toxin, a tumor-associated antigen, a transmembrane domain protein, a G-protein coupled receptor, an ion channel, a hepatitis C virus antigen, a hepatitis B virus antigen, a MERS-CoV antigen, a Zika virus antigen, an influenza virus antigen, a malaria antigen; and/or   f. any one of the malaria antigens selected from MAL13P1.225, PF13_0203, PF11_0164, PFL0375W, PFI1270W, PF10_0104, PF13_0128, PF11_0058, PFA0490W, PF07_0087, PFB0570W, PF13_0180, PF11_0065, PF14_0678, PF13_0125, PF13_0141, PF14_0117, PF11_0098, PF14_0660, PFD0240C, PFE0080C, PFA0660W, PF11_0352, PF11_0055, PFB0475C, PF11_0302, PFA0210C, PF07_0089, PF14_0060, MAL8P1.17, MAL7P1.77, PF11_0099, PF07_0068, PF13_0201, PF07_0094, PF07_0006, PFL0770W, PFI1645C, PF14_0166, PFE0815W, PF11_0174, PFE0475W, PF11_0074, PFL1385C, PF14_0102, PF11_0212, PF07_0129, PFL2570W, PFL1070C, PFB0695C, PF11_0175, MAL13P1.22, PFL0035C, PFI0685W, PFD0425W, PF14_0293, PF14_0344, PFL0560C, PF07_0035, PFL1675C, PFC0435W, PFI1445W, PF13_0354, PFC0110W, PFC0120W, PF08_0078, PF14_0614, PF14_0051, PF11_0076, MAL13P1.262, PFC0640W, PFL2520W, PFC0282W, PF08_0004, PF11_0224, PF13_0272, MAL13P1.171, PFE1340W, PF14_0369, PF14_0178, PFL0870W, PFC0210C, PFI0500W, PF11_0069, PFD0355C, PFI0880C, PF11_0251, PFC0065C, PFC0925W, MAL13P1.121, PF13_0133, PFL2015W, PFD0440W, PFC0330W, PFD0430C, PF14_0462, PF07_0100, MAL7P1.23, PFL1835W, PF07_0047, PF14_0250, PFE0905W, PFA0180W, PFL1210W, PF14_0363, PFB0400W, PFI0920C, PF14_0094, PFA0125C, PF10_0372, PFL2315C, MAL13P1.271, MAL7P1.31, PF10_0317, PF07_0070, MAL7P1.64, PFI0935W, PFA0160C, PFB0465C, PF10_0208, PFB0760W, MAL13P1.206, PF14_0541, PFL0790W, PFL2410W, PF14_0440, PF11_0333, PF10_0242, PFC0590C, PF14_0342, MAL7P1.92, PF14_0593, MAL13P1.49, MAL13P1.172, PF08_0006, PFD1035W, PF11_0052, MAL13P1.79, PF10_0295, PFL1745C, PF14_0677, PFL0600W, PF08_0108, PF08_0081, PF14_0620, PFE0710W, PF11_0270, MAL7P1.149, PFC0810C, PF14_0249, PF13_0116, MAL13P1.60, PF11_0246, PFL2505C, PFB0405W, PF11_0256, PF08_0047, PFC0835C, PFI0605C, PF10_0127, PF11_0344, PF10_0130, PFL2395C, PF08_0008, PF14_0201, PFI1475W, PF13_0182, PF14_0495, or PF13_0277; and/or   g. any one of the Targets selected from A4GCL9, A8T655, B2R7F8, B4DEZ9, B4DTR1, D4HNR6, D5RWT1, F8WCM5, G8EJ09, I6YE93, K9N5Q8, O00141, O00189, O00206, O00220, O00253, O00294, O00300, O00522, O00585, O00754, O14522, O14763, O14773, O14788, O14793, O14807, O14836, O14896, O14931, O14936, O14976, O14994, O15020, O15118, O15230, O15305, O15357, O15372, O15394, O15455, O15520, O43155, O43291, O43323, O43424, O43557, O43570, O43707, O43854, O43897, O43933, O60229, O60494, O60565, O60602, O60706, O60840, O60882, O75197, O75367, O75369, O75436, O75581, O75665, O75695, O75712, O75874, O75888, O75923, O75970, O76041, O76090, O94804, O95150, O95256, O95342, O95407, O95445, O95452, O95633, O95760, O95967, P00167, P00338, P00387, P00403, P00439, P00441, P00451, P00488, P00492, P00519, P00533, P00568, P00734, P00740, P00742, P00746, P00747, P00748, P00751, P00797, P00918, P00966, P00995, P01008, P01009, P01019, P01024, P01031, P01032, P01034, P01111, P01127, P01130, P01137, P01138, P01185, P01241, P01308, P01344, P01374, P01375, P01579, P01583, P01584, P01588, P01589, P01730, P01854, P01889, P01903, P01906, P01909, P01911, P01912, P01920, P02452, P02458, P02461, P02462, P02489, P02647, P02649, P02671, P02675, P02679, P02708, P02730, P02760, P02766, P02768, P02792, P03147, P03372, P03420, P03441, P03446, P03448, P03456, P03457, P03471, P03472, P03474, P03476, P03477, P03478, P03886, P03950, P03951, P04040, P04049, P04062, P04070, P04080, P04155, P04156, P04179, P04180, P04233, P04234, P04275, P04424, P04440, P04578, P04626, P04629, P04661, P04792, P04839, P05019, P05062, P05067, P05089, P05091, P05106, P05107, P05112, P05113, P05121, P05129, P05155, P05156, P05186, P05231, P05386, P05540, P05556, P05961, P05997, P06127, P06213, P06239, P06280, P06340, P06396, P06681, P06684, P06737, P06744, P06804, P06865, P07204, P07225, P07288, P07339, P07477, P07585, P07602, P07686, P07911, P07949, P07954, P08034, P08100, P08118, P08133, P08183, P08235, P08236, P08237, P08246, P08253, P08326, P08473, P08514, P08519, P08567, P08572, P08575, P08581, P08603, P08617, P08708, P08709, P08887, P09417, P09467, P09603, P09619, P09668, P09936, P09960, P0C0L4, P0C0L5, P0DJI8, P0DJI9, P10071, P10321, P10448, P10619, P10721, P10809, P10828, P10912, P11021, P11132, P11166, P11168, P11177, P11215, P11217, P11224, P11230, P11233, P11310, P11362, P11413, P11532, P11586, P11597, P11836, P12023, P12111, P12259, P12318, P12643, P12644, P12821, P12830, P12955, P13103, P13224, P13500, P13569, P13612, P13637, P13716, P13726, P13747, P13765, P13804, P13807, P14138, P14174, P14207, P14210, P14222, P14416, P14672, P14770, P14778, P14780, P14784, P14867, P14921, P14923, P15018, P15259, P15260, P15289, P15291, P15313, P15328, P15391, P15509, P15529, P15692, P15814, P15848, P15907, P15924, P15941, P16109, P16144, P16154, P16278, P16401, P16410, P16422, P16442, P16473, P16871, P16930, P17050, P17174, P17693, P17787, P17858, P17900, P17927, P17948, P18074, P18177, P18206, P18510, P19079, P19235, P19256, P19320, P19438, P19694, P19838, P19876, P20023, P20036, P20138, P20151, P20273, P20701, P20702, P20749, P20807, P20849, P20929, P20933, P21333, P21359, P21439, P21580, P21583, P21709, P21781, P21802, P21817, P21860, P21912, P22105, P22223, P22301, P22362, P22413, P22460, P22735, P22748, P23284, P23415, P23468, P23510, P23560, P23945, P23975, P24723, P24855, P25024, P25025, P25054, P25189, P25445, P26010, P26136, P26927, P27169, P27487, P27909, P27930, P27958, P28062, P28068, P28472, P28482, P28799, P28907, P28908, P29033, P29120, P29216, P29320, P29323, P29400, P29459, P29460, P29590, P29597, P29965, P30044, P30203, P30301, P30460, P30530, P30556, P31371, P31785, P31994, P31995, P31997, P32297, P32754, P33681, P33795, P34059, P34130, P35221, P35222, P35225, P35442, P35462, P35475, P35555, P35579, P35609, P35625, P35670, P35749, P35754, P35968, P35991, P36222, P36871, P36888, P36894, P36896, P36897, P36915, P36941, P36955, P37023, P37173, P38117, P39019, P39023, P39060, P39905, P40225, P40238, P40259, P40394, P41159, P41180, P41181, P41250, P42081, P42261, P42263, P42336, P42568, P42658, P42702, P42768, P43026, P43034, P43235, P43258, P43259, P43260, P43681, P44815, P45381, P45954, P46094, P46379, P46459, P46531, P46783, P46934, P46952, P47756, P48023, P48061, P48167, P48436, P48544, P48643, P49257, P49419, P49588, P49683, P49747, P49768, P49789, P49810, P50440, P50454, P50570, P50897, P50993, P51149, P51159, P51532, P51570, P51608, P51648, P51681, P51688, P51690, P51693, P51787, P51800, P51812, P51813, P52788, P52803, P53004, P53420, P53634, P54098, P54577, P54764, P54802, P54803, P54819, P55008, P55072, P55075, P55268, P55286, P55291, P55789, P56537, P57075, P57735, P58335, P59594, P60033, P60484, P60568, P60900, P60953, P61457, P61626, P61769, P61812, P61916, P62070, P62081, P62736, P62854, P62913, P63092, P63252, P67870, P68032, P68036, P68133, P68871, P69905, P78324, P78363, P78380, P78504, P78509, P78545, P80098, P82279, P84022, P98082, P98160, P98161, Q00653, Q00973, Q01484, Q01955, Q01968, Q02223, Q02297, Q02388, Q02413, Q02487, Q03154, Q03403, Q03426, Q03591, Q04446, Q04609, Q04656, Q04721, Q04771, Q04844, Q05397, Q05495, Q05586, Q06124, Q06187, Q07001, Q07108, Q07599, Q07954, Q07FI5, Q08048, Q08334, Q08345, Q08881, Q09428, Q09470, Q0A2R1, Q0A448, Q0ED31, Q0Z7S6, Q0ZME7, Q0ZNA6, Q12791, Q12809, Q12879, Q12965, Q13002, Q13009, Q13018, Q13093, Q13126, Q13224, Q13231, Q13253, Q13410, Q13421, Q13424, Q13478, Q13496, Q13501, Q13563, Q13614, Q13620, Q13651, Q13698, Q13753, Q13822, Q13835, Q13873, Q13936, Q14019, Q14103, Q14108, Q14114, Q14117, Q14126, Q14204, Q14315, Q14376, Q14563, Q14571, Q14573, Q14654, Q14833, Q14956, Q14974, Q15027, Q15046, Q15109, Q15116, Q15149, Q15223, Q15262, Q15303, Q15465, Q15582, Q15746, Q15822, Q15831, Q15833, Q15848, Q16288, Q16348, Q16515, Q16552, Q16566, Q16787, Q16827, Q189K3, Q189K5, Q18PE1, Q1HLC5, Q1HP67, Q1PHM6, Q28090, Q289M7, Q29974, Q29980, Q29983, Q2M1P5, Q2N0S5, Q2N0S6, Q30154, Q30201, Q32ZE1, Q3T906, Q3UV74, Q3ZC95, Q3ZLH8, Q46342, Q49DS8, Q4KMG0, Q53SF7, Q59FL8, Q59H50, Q5D862, Q5JWF2, Q5T2T1, Q5VV43, Q5VWQ8, Q5VYX0, Q67333, Q69994, Q6DQ33, Q6IQ55, Q6KF10, Q6PHW0, Q6Q1S2, Q6TXC0, Q6VMK1, Q6XV28, Q71U36, Q75760, Q7L0Q8, Q7LG56, Q7Z6G8, Q7Z6Z7, Q82559, Q84850, Q86UR5, Q86UX7, Q8IU80, Q8IWT1, Q8IZI9, Q8IZJ0, Q8IZP0, Q8IZU9, Q8J581, Q8JDI3, Q8N0W4, Q8N157, Q8N1C3, Q8N5C8, Q8N608, Q8NBP7, Q8NCM8, Q8NFY4, Q8NG31, Q8Q7Z9, Q8TDA6, Q8TDX5, Q8TEW8, Q8TF72, Q8WWA0, Q8WX93, Q8WXD0, Q8WXH0, Q8WXI7, Q91MA7, Q92185, Q92542, Q92597, Q92692, Q92736, Q92743, Q92765, Q92766, Q92796, Q92834, Q92838, Q92887, Q92956, Q93052, Q93099, Q969J5, Q96B97, Q96F46, Q96FE5, Q96HC4, Q96J02, Q96J92, Q96MS0, Q96P20, Q96PD4, Q96PX8, Q96QZ7, Q993A8, Q99466, Q99497, Q99877, Q99972, Q99985, Q9BQB4, Q9BUL8, Q9BWV1, Q9BXJ0, Q9BXM0, Q9BXR6, Q9BZ11, Q9C000, Q9D1H1, Q9DBR4, Q9EQF4, Q9GZV9, Q9H0F7, Q9H0U3, Q9H221, Q9H222, Q9H251, Q9H3Z4, Q9H4F8, Q9H6X2, Q9HB21, Q9HBA0, Q9HBE4, Q9HCE7, Q9HCK4, Q9HD26, Q9HDB5, Q9IGQ6, Q9J3E7, Q9NPF7, Q9NQ25, Q9NQ38, Q9NQC7, Q9NS40, Q9NVA2, Q9NVD7, Q9NY72, Q9NZ08, Q9NZN1, Q9NZQ7, Q9NZV8, Q9P1W8, Q9UBP0, Q9UBQ7, Q9UBV7, Q9UBX5, Q9UGJ0, Q9UGM3, Q9UHF4, Q9UHG0, Q9UHN1, Q9UHP7, Q9UIR5, Q9UKL4, Q9UKM7, Q9UKQ2, Q9ULV1, Q9ULV5, Q9UM47, Q9UM54, Q9UNA1, Q9UNG2, Q9UPW8, Q9W7Y7, Q9WFX3, Q9WMX2, Q9Y275, Q9Y281, Q9Y2Q5, Q9Y496, Q9Y4CO, Q9Y4J8, Q9Y5K2, Q9Y5K6, Q9Y6D5, Q9Y6N8, or Q9Y6Q6; and/or   h. a tumor associated antigen selected from MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9, MAGE-A10, MAGE-A11, MAGE-A12, GAGE-I, GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7, GAGE-8, BAGE-I, RAGE-1, LB33/MUM-1, PRAME, NAG, MAGE-Xp2 (MAGE-B2), MAGE-Xp3 (MAGE-B3), MAGE-Xp4 (MAGE-B4), MAGE-C1/CT7, MAGE-C2, NY-ESO-I, LAGE-I, SSX-I, SSX-2(HOM-MEL-40), SSX-3, SSX-4, SSX-5, SCP-I and XAGE, melanocyte differentiation antigens, p53, ras, CEA, MUC1, PMSA, PSA, tyrosinase, Melan-A, MART-1, gp100, gp75, alpha-actinin-4, Bcr-Abl fusion protein, Casp-8, beta-catenin, cdc27, cdk4, cdkn2a, coa-1, dek-can fusion protein, EF2, ETV6-AML1 fusion protein, LDLR-fucosyltransferaseAS fusion protein, HLA-A2, HLA-A11, hsp70-2, KIAA0205, Mart2, Mum-2, and 3, neo-PAP, myosin class I, OS-9, pml-RAR alpha fusion protein, PTPRK, K-ras, N-ras, Triosephosphate isomerase, GnTV, Herv-K-mel, NA-88, SP17, and TRP2-Int2, (MART-I), E2A-PRL, H4-RET, IGH-IGK, MYL-RAR, Epstein Barr virus antigens, EBNA, human papillomavirus (HPV) antigens E6 and E7, TSP-180, MAGE-4, MAGE-5, MAGE-6, p185erbB2, p180erbB-3, c-met, nm-23H1, PSA, TAG-72-4, CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras, alpha-fetoprotein, 13HCG, BCA225, BTAA, CA 125, CA 15-3 (CA 27.29\BCAA), CA 195, CA 242, CA-50, CAM43, CD68\KP1, CO-029, FGF-5, G250, Ga733 (EpCAM), HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB\170K, NY-CO-1, RCAS1, SDCCAG16, TA-90 (Mac-2 binding protein\cyclophilin C-associated protein), TAAL6, TAG72, TLP, TPS, tyrosinase related proteins, TRP-1, TRP-2, or cytomegalovirus phosphoprotein 65 (pp65).   
     
     
         50 . The isolated mixture of polypeptides of  claim 43 , wherein the peptidogenic proteins are derived:
 a. from the same starting protein; or   b. from multiple starting proteins; or   
       from multiple related starting proteins. 
     
     
         51 . The isolated mixture of polypeptides of  claim 43 , wherein the polynucleotides:
 c. are synthesized in vitro; or   d. comprise DNA; or   e. comprise in vitro transcribed (IVT) mRNA; or   f. comprise IVT mRNA comprising a poly(A) tail; or   g. comprise IVT mRNA comprising a 5′ Cap,   
       optionally wherein the polynucleotides are:
 1) not associated with any targeting components; or 
 2) are associated with a targeting component capable of targeting the polynucleotides to a cell or an organ; or 
 3) are associated with a targeting component capable of targeting the polynucleotides to a cell or an organ, wherein the targeting component is a vector. 
 
     
     
         52 . The isolated mixture of polypeptides of  claim 43 , wherein the mixture of peptidogenic proteins further comprises the starting protein. 
     
     
         53 . The isolated mixture of polypeptides of  claim 43 , wherein each of the peptidogenic proteins has increased conformational dynamics as compared to the starting protein. 
     
     
         54 . An animal comprising a mixture of at least two different peptidogenic proteins encoded by the isolated polynucleotide or set of polynucleotides of  claim 32 , wherein the starting protein is heterologous to the animal. 
     
     
         55 . The animal of  claim 54 , wherein the animal is a mouse, rabbit, rat, llama, camel, or cow.