US010314901B2

(12) United States Patent ( 10 ) Patent No.: US 10 ,314 , 901 B2

Chen et al. (45) Date of Patent: * Jun. 11, 2019
(54 ) MALARIA ANTIGENS AND METHODS OF OTHER PUBLICATIONS
USE
Agnandji et al., “ First Results of Phase 3 Trial of RTS ,S /AS01
(71) Applicant: GenVec, Inc., Gaithersburg , MD (US) Malaria Vaccine in African Children ,” N . Engl. J. Med ., 365 :
1863- 1875 (2011).
Bergmann -Leitner et al., “ Immunization with Pre -Erythrocytic Anti
(72 ) Inventors: Ping Chen , Potomac , MD (US); gen CelTOS from Plasmodium falciparum Elicits Cross - Species
Duncan McVey, Derwood , MD (US ); Protection against Heterologous Challenge with Plasmodium berghei,”
Douglas E . Brough , Gaithersburg , MD PLoS One, 5 ( 8 ): 1 - 9 (2010 ).
(US ); Joseph Bruder , Gaithersburg , Bowie et al., “ Deciphering the Message in Protein Sequences :
MD (US) Tolerance to Amino Acid Substitutions ,” Science , 247: 1306 - 1310
( 1990 ).
(73 ) Assignee : GenVec , Inc., Gaitherburg, MD (US) Carlton et al., “ Genome sequence and comparative analysis of the
model rodent malaria parasite Plasmodium yoelii yoelii,” Nature ,
419 : 512 -519 (2002 ) .
( * ) Notice : Subject to any disclaimer, the term of this Carlton et al., “ Comparative genomics of the neglected human
patent is extended or adjusted under 35 malaria parasite Plasmodium vivax, ” Nature, 455 ( 7214 ): 757 -763
U . S .C . 154 (b ) by 0 days . (2008 ).
This patent is subject to a terminal dis Carvalho et al., “Malaria vaccine : candidate antigens,mechanisms,
claimer . constraints and prospects,” Scand. J. Immunol., 56 : 327 -343 (2002).
Cesares et al., “ The RTS, S malaria vaccine," Vaccine , 28 : 4880
4894 ( 2010 ).
(21) Appl. No.: 16 /008,852 Clyde et al., “ Immunization of man against sporozite -induced
falciparum malaria,” Am . J. Med . Sci., 266 ( 3 ): 169 - 177 ( 1973 ).
(22) Filed : Jun. 14 , 2018 Crompton et al., " Advances and challenges in malaria vaccine
development,” J. Clin . Invest., 120 : 4168 -4178 (2010 ) .
Dharia et al., “Whole - genome sequencing and microarray analysis
(65 ) Prior Publication Data of ex vivo Plasmodium vivax reveal selective pressure on putative
US 2018 /0344833 A1 Dec . 6 , 2018 drug resistance genes,” Proc. Natl. Acad . Sci. USA, 107 : 20045
20050 ( 2010 ).
Gardner et al., “ Genome sequence of the human malaria parasite
Plasmodium falciparum ,” Nature, 419 : 498 -511 (2002).
Related U .S . Application Data Graves and Gelband , “ Vaccines for preventing malaria ,” Cochrane
(60 ) Continuation of application No. 15/800 ,975 , filed on Database Syst. Rev., 1 : CD000129 ( 2003) .
Nov. 1, 2017 , now Pat. No . 10 ,022 ,432 , which is a Greenspan et el., “ Defining epitopes: It's not as easy as it seems,"
division of application No . 14 /441, 988 , filed as Nature Biotechnology, 17 : 936 - 937 ( 1999 ).
application No. PCT /US2013 / 069620 on Nov. 12 , Greenwood and Alonso, “Malaria vaccine trials,” Chem . Immunol.,
80 : 366 -395 (2002 ).
2013 , now Pat. No. 9, 833 ,502 . Hoffman et al., “ Protection of humans against malaria by immuni
zation with radiation -attenuated Plasmodium falciparum sporozoites,"
J. Infect. Dis., 185 : 1155 - 1164 (2002 ).
(60 ) Provisional application No. 61/ 725,248, filed on Nov. Ivanov et al., “ The adenosine deaminases of Plasmodium vivax and
12 , 2012 . Plasmodium falciparurn exhibit surprising differences in ligand
specificity ,” Journal of Molecular Graphics and Modelling, 35 :
43 -48 ( 2012 ).
( 51 ) Int . Ci. Kester et al.,“ Randomized , double -blind , phase 2a trial of falciparum
A61K 39/00 ( 2006 .01) malaria vaccines RTS , S / ASO1B and RTS, S / ASO2A in malaria -naive
A61K 39/015 ( 2006 .01 ) adults : safety, efficacy, and immunologic associates of protection ,"
(52) U .S. CI. J. Infect. Dis ., 200 : 337 - 346 (2009 ) .
CPC .... A61K 39 /015 (2013. 01 ); A61K 2039 /5256 (Continued )
(2013 .01); A61K 2039 /53 (2013 .01); YO2A
50 /412 (2018 .01) Primary Examiner — Brian Gangle
(58 ) Field of Classification Search (74 ) Attorney , Agent, or Firm — Leydig, Voit & Mayer ,
None Ltd .
See application file for complete search history. (57 ) ABSTRACT
(56 ) References Cited The invention is directed to a composition comprising one or
more polypeptides or one or more nucleic acid sequences
U . S . PATENT DOCUMENTS that can induce a protective immune response against Plas
modium species that infect humans. The invention also is
2008/0248060 Al 10 /2008 Bruder et al. directed to a method of using such compositions to induce
a protective immune response against a Plasmodium parasite
FOREIGN PATENT DOCUMENTS in a mammal.
WO
wo
WO 2007 /027860 A2
WO 2007/041216 A2
3 / 2007
4 /2007 20 Claims,No Drawings
WO WO 2008 /086386 A2 7 /2008 Specification includes a Sequence Listing .
 US 10,Page
314 ,2 901 B2
( 56 ) References Cited Rieckmann et al., “ Letter: Sporozoite induced immunity in man
against an Ethiopian strain of Plasmodium falciparurn ,” Trans R .
Soc. Trop . Med. Hyg., 68 : 258 ( 1974 ).
OTHER PUBLICATIONS Sanni et al.,Methods in Molecular Medicine, 72: Malaria Methods
and Protocols : 57 - 76 (2002 ).
Kester et al., “ A phase I/IIa safety, immunogenicity, and efficacy Schofield et al., “ Gamma interferon , CD8 + T cells and antibodies
bridging randomized study of a two-dose regimen of liquid and required for immunity to malaria sporozoites,” Nature , 330 (6149 ):
664 -666 ( 1987 ) .
lyophilized formulations of the candidate malaria vaccine RTS , S / Schwartz et al., “ A review of malaria vaccine clinical projects based
ASO2A in malaria -naïve adults ," Vaccine, 25 : 5359 - 5366 ( 2007) . on the WHO rainbow table,” Malaria Journal, 11 ( 1) : 11 (2012 ).
Kester et al., “ Phase 2a trial of 0 , 1 , and 3 month and 0 , 7 , and 28 Stoute et al., “ A preliminary evaluation of a recombinant
day immunization schedules of malaria vaccine RTS ,S /AS02 in circumsporozoite protein vaccine against Plasmodium falciparurn
malaria -naïve adults at the Walter Reed Army Institute of Research ," malaria . RTS ,S Malaria Vaccine Evaluation Group ,” N . Engl. J.
Vaccine , 26 : 2191 - 2202 (2008 ). Med ., 336 : 86 -91 ( 1997 ).
Kumar et al., “ The circumsporozoite protein is an immunodominant Tyagi et al., “ Vaccination Strategies against Malaria : novel carri
protective antigen in irradiated sporozoites,” Nature, 444 : 937 er(s ) more than a tour de force,” J. Control Release, 162( 1) : 242-254
(2012 ).
( 2006 ). UNIPROT Accession No. A5KE01 , “ Adenosine deaminase, puta
Mehlin et al., “Heterologous expression of proteins from Plasmo tive .” ( 2007) .
dium falciparum : Results from 1000 genes," Molecular and Bio UNIPROT Accession No. Q8IJA9, “ Adenosine deaminase , puta
chemical Parasitology, 148 ( 2 ) : 144 - 160 (2006 ) . tive .” ( 2003) .
Moore et al., “Malaria vaccines : where are we and where are we Van Braeckel -Budimir et al., " CD8 T -cell -mediated protection against
going ?” Lancet Infect. Dis., 2 : 737 - 743 (2002 ). liver -stage malaria : lessons from a mouse model,” Frontiers in
Moorthy and Hill , " Malaria vaccines,” Br. Med . Bull., 62 : 59 -72 Microbiology, 5 ( 272 ): 1 - 9 (2014 ) .
Vaughan et al., “Malaria vaccine development: persistent chal
( 2002 ) . lenges," Curr Opin ., Immunol., 24 ( 3 ): 324 -331 (2012 ) .
Neafsey et al, “ The malaria parasite Plasmodium vivax exhibits Voza et al., “ Intradermal immunization of mice with radiation
greater genetic diversity than Plasmodium falciparum ,” Nature attenuated sporozoites of Plasmodium yoelii induces effective pro
Genetics, 44 : 1046 - 1050 (2012 ). tective immunity," Malaria Journal, 9 : 362 ( 2010 ) .
Nussenzwig and Nussenzweig , “ Rationale for the development of Weiss et al., “ CD8+ T cells ( cytotoxic / suppressors ) are required for
an engineered sporozoite malaria vaccine,” Adv. Immunol., 45 : protection in mice immunized with malaria sporozoites,” Proc.
283 -334 ( 1989 ). Natl . Acad . Sci. USA , 85 : 573 ( 1988 ).
Nussenzwig et al., " Protective immunity produced by the injection Yadav et al., “ Purification and characterization of plasmodium
of x - irradiated sporozoites of plasmodium berghei," Nature, 216 : yoleii adenosine deaminase ,” Experimental Parasitology, 129 (4 ):
160- 162 ( 1967) . 368 -374 ( 2011) .
Reyes -Sandoval et al., “ Single - dose immunogenicity and protective Zeeshan et al., “ Genetic variation in the Plasmodium falciparum
efficacy of simian adenoviral vectors against Plasmodium berghei,” circumsporozoite protein in India and its relevance to RTS,S malaria
European Journal of Immunology, 38 ( 1 ): 732 - 741 (2008). vaccine ,” PLOS ONE , 7 (8 ): e43430 (2012 ).
Richie and Saul, “ Progress and challenges for malaria vaccines," International Preliminary Report on Patentability, Application No.
Nature, 415 : 694 -701 ( 2002 ). PCT/US2013/069620 , dated May 21, 2015 .
 US 10 ,314 , 901 B2
MALARIA ANTIGENS AND METHODS OF rates. Vaccines are the most cost effective and efficient
USE therapeutic interventions for infectious diseases . In this
regard , vaccination has the advantage of administration prior
CROSS -REFERENCE TO RELATED to military deployment and likely reduction in non - compli
APPLICATIONS 5 ance risks . However, decades of research and development
directed to a malaria vaccine have not proven successful.
This patent application is a continuation of U .S . patent Recent efforts have focused on developing vaccines against
application Ser. No . 15 /800 , 975 , now U . S . Pat. No . 10 ,022 , several specific malaria genes and delivery vector systems
432. which was filed Nov . 1 . 2017 . which is a divisional of including adenovirus, poxvirus, and plasmids . The current
U . S . patent application Ser. No. 14 /441 . 988. filed May 11 . 10 status ofmalaria vaccine development and clinical trials is
2015 , now U . S . Pat. No . 9 . 833 ,502, which was filed as the reviewed in , for example, Vaughan et al., Curr. Opin .,
U .S . national stage of PCT /US2013 /069620 , filed Nov. 12 , Immunol., 24 ( 3 ): 324- 331 ( 2012 ); Schwartz et al., Malaria
2013 , which claims the benefit of U . S . Provisional Patent Journal, 11: 11 (2012 ); Tyagi et al., J. Control Release,
Application No . 61/725 . 248 . filed Nov . 12 . 2012 . all of 162 ( 1 ) : 242 - 254 ( 2012 ); Graves and Gelband , Cochrane
which are incorporated by reference in their entirety herein . 15 Database Syst. Rev., 1 : CD000129 ( 2003 ); Moore et al.,
Lancet Infect. Dis., 2 : 737 - 743 ( 2002 ); Carvalho et al.,
STATEMENT REGARDING FEDERALLY Scand . Immunol., 56 : 327 -343 (2002 ); Moorthy and Hill , Br.
SPONSORED RESEARCH AND Med . Bull ., 62: 59 - 72 (2002 ); Greenwood and Alonso ,
DEVELOPMENT Chem . Immunol., 80 : 366 - 395 (2002 ) ; and Richie and Saul,
20 Nature, 415 : 694 - 701 ( 2002).
This invention was made with Government support under An unprecedented quantity of genomic data has emerged
Grant Number 1R43A01084269 -01 awarded by the from the sequencing and functional genomic analysis of
National Institute of Allergy and Infectious Diseases (NI- many disease - causing organisms, including malaria . Indeed ,
AID ). The Government has certain rights in this invention . it has been determined that the parasite Plasmodium falci
25 parum encodes an estimated 5 , 268 putative proteins (see
INCORPORATION - BY -REFERENCE OF Gardner et al., Nature, 419 : 498 -511 ( 2002 )) . This genetic
MATERIAL SUBMITTED ELECTRONICALLY information can be exploited for the systematic discovery of
novel antigens for vaccine development. In the past, target
Incorporated by reference in its entirety herein is a com - antigens for genetic vaccines have been identified based
puter-readable nucleotide /amino acid sequence listing sub - 30 mainly on their abundance in the pathogen of interest and
mitted concurrently herewith and identified as follows: One their susceptibility to neutralization by antibodies generated
828,251 Byte ASCII ( Text) file named “ 739623 _ ST25. txt,” in infected individuals and animal models . This approach
created on Jun . 14 , 2018 . has failed to yield effective vaccines against many of the
most devastating infectious diseases.
BACKGROUND OF THE INVENTION 35 With regard to malaria , less than 5 % of the Plasmodium
falciparum genome is represented by antigens currently in
Malaria is one of the most devastating parasitic diseases clinical development. However, a number of potential vac
affecting humans . The Centers for Disease Control (CDC ) cine candidates targeted against pre - erythrocytic , erythro
estimate that over three billion people live in areas at risk of cytic and sexual stages of P. falciparum are under various
malaria transmission in 106 countries and territories ( e . g ., 40 stages of clinical development (see , e . g ., Crompton et al., J .
parts of Africa , Asia , the Middle East, Central and South Clin . Invest., 120 : 4168 -4178 (2010 ) ) . The RTS , S vaccine is
America , Hispaniola , and Oceania ). The World Health Orga - the most clinically advance malaria vaccine . RTS, S is a
nization (WHO ) and CDC also estimate that in 2010 , pre -erythrocytic stage vaccine based on the P . falciparum
malaria caused over 200 million clinical episodes 655,000 circumsporozoite protein (CSP), and provides protective
deaths, with the majority of deaths occurring in Africa . 45 efficacy in phase II clinical trials of 30 - 50 % against patho
About 86 % of the malaria deaths in 2010 occurred in gen challenge (see , e .g., Cesares et al., Vaccine , 28 : 4880
children . On average , 1, 500 cases of malaria are reported 4894 ( 2010 ); Stoute et al., N . Engl. J. Med ., 336 : 86 - 91
annually in the United States, and malaria is a major health (1997 ); Kester et al., Vaccine, 26 : 2191 -2202 (2008 ); Kester
concern to U .S . military personnel deployed to tropical et al., J. Infect. Dis., 200 : 337-346 (2009) ; Kester et al .,
regions of the world . For example , in August 2003 , 28 % of 50 Vaccine, 25 : 5359 -5366 (2007 ); and Zeeshan et al., PLOS
the 26th Marine Expeditionary Unit and Joint Task Force ONE , 7 (8 ): e43430 ( 2012 )). Initial results of phase III
briefly deployed to Monrovia , Liberia , were infected with clinical trials show that the RTS, S vaccine provides protec
the malaria parasite Plasmodium falciparum . In addition , tive efficacies of 56 % and 47 % against clinical and severe
one 157 -man Marine Expeditionary Unit sustained a 44 % malaria , respectively, in African children age 5 to 17 months
malaria casualty rate over a 12 -day period while stationed at 55 (see, e. g., Agnandji et al ., N . Engl. J. Med., 365: 1863- 1875
Robert International Airport in Monrovia . In all conflicts ( 2011 )). The protection afforded by this protein -based vac
during the past century conducted in malaria endemic areas, cine , however, is short lived ( 3 - 8 weeks).
malaria has been the leading cause of casualties, exceeding Other recent efforts at developing a malaria vaccine have
enemy-inflicted casualties in its impact on “ person -days” focused on several specific genes and their delivery using
lost from duty . 60 various different vector systems including adenovirus, pox
To combatmalaria during U .S . military operations, pre - virus , and plasmid DNA . It is not apparent, however ,
ventive drugs , insect repellants, and barriers have been used whether these recombinant vaccines are effective against
with some success, but developing drug resistance by the malaria, or if they encode the most potent protective anti
malaria parasite and insecticide resistance by mosquito gens . It is clear that protective antigens do exist for the
vectors has limited the efficacy of these agents . Moreover, 65 malaria pathogen Plasmodium falciparum , as evidenced by
the logistical burden and side effects associated with the use the ability of irradiated sporozoites to induce cellular
of these agents often is associated with high non -compliance immune responses in human subjects and robust sterile
 US 10 ,314 , 901 B2
protection against parasite challenge (see , e.g ., Nussenzweig 31 ), (c ) the amino acid sequence of HISCKFLNIDSKID
and Nussenzweig , Adv. Immunol., 45 : 283-334 ( 1989), and KRSGKVVEENPK (SEQ ID NO : 32 ), or ( d ) an amino acid
Hoffman et al., J. Infect. Dis., 185 : 1155 -1164 (2002)). sequence which comprises at least 20 contiguous amino acid
Thus, there remains a need for compositions containing residues of the sequence LEPKKPMVVETFTEYPPLGR
improved antigens that induce potent protective immunity 5 FAIRDMRQTIAVGIIK (SEQ ID NO : 33 ), and wherein
against challenge with malaria -causing parasites. The inven - each of the one or more isolated polypeptides induces a
tion provides such a composition . This and other advantages protective immune response against Plasmodium falciparum
of the invention will become apparent from the detailed and /or Plasmodium vivax in a mammal.
description provided herein . The invention provides a composition comprising a phar
10 maceutically acceptable carrier and one or more isolated
BRIEF SUMMARY OF THE INVENTION polypeptides, wherein each of the one or more isolated
polypeptides comprises (a ) the amino acid sequence of
The invention provides a composition comprising a phar GKVAIILXGKHMGKRCIITK (SEQ ID NO : 40 ), wherein
maceutically acceptable carrier and one or more isolated X is a threonine ( T ) residue or a serine (S ) residue , (b ) the
polypeptides, wherein each of the one or more isolated 15 amino acid sequence of GKHMGKRCIITKXLXSGLLAV
polypeptides comprises (a ) the amino acid sequence of VGPYE (SEQ ID NO : 41), wherein X is an isoleucine (1)
KKERYXIWXRXPKXELHCHLD (SEQ ID NO : 1), residue, a valine (V ) residue, an asparagine (N ) residue, or
wherein X is a lysine (K ) residue, a glutamic acid ( E ) a threonine ( T ) residue, (c ) the amino acid sequence of
residue, an arginine (R ) residue , an isoleucine (I) residue , a SGLLAVVGPYEXNGVPLKRV (SEQ ID NO : 42),
leucine (L ) residue , a cysteine (C ) residue, or a valine ( V ) 20 wherein X is a valine (V ) residue or an isoleucine (I) residue ,
residue , (b ) the amino acid sequence of KYKEGVVLME - and wherein each of the one or more isolated polypeptides
FRYSP (SEQ ID NO : 2 ), (c ) an amino acid sequence induces a protective immune response against Plasmodium
comprising at least 20 contiguous amino acid residues of the falciparum and / or Plasmodium vivax in a mammal.
sequence EDLAKXAVXXKYKEGVVLMEFRYSP (SEQ The invention provides a composition comprising a phar
ID NO : 3 ), wherein X is a histidine ( H ) residue , a tryptophan 25 maceutically acceptable carrier and one or more isolated
( W ) residue, a phenylalanine ( F ) residue , an isoleucine (1 ) polypeptides , wherein each of the one or more isolated
residue, an asparagine (N ) residue , or a glutamic acid (E ) polypeptides comprises (a ) an amino acid sequence com
residue , or ( d ) an amino acid sequence comprising at least 20 prising at least 20 contiguous amino acid residues of the
contiguous amino acid residues of the sequence KSMDTH - sequence LEKKMQLKKEGKLLSAKAKEEKKK (SEQ ID
PIRXLYDAGVKVSVNSDDPGMFL (SEQ ID NO : 4 ), 30 NO : 55 ), (b ) an amino acid sequence comprising at least 21
wherein X is a glutamine ( Q ) residue, a methionine (M ) contiguous amino acid residues of the sequence IVCIL
residue , or a lysine ( K ) residue , and wherein each of the one GHVDTGKTKLLDKLRHTNVQDNEAGGITQQI
or more isolated polypeptides induces a protective immune GATFFPKD (SEQ ID NO : 56 ), (c ) an amino acid sequence
response against Plasmodium falciparum and /or Plasmo comprising at least 21 contiguous amino acid residues of the
dium vivax in a mammal. 35 sequence SKGIMIIDTPGHESFYNLRKRGSSLCDIAIL
The invention provides a composition comprising a phar- VIDLMHGLEQQTKESIQILKQRNCPFVIA
maceutically acceptable carrier and one or more isolated LNKIDRLYMW (SEQ ID NO : 57), ( d ) an amino acid
polypeptides, wherein each of the one or more isolated sequence comprising at least 20 contiguous amino acid
polypeptides comprises (a ) an amino acid sequence com residues of the sequence KLECTVLEVKNIEGLGTTID
prising at least 20 contiguous amino acid residues of the 40 VILTNG (SEQ ID NO : 58 ), ( e ) the amino acid sequence of
sequence MKKDREPIDEDEMRITSTGRMTNYVNY GVGLYVMASTLGSLEALLIFL (SEQ ID NO : 59 ), and (f)
GAKILG (SEQ ID NO : 20 ), (b ) an amino acid sequence an amino acid sequence comprising at least 20 contiguous
comprising at least 20 contiguous amino acid residues of the amino acid residues of the sequence MTDVSDVFNHVD
sequence KIKATGNAIGKAVTLAEIIKRRFKGLHQIT KXGVGLYVMASTLGSLEALLIFL (SEQ ID NO : 60 ),
(SEQ ID NO : 21), or (c ) an amino acid sequence comprising 45 wherein X is a serine ( S ) residue or a threonine ( T ) residue ,
SEQ ID NO : 22 , and wherein each of the one or more and wherein each of the one or more isolated polypeptides
isolated polypeptides induces a protective immune response induces a protective immune response against Plasmodium
against Plasmodium falciparum and/ or Plasmodium vivax in falciparum and /or Plasmodium vivax in a mammal.
a mammal. The invention provides a composition comprising a phar
The invention provides a composition comprising a phar- 50 maceutically acceptable carrier and one or more isolated
maceutically acceptable carrier and one or more isolated polypeptides, wherein each of the one or more isolated
polypeptides, wherein each of the one or more isolated polypeptides comprises (a ) the amino acid sequence of
polypeptides comprises (a ) an amino acid sequence which NLFFAKQVIPNACATQAILSI (SEQ ID NO : 69),or (b ) the
comprises at least 40 contiguous amino acid residues of the amino acid sequence of NFDSXMKGLTLSNCXFLRNIHN
sequence MGKEKTHINLVVIGHVDSGKSTTTGHII - 55 (SEQ ID NO : 70 ), wherein X is a serine ( S ) residue , a
YKLGGIDRRTIEKFEKESAEMGKGSFKYA WVLDKL threonine ( T ) residue , or an asparagine ( N ) residue, and
KAERERGITIDIALWKFETPRYFFTVIDAPGHKDFIKN wherein each of the one or more isolated polypeptides
MITGTSQADVALLV induces a protective immune response against Plasmodium
VPAEVGGFEGAFSKEGOTKEHALLAFTLGVKQIV . falciparum and /or Plasmodium vivax in a mammal.
VGVNKMDTVKYSEDRYEEIKKE V (SEQ ID NO : 30 ), 60 The invention provides a composition comprising a phar
( b ) an amino acid sequence which comprises at least 55 maceutically acceptable carrier and one or more isolated
contiguous amino acid residues of the sequence DYLKK - polypeptides, wherein each of the one or more isolated
VGYQADKVDFIPISGFEGDNLIEKSDKTPWYKGRTLI- polypeptides comprises (a ) the amino acid sequence of
EALDTMEPPKRPYDK PLRIPLOGVYKIGGIGT SGWKFEEQEGDVNMVLTKNVD (SEQ ID NO : 80 ), (b )
VPVGRVETGILKAGMVLNFAPSAVVSECKSVEMHKE 65 an amino acid sequence comprising at least 20 contiguous
VLEE ARPGDNIGFNVKNVSVKEIKRGYVASDT amino acid residues of the sequence IIDFQLVSPFQAEGE
KNEPAKGCSKFTAQVIILNHPGEIKNGY ( SEQ ID NO : NEAQAEMTDFSVTVEKPN (SEQ ID NO : 81), (c ) an
 US 10 ,314 , 901 B2
amino acid sequence comprising at least 20 contiguous 211, or SEQ ID NO : 212 , and wherein each of the one or
amino acid residues of the sequence GGITFYCT- more isolated polypeptides induces a protective immune
TLQNDEKFRYMIGNVKYYKNEEGKNSVS (SEQ ID response against Plasmodium falciparum and /or Plasmo
NO : 82), and (d ) an amino acid sequence comprising at least dium vivax in a mammal.
21 contiguous amino acid residues of the sequence YNGPE - 5
FEDLDDSLQTSLDEWLANLGVDSELCDFIDSCSID DETAILED DESCRIPTION OF THE
KEQREYM (SEQ ID NO : 83), and wherein each of the one INVENTION
or more isolated polypeptides induces a protective immune
response against Plasmodium falciparum and / or Plasmo The invention is predicated , at least in part , on the
dium vivax in a mammal. 10 identification of antigenic polypeptides from Plasmodium
The invention provides a composition comprising a phar- parasites that provide protective immunity in mammals
maceutically acceptable carrier and one or more isolated against challenge with malaria -causing parasites. In this
polypeptides , wherein each of the one or more isolated respect, the invention provides a composition comprising a
polypeptides comprises the amino acid sequence ofSEQ ID pharmaceutically acceptable carrier and one ormore isolated
NO : 90 , SEQ ID NO : 91, SEQ ID NO : 92, SEQ ID NO : 93 , 15 polypeptides or nucleic acid sequences, each of which
or SEQ ID NO : 202, and wherein each of the one or more comprises or encodes, respectively , a Plasmodium amino
isolated polypeptides induces a protective immune response acid sequence , wherein the Plasmodium amino acid
against Plasmodium falciparum and/ or Plasmodium vivax in sequence induces a protective immune response against
a mammal. Plasmodium falciparum and /or Plasmodium vivax in a mam
The invention provides a composition comprising a phar - 20 mal . The one or more polypeptides are “ isolated ” in that they
maceutically acceptable carrier and one or more isolated are removed from their natural environment (i.e ., a Plasmo
polypeptides, wherein each of the one or more isolated dium parasite ).
polypeptides comprises the amino acid sequence ofSEQ ID Each of the one or more Plasmodium amino acid
NO : 98 , SEQ ID NO : 99 , or SEQ ID NO : 100 , and wherein sequences is a Plasmodium antigen . An " antigen ” is a
each of the one or more isolated polypeptides induces a 25 molecule that triggers an immune response in a mammal. An
protective immune response against Plasmodium falciparum “ immune response " can entail, for example , antibody pro
and/ or Plasmodium vivax in a mammal. duction and /or the activation of immune effector cells . An
The invention provides a composition comprising a phar - antigen in the context of the invention can comprise any
maceutically acceptable carrier and one or more isolated subunit , fragment, or epitope of any proteinaceous or non
polypeptides, wherein each of the one or more isolated 30 proteinaceous ( e.g ., carbohydrate or lipid ) molecule which
polypeptides comprises (a ) the amino acid sequence of provokes an immune response in a mammal. By " epitope"
LLKHGWCEMLKGGVIMDVKX (SEQ ID NO : 104), is meant a sequence of an antigen that is recognized by an
wherein X is an asparagine ( N ) residue or a serine (S ) antibody or an antigen receptor. Epitopes also are referred to
residue , (b ) an amino acid sequence comprising at least 20 in the art as “ antigenic determinants .”
contiguous amino acid residues of the sequence VEQAKI- 35 A Plasmodium antigen in the context of the invention can
AEXAGAIGVMVLENIPSELR (SEQ ID NO : 105 ), comprise any proteinaceous Plasmodium molecule or por
wherein X is a lysine (K ) residue or a glutamic acid (E ) tion thereof that provokes a Plasmodium -related immune
residue, (c ) an amino acid sequence comprising at least 20 response in a mammal. A “ Plasmodium molecule ” is a
contiguous amino acid residues of the sequence SINVLAK - molecule that is a part of a Plasmodium parasite , is encoded
VRIGHFVEAQILEELK (SEQ ID NO : 106 ), (d ) an amino 40 by a nucleic acid sequence of a Plasmodium parasite, or is
acid sequence comprising at least 27 contiguous amino acid derived from or synthetically based upon any such molecule .
residues of the sequence KHKFKTPFVCGCTNLGEALR - Administration of a Plasmodium antigen that provokes an
RXSEGASMIRTKGEAGTGNII (SEQ ID NO : 107), immune response in accordance with the invention prefer
wherein X is an isoleucine (I) residue or a methionine (M ) ably leads to protective immunity against Plasmodium . In
residue , ( e ) an amino acid sequence comprising at least 20 45 this regard , an “ immune response " to Plasmodium is an
contiguous amino acid residues of the sequence immune response to any one or more Plasmodium antigens .
ATPADAAMCMQLGMDGVFVGSGIFESENP (SEQ ID The one or more Plasmodium amino acid sequences can
NO : 108 ), or (f) an amino acid sequence comprising at least be obtained or derived from any Plasmodium species. Pref
20 contiguous amino acid residues of the sequence LPV - erably, the one or more Plasmodium amino acid sequences
VNFAAGGXATPADAAMCMQLGMDGVFVGS- 50 are derived or obtained from a Plasmodium species that
GIFESENP (SEQ ID NO : 109 ), wherein X is an isoleucine infects humans and causes malaria . Human - infecting Plas
( I) residue or a valine ( V ) residue, and wherein each of the modium species include P . malariae , P . ovale, P. knowlesi, P .
one or more isolated polypeptides induces a protective vivax , and P . falciparum . P. vivax and P . falciparum are the
immune response against Plasmodium falciparum and/or most common species, while P. falciparum is the most
Plasmodium vivax in a mammal . 55 deadly species of Plasmodium in human .
The invention provides composition comprising a phar - Alternatively, each of the one or more Plasmodium amino
maceutically acceptable carrier and one or more isolated acid sequences can be an ortholog of an amino acid sequence
nucleic acid sequences, wherein each of the one or more from a human -infecting Plasmodium species . “ Orthologs"
isolated nucleic acid sequences encodes a polypeptide com - or " orthologous genes ” are nucleic acid or amino acid
prising the amino acid sequence of any one of SEQ ID NO : 60 sequences that evolved from a common ancestral gene by
1 -SEO ID NO : 16 , SEQ ID NO : 20 -SEQ ID NO : 26 , SEQ speciation , and typically retain the same function in the
ID NO : 30 -SEQ ID NO : 36 , SEQ ID NO : 40 -SEQ ID NO : course of evolution . In other words, when a species diverges
51, SEQ ID NO : 55 - SEQ ID NO : 65, SEQ ID NO : 69 -SEQ into two separate species, the copies of a single gene in the
ID NO : 76 , SEQ ID NO : 80 -SEQ ID NO : 86 , SEQ ID NO : two resulting species are said to be " orthologous.” Plasmo
90 -SEQ ID NO : 93 , SEQ ID NO : 98 -SEQ ID NO : 100 , SEQ 65 dium species that infect non - human animals and contain
ID NO : 104 -SEQ ID NO : 118 , SEQ ID NO : 202, SEQ ID orthologous genes include, for example , rodent-infecting
NO : 205 , SEQ ID NO : 207 -SEQ ID NO : 209, SEQ ID NO : species such as P. vinckei, P. chabaudi, P . yoelii , and P .
 US 10 ,314 , 901 B2
berghei; and non -human primate - infecting species such as P . or a glutamic acid (E ) residue. In this respect , the isolated
knowlesi, P . cynomolgi, P. simiovale , P. fieldi, P. inui, P. polypeptide can comprise, for example, an amino acid
brasilianum , P. billbrayi, P . billcollinsi, P . bouillize , P. sequence comprising at least 20 contiguous amino acid
brasilianum , P . bucki, P. cercopitheci, P. coatneyi, P . cou - residues of the sequence EDLAKWAVIEKYKEGVVLME
langesi, P . eylesi, P. fieldi, P. foleyi, P . fragile , P . inui, P . 5 FRYSP (SEO ID NO : 8 ). EDLAKHAVFNKYKEGVV
gaboni, P. georgesi , P. girardi, and P . gonderi. In order to LMEFRYSP (SEO ID NO : 9 ). or EDLAKHAVENKYKEG
advance vaccine discovery , the genomes of a number of VVLMEFRYSP (SEQ ID NO : 10 ). In particular ,the isolated
Plasmodium species have been sequenced . For example , the polypeptide can comprise the amino acid sequence of
complete P . falciparum genome has been sequenced and is EDLAKWAVIEKYKEGVVLMEFRYSP (SEO ID NO : 8 ),
disclosed in Gardner et al., Nature, 419 : 498 -511 (2002). 10 EDLAKHAVFNKYKEGVVLMEFRYSP (SEQ ID NO : 9 ),
The complete P. vivax genome has been sequenced and is
disclosed in Neafsey et al., Nature Genetics, 44: 1046 - 1050 or EDLAKHAVFNKYKEGVVLMEFRYSP (SEQ ID NO :
( 2012); Carlton et al., Nature , 455 : 757 -763 (2008 ); and tide 10 ). When the composition comprises an isolated polypep
Dharia et al., Proc. Natl. Acad . Sci. USA , 107: 20045 - 20050 comprising at least 20 contiguous amino acid residues
( 2010 ). In addition , the complete P. voelii genome sequence 15 ( e . g ., 20 , 21, 22 , 23 , 24, 25 , 26 , 27 , 28 , or 29 contiguous
is disclosed in Carlton et al., Nature, 419 : 512 - 9 (2002 ). amino acid residues ) of SEQ ID NO : 4 , the isolated poly
Mouse models ofmalaria infection , in which P. voelii is the peptide can comprise , for example , at least 20 contiguous
infecting parasite , have been generated (see , e .g ., Wiersch et amino acid residues of the sequence KSMDTHPIRK
al., Methods in Molecular Medicine, 72 : Malaria Methods LYDAGVKVSVNSDDPGMFL (SEQ ID NO : 11 ) , KSM
and Protocols : 57 - 76 (2002 ); and Voza et al., Malaria 20 DTHPIRMLYDAGVKVSVNSDDPGMFL (SEQ ID NO :
Journal, 9 : 362 ( 2010 )), and have been established as 12 ), or KSMDTHPIRQLYDAGVKVSVNSDDPGMFL
reliable models for pre - erythrocytic stage P . falciparum (SEQ ID NO : 13 ). In particular, the isolated polypeptide can
vaccine development (see , e. g., Nussenzweig et al., Nature, comprise the amino acid sequence of KSMDTHPIRK
216 : 160 (1967 ) ; Clyde et al., Am . J. Med . Sci., 266 : 169 LYDAGVKVSVNSDDPGMFL (SEQ ID NO : 11 ) , KSM
( 1973 ); Roeckmann et al., Trans R . Soc . Trop . Med . Hyg ., 25 DTHPIRMLYDAGVKVSVNSDDPGMFL (SEQ ID NO :
68 : 258 (1974 ); Hoffman et al., J. Inf. Dis., 185 : 1155 12 ), or KSMDTHPIRQLYDAGVKVSVNSDDPGMFL
( 2002 ); Schofield et al ., Nature , 330 : 64 ( 1987 ); Weiss et al., (SEO ID NO : 13 ).
Proc . Natl. Acad . Sci. USA , 85 : 573 ( 1988 ); Kumar et al., The composition can comprise one, two, three , or all four,
Nature, 444 : 937 (2006 ); Stoute et al., N . Eng. J. Med ., 336 : of the aforementioned polypeptides alone or in any combi
86 ( 1997 ) ) . 30 nation . In this respect, the composition can comprise any
In one embodiment, the composition comprises a phar- combination of any two of the aforementioned sequences,
maceutically acceptable carrier and one or more isolated any combination of any three of the aforementioned
polypeptides, wherein each of the one or more isolated sequences, or all four of the aforementioned sequences. In a
polypeptides comprises : (a ) the amino acid sequence of preferred embodiment, the composition comprises an iso
KKERYXIWXRXPKXELHCHLD (SEQ ID NO : 1 ), 35 lated polypeptide comprising the amino acid sequence of
wherein X is a lysine (K ) residue, a glutamic acid ( E ) SEQ ID NO : 14 , SEQ ID NO : 15 , SEQ ID NO : 16 , or SEQ
residue , an arginine (R ) residue, an isoleucine (I) residue , a ID NO : 209.
leucine (L ) residue, a cysteine (C ) residue, or a valine (V ) In another embodiment, the composition comprises a
residue, (b ) the amino acid sequence of KYKEGVVLME pharmaceutically acceptable carrier and one or more isolated
FRYSP (SEQ ID NO : 2 ) , (c ) an amino acid sequence 40 polypeptides, wherein each of the one or more isolated
comprising at least 20 contiguous amino acid residues of the polypeptides comprises ( a ) an amino acid sequence com
sequence EDLAKXAVXXKYKEGVVLMEFRYSP (SEQ prising at least 20 contiguous amino acid residues ( e. g ., 20 ,
ID NO : 3 ), wherein X is a histidine (H ) residue, a tryptophan 21, 22 , 23 , 24 , 25 , 26 , 27, 28 , 29, 30 , 31, 32 , or 33
( W ) residue , a phenylalanine (F ) residue , an isoleucine (1) contiguous amino acid residues ) of the sequence MKK
residue , an asparagine (N ) residue, or a glutamic acid (E ) 45 DREPIDEDEMRITSTGRMTNYVNYGAKILG (SEQ ID
residue , or (d ) an amino acid sequence comprising at least 20 NO : 20 ), (b ) an amino acid sequence comprising at least 20
contiguous amino acid residues of the sequence KSMDTH - contiguous amino acid residues ( e . g ., 20 , 21 , 22 , 23 , 24 , 25 ,
PIRXLYDAGVKVSVNSDDPGMFL (SEQ ID NO : 4 ), 26 , 27 , 28 , 29, or 30 contiguous amino acid residues ) of the
wherein X is a glutamine ( Q ) residue, a methionine (M ) sequence KIKATGNAIGKAVTLAEIIKRRFKGLHQIT
residue, or a lysine ( K ) residue. 50 (SEQ ID NO : 21), or (c ) the amino acid sequence of SEQ ID
When the composition comprises an isolated polypeptide NO : 22.
comprising the amino acid sequence of SEQ ID NO : 1, X When the composition comprises an isolated polypeptide
can be any suitable amino acid residue, but preferably is a comprising an amino acid sequence comprising at least 20
lysine (K ) residue, a glutamic acid ( E ) residue , an arginine contiguous amino acid residues of SEQ ID NO : 20 , the
(R ) residue, an isoleucine (1) residue, a leucine ( L ) residue , 55 isolated polypeptide can comprise, for example , the amino
a cysteine (C ) residue, or a valine ( V ) residue . In this respect, acid sequence of SEQ ID NO : 20 . When the composition
the isolated polypeptide can comprise , for example , the comprises an isolated polypeptide comprising an amino acid
amino acid sequence of KKERYEIWRRIPKVELHCHLD sequence comprising at least 20 contiguous amino acid
(SEQ ID NO : 5 ) , KKERYKIWKRLPKCELHCHLD (SEQ residues of SEQ ID NO : 21 , the isolated polypeptide can
ID NO : 6 ), or KKERYKIWKRIPKCELHCHLD (SEQ ID 60 comprise , for example, the amino acid sequence of SEQ ID
NO : 7). When the composition comprises an amino acid NO : 21 . When the composition comprises an isolated poly
sequence comprising at least 20 contiguous amino acid peptide comprising the amino acid sequence of SEQ ID NO :
residues (e .g., 20 , 21, 22 , 23 , 24 , or 25 contiguous amino 22 , the isolated polypeptide can comprise , for example , the
acid residues ) of the sequence SEQ ID NO : 3 , X can be any amino acid sequence of KDAGYQPPLDEKYVKEMX
suitable amino acid residue , but preferably is a histidine ( H ) 65 PEEIVN (SEQ ID NO : 23), wherein X can be any suitable
residue, a tryptophan ( W ) residue, a phenylalanine ( F ) amino acid residue, but is preferably a serine (S ) residue or
residue, an isoleucine (I) residue, an asparagine (N ) residue, a threonine ( T ) residue (i.e., KDAGYQPPLDEKYVKEM
 US 10 ,314 ,901 B2
10
SPEEIVN (SEQ ID NO : 211) or KDAGYQP- VGPYE (SEQ ID NO : 41), wherein X is an isoleucine (I)
PLDEKYVKEMTPEEIVN ( SEQ ID NO : 212 )). residue, a valine (V ) residue, an asparagine (N ) residue, or
The composition can comprise one , two, or all three of the a threonine ( T ) residue, (c ) the amino acid sequence of
aforementioned polypeptides alone or in any combination . SGLLAVVGPYEXNGVPLKRV (SEQ ID NO : 42 ),
In this respect, the composition can comprise any combina - 5 wherein X is a valine (V ) residue or an isoleucine (I) residue .
tion of any two of the aforementioned sequences , or all three When the composition comprises an isolated polypeptide
of the aforementioned sequences. In a preferred embodi comprising the amino acid sequence of GKVAIILXG
ment, the composition comprises an isolated polypeptide KHMGKRCIITK (SEQ ID NO : 40), X can be any suitable
comprising the amino acid sequence of SEQ ID NO : 24 , amino acid residue , but preferably is a threonine ( T ) residue
SEQ ID NO : 25 , or SEQ ID NO : 26 . 10 or a serine (S ) residue. In this respect, the isolated polypep
In another embodiment, the composition comprises a tide can comprise , for example , the amino acid sequence of
pharmaceutically acceptable carrier and one or more isolated GKVAIILTGKHMGKRCIITK (SEQ ID NO : 43 ) or
polypeptides , wherein each of the one or more isolated GKVAIILSGKHMGKRCIITK (SEQ ID NO : 44 ). When the
polypeptides comprises ( a ) an amino acid sequence which composition comprises an isolated polypeptide comprising
comprises at least 40 contiguous amino acid residues of the 15 the amino acid sequence of GKHMGKRCIITKXLXSGL
sequence MGKEKTHINLVVIGHVDSGKSTTTGHII- LAVVGPYE (SEQ ID NO : 41) , X can be any suitable amino
YKLGGIDRRTIEKFEKESAEMGKGSFKYA WVLDKL - acid residue, but preferably is an isoleucine (I) residue , a
KAERERGITIDIALWKFETPRYFFTVIDAPGHKDFIKN valine (V ) residue, an asparagine (N ) residue , or a threonine
MITGTSQADVALLV ( T ) residue. In this respect, the isolated polypeptide can
VPAEVGGFEGAFSKEGQTKEHALLAFTLGVKQIV - 20 comprise, for example , the amino acid sequence of
VGVNKMDTVKYSEDRYEEIKKE V (SEQ ID NO : 30 ) GKHMGKRCIITKILNSGLLAVVGPYE ( SEQ ID NO : 45 )
(b ) an amino acid sequence which comprises at least 55 or GKHMGKRCIITKVLTSGLLAVVGPYE (SEQ ID NO :
contiguous amino acid residues of the sequence DYLKK 46 ). When the composition comprises an isolated polypep
VGYQADKVDFIPISGFEGDNLIEKSDKTPWYKGRTLI tide comprising the amino acid sequence of SGLLAVVG
EALDTMEPPKRPYDK PLRIPLQGVYKIGGIGT- 25 PYEXNGVPLKRV (SEQ ID NO : 42 ), X can be any suitable
VPVGRVETGILKAGMVLNFAPSAVVSECKSVEMHKE amino acid residue , but preferably is a valine ( V ) residue or
VLEE ARPGDNIGFNVKNVSVKEIKRGYVASDT- an isoleucine ( 1) residue. In this respect, the isolated poly
KNEPAKGCSKFTAQVIILNHPGEIKNGY (SEQ ID NO : peptide can comprise , for example , the amino acid sequence
31), (c ) the amino acid sequence of HISCKFLNIDSKID of SGLLAVVGPYEVNGVPLKRV (SEQ ID NO : 47 ) or
KRSGKVVEENPK (SEQ ID NO : 32 ), or ( d ) an amino acid 30 SGLLAVVGPYEINGVPLKRV (SEQ ID NO : 48 ).
sequence which comprises at least 20 contiguous amino acid The composition can comprise one , two , or all three of the
residues of the sequence LEPKKPMVVETFTEYPPLGR - aforementioned polypeptides alone or in any combination .
FAIRDMRQTIAVGIIK (SEQ ID NO : 33 ). In this respect , the composition can comprise any combina
When the composition comprises an isolated polypeptide tion of any two of the aforementioned sequences, or all three
comprising at least 40 contiguous amino acid residues ( e . g ., 35 of the aforementioned sequences . In a preferred embodi
40 , 45 , 50 , 55 , 60 , 65, 70 , 75, 80 , 85 , 90 , 95 , 100 , 105 , 110 , ment, the composition comprises an isolated polypeptide
115 , 120 , 125 , 130 , 135 , 140 , 145, 150 155, 160, 165 , 170 , comprising the amino acid sequence of SEQ ID NO : 49,
or 172 contiguous amino acid residues ) of SEQ ID NO : 30 , SEQ ID NO : 50, or SEQ ID NO : 51 .
the isolated polypeptide can comprise , for example , SEQ ID In another embodiment, the composition comprises a
NO : 30 . When the composition comprises an isolated poly - 40 pharmaceutically acceptable carrier and one ormore isolated
peptide comprising at least 55 contiguous amino acid resi- polypeptides, wherein each of the one or more isolated
dues ( e. g ., 55 , 60 , 65 , 70 , 75 , 80 , 85 , 90 , 95 , 100 , 105 , 110 , polypeptides comprises (a ) an amino acid sequence com
115 , 120 , 125 , 130 , 135 , 140 , 145 , 150 155 , 160 , 165 , or 170 prising at least 20 contiguous amino acid residues of the
contiguous amino acid residues ) of SEQ ID NO : 31, the sequence LEKKMQLKKEGKLLSAKAKEEKKK (SEQ ID
isolated polypeptide can comprise , for example, SEQ ID 45 NO : 55 ), (b ) an amino acid sequence comprising at least 21
NO : 31. When the composition comprises an isolated poly - contiguous amino acid residues of the sequence IVCIL
peptide comprising at least 20 contiguous amino acid resi- GHVDTGKTKLLDKLRHTNVODNEAGGITQQI
dues (e.g., 20 , 21, 22 , 23, 24 , 25, 26 , 27 , 28 , 29, 30 , 31, 32 GATFFPKD (SEQ ID NO : 56 ), (c ) an amino acid sequence
or 33 contiguous amino acid residues) of SEQ ID NO : 33 , comprising at least 21 contiguous amino acid residues of the
the isolated polypeptide can comprise , for example, SEQ ID 50 sequence the amino acid sequence of SKGIMIIDTPGHES
NO : 33 . FYNLRKRGSSLCDIAILVIDLMHGLEQQTKESIQ
The composition can comprise one , two, three or all four ILKQRNCPF VIA LNKIDRLYMW (SEQ ID NO : 57 ), (d )
of the aforementioned polypeptides alone or in any combi- an amino acid sequence comprising at least 20 contiguous
nation . In this respect, the composition can comprise any amino acid residues of the sequence KLECTV
combination of any two of the aforementioned sequences , 55 LEVKNIEGLGTTIDVILTNG (SEQ ID NO : 58 ), (e ) the
any combination of any three of the aforementioned amino acid sequence of GVGLYVMASTLGSLEALLIFL
sequences, or all four of the aforementioned sequences. In a (SEQ ID NO : 59 ), and ( f) an amino acid sequence compris
preferred embodiment, the composition comprises an iso - ing at least 20 contiguous amino acid residues of the
lated polypeptide comprising the amino acid sequence of sequence MTDVSDVFNHVDKXGVGLYVMAS
SEQ ID NO : 34 , SEQ ID NO : 35 , or SEQ ID NO : 36 . 60 TLGSLEALLIFL (SEQ ID NO : 60 ), wherein X is a serine
In another embodiment , the composition comprises a (S ) residue or a threonine ( T ) residue.
pharmaceutically acceptable carrier and one ormore isolated When the composition comprises an isolated polypeptide
polypeptides, wherein each of the one or more isolated comprising at least 20 contiguous amino acid residues (e .g .,
polypeptides comprises (a ) the amino acid sequence of 20 , 21, 22, 23 , or 24 contiguous amino acid residues ) ofSEQ
GKVAIILXGKHMGKRCIITK (SEQ ID NO : 40 ), wherein 65 ID NO : 55 , the isolated polypeptide can comprise , for
X is a threonine ( T ) residue or a serine ( S ) residue , ( b ) the example , SEQ ID NO : 55 . When the composition comprises
amino acid sequence of GKHMGKRCIITKXLXSGLLAV - an isolated polypeptide comprising an amino acid sequence
 US 10 ,314 , 901 B2
11 12
comprising at least 21 contiguous amino acid residues ( 22 , SGWKFEEQEGDVNMVLTKNVD (SEQ ID NO : 80 ), (b )
23 , 24 25 , 26 , 27 , 28 , 29 , 30 , 31, 32 , 33 , 34 , 35 , 36 , 37, 38 , an amino acid sequence comprising at least 20 contiguous
39 , 40 , 41, 42, 43 , or 44 contiguous amino acid residues) of amino acid residues of the sequence IIDFQLVSPFQAEGE
SEQ ID NO : 56 , the isolated polypeptide can comprise, for NEAQAEMTDFSVTVEKPN (SEQ ID NO : 81), (c ) an
example , SEQ ID NO : 56 . When the composition comprises 5 amino acid sequence comprising at least 20 contiguous
an isolated polypeptide comprising at least 21 contiguous amino acid residues of the sequence GGITFYCT
amino acid residues (e . g ., 21, 25, 30 , 35 , 40 , 45 , 50 , 55, 60 , TLONDEKFRYMIGNVKYYKNEEGKNSVS (SEQ ID
65, or 70 contiguous amino acid residues) of SEQ ID NO : NO : 82), and (d ) an amino acid sequence comprising at least
57 , the isolated polypeptide can comprise , for example , SEQ 21 contiguous amino acid residues of the sequence YNGPE
ID NO : 57. When the composition comprises an isolated 10 FEDLDDSLQTSLDEWLANLGVDSELCDFIDSCSID
polypeptide comprising at least 20 contiguous amino acid KEQREYM (SEQ ID NO : 83 ).
residues ( e.g ., 20 , 21 , 22, 23 , 24 , 25 , or 26 contiguous amino When the composition comprises an isolated polypeptide
acid sequences ) of SEQ ID NO : 58, the isolated polypeptide comprising at least 20 contiguous amino acid residues ( e . g .,
can comprise, for example , SEQ ID NO : 58 . When the 20 , 21 , 22, 23 , 24 25 , 26 , 27, 28 , 29 , 30 , 31 , 32, or 33
composition comprises an isolated polypeptide comprising 15 contiguous amino acid residues ) of SEQ ID NO : 81 , the
at least 20 contiguous amino acid residues (e. g., 20 , 21 , 22 , isolated polypeptide can comprise, for example, SEQ ID
23 , 24 25 , 26 , 27 , 28 , 29, 30 , 31 , 32, 33 , 34, or 35 contiguous NO : 81. When the composition comprises an isolated poly
amino acid residues ) of SEQ ID NO : 60 , X can be any peptide comprising at least 20 contiguous amino acid resi
suitable amino acid residue, but preferably is a serine (S ) dues ( e.g ., 20 , 21, 22, 23, 24 25 , 26 , 27 , 28 , 29 , 30 , 31, 32 ,
residue or a threonine ( T ) residue . In this respect, the 20 33, 34 , 35 or 36 contiguous amino acid residues) of SEQ ID
isolated polypeptide can comprise , for example , an isolated NO : 82 , the isolated polypeptide can comprise , for example ,
polypeptide comprising at least 20 contiguous amino acid SEQ ID NO : 82 . When the composition comprises an
residues of the sequence MTDVSDVFNHVDKSGVGLYV - isolated polypeptide comprising at least 21 contiguous
MASTLGSLEALLIFL (SEQ ID NO : 61) or MTDVSDVF - amino acid residues of SEQ ID NO : 83 , ( e .g ., 21, 22 , 23 , 24
NHVDKTGVGLYVMASTLGSLEALLIFL (SEQ ID NO : 25 25 , 26 , 27 , 28 , 29 , 30 , 31, 32 , 33 , 34 , 35 , 36 , 37, 38 , 39 , 40 ,
62 ). In particular, the isolated polypeptide can comprise , for 41, 42, 43 , 44, 45 , 46 , or 47 contiguous amino acid residues ),
example , the amino acid sequence of MTDVSDVFN - the isolated polypeptide can comprise, for example , SEQ ID
HVDKSGVGLYVMASTLGSLEALLIFL (SEQ ID NO : 61) NO : 83 .
or MTDVSDVFNHVDKTGVGLYVMASTLGSLEAL - The composition can comprise one, two , or all three ofthe
LIFL (SEQ ID NO : 62 ). 30 aforementioned polypeptides alone or in any combination .
The composition can comprise one, two , three , four, five , In this respect, the composition can comprise any combina
or all six of the aforementioned polypeptides alone or in any tion of any two of the aforementioned sequences , or all three
combination . In this respect , the composition can comprise of the aforementioned sequences . In a preferred embodi
any combination of any two of the aforementioned ment, the composition comprises an isolated polypeptide
sequences , any combination of any three of the aforemen - 35 comprising the amino acid sequence of SEQ ID NO : 84 ,
tioned sequences, any combination of any four of the SEQ ID NO : 85 , or SEQ ID NO : 86 .
aforementioned sequences, any combination of any five of In another embodiment, the composition comprises a
the aforementioned sequences , or all six of the aforemen - pharmaceutically acceptable carrier and one or more isolated
tioned sequences . In a preferred embodiment, the composi- polypeptides , each of which comprises the amino acid
tion comprises an isolated polypeptide comprising the amino 40 sequence of SEQ ID NO : 90 , SEQ ID NO : 91, SEQ ID NO :
acid sequence of SEQ ID NO : 63, SEQ ID NO : 64 , or SEQ 92, SEQ ID NO : 93 , or SEQ ID NO : 202. The invention also
ID NO : 65 . provides a composition comprising a pharmaceutically
In another embodiment, the composition comprises a acceptable carrier and one or more isolated polypeptides ,
pharmaceutically acceptable carrier and one ormore isolated each of which comprises the amino acid sequence of SEQ ID
polypeptides, wherein each of the one or more isolated 45 NO : 98 , SEQ ID NO : 99 , or SEQ ID NO : 100 .
polypeptides comprises (a ) the amino acid sequence of In another embodiment, the composition comprises a
NLFFAKQVIPNACATQAILSI (SEQ ID NO : 69), and (b ) pharmaceutically acceptable carrier and one or more isolated
the amino acid sequence of NFDSXMKGLTLSNCXFL - polypeptides, wherein each of the one or more isolated
RNIHN (SEQ ID NO : 70 ) , wherein X is a serine (S ) residue , polypeptides comprises (a ) the amino acid sequence of
a threonine ( T ) residue, or an asparagine ( N ) residue . 50 LLKHGWCEMLKGGVIMDVKX (SEO ID NO : 104 ) ,
When the composition comprises the amino acid wherein X is an asparagine (N ) residue or a serine ( S )
sequence of SEQ ID NO : 70, X can be any suitable amino residue, (b ) an amino acid sequence comprising at least 20
acid residue , but preferably is a serine ( S ) residue , a threo contiguous amino acid residues of the sequence VEQAKI
nine ( T ) residue , or an asparagine (N ) residue . In this AEXAGAIGVMVLENIPSELR (SEQ ID NO : 105 ),
respect, the isolated polypeptide can comprise, for example , 55 wherein X is a lysine (K ) residue or a glutamic acid ( E )
the amino acid sequence of NFDSTMKGLTLSNCNFL - residue, (c) an amino acid sequence comprising at least 20
RNIHN (SEQ ID NO : 71), NFDSSMKGLTLSNCTFL - contiguous amino acid residues of the sequence SINVLAK
RNIHN (SEQ ID NO : 72 ), or NFDSSMKGLTLSNCNFL VRIGHFVEAQILEELK (SEQ ID NO : 106 ), (d ) an amino
RNIHN (SEQ ID NO : 73 ). The composition can comprise acid sequence comprising at least 27 contiguous amino acid
one or both of the aforementioned polypeptides . In a pre - 60 residues of the sequence KHKFKTPFVCGCTNLGEALR
ferred embodiment, the composition comprises an isolated RXSEGASMIRTKGEAGTGNII (SEQ ID NO : 107 ),
polypeptide comprising the amino acid sequence of SEQ ID wherein X is an isoleucine (I) residue or a methionine (M )
NO : 74, SEQ ID NO : 75 , or SEQ ID NO : 76 . residue , (e ) an amino acid sequence comprising at least 20
In another embodiment, the composition comprises a contiguous amino acid residues of the sequence
pharmaceutically acceptable carrier and one ormore isolated 65 ATPADAAMCMQLGMDGVFVGSGIFESENP (SEQ ID
polypeptides, wherein each of the one or more isolated NO : 108 ), or (f ) an amino acid sequence comprising at least
polypeptides comprises (a ) the amino acid sequence of 20 contiguous amino acid residues of the sequence LPV
 US 10 ,314 , 901 B2
13 14
VNFAAGGXATPADAAMCMQLGMDGVFVGS The composition can comprise one, two, three , four, five ,
GIFESENP (SEQ ID NO : 109 ), wherein X is an isoleucine or all six of the aforementioned polypeptides alone or in any
(I) residue or a valine (V ) residue . combination . In this respect, the composition can comprise
When the composition comprises an isolated polypeptide any combination of any two of the aforementioned
comprising the amino acid sequence of LLKHGWCEMLK - 5 sequences, any three of the aforementioned sequences , any
GGVIMDVKX (SEQ ID NO : 104 ), X can be any suitable four of the aforementioned sequences , any five of the
amino acid residue, but preferably is an asparagine (N ) aforementioned sequences, or all six of the aforementioned
residue or a serine (S ) residue . In this respect, the isolated sequences . In a preferred embodiment, the composition
polypeptide can comprise, for example , the amino acidID 10 comprises an isolated polypeptide comprising the amino
sequence of LLKHGWCEMLKGGVIMDVKN (SEQ ID acid sequence of SEQ ID NO : 116 , SEQ ID NO : 117 , SEQ
NO : 110 ), or LLKHGWCEMLKGGVIMDVKS (SEQ ID ID NO : 118 , or SEQ ID NO : 205 .
NO : 111). When the composition comprises an isolated The polypeptides of the inventive composition can be
prepared by any method , such as by synthesizing the poly
polypeptide comprising at least 20 contiguous amino acid peptide or by expressing a nucleic acid encoding an appro
residues ( e.g ., 20 , 21 , 22, 23 , 24 , 25 , or 26 contiguous amino
amino 15 priate amino acid sequence in a cell and harvesting the
acid residues) of SEQ ID NO : 105 , X can be any suitable peptide from the cell or media containing the cell . A
amino acid residue, but preferably is a lysine (K ) residue or combination of such methods also can be used . Methods of
a glutamic acid (E ) residue . In this respect, the isolated de novo synthesizing polypeptides and methods of recom
polypeptide can comprise , for example , at least 20 contigu - binantly producing polypeptides are known in the art (see ,
ous amino acid residues of the sequence VEQAKIAEKA - 20 e.g ., Chan et al ., Fmoc Solid Phase Peptide Synthesis,
GAIGVMVLENIPSELR (SEQ ID NO : 112 ), or VEQAKI- Oxford University Press, Oxford , United Kingdom (2005) ;
AEEAGAIGVMVLENIPSELR (SEQ ID NO : 113 ). In Reid , R . (ed .), Peptide and Protein Drug Analysis, Marcel
particular, the isolated polypeptide can comprise , for Dekker, Inc. (2000 ); Westwood et al. ( ed .), Epitope Map
example , the amino acid sequence of VEQAKIAEKA - ping , Oxford University Press , Oxford , United Kingdom
GAIGVMVLENIPSELR (SEQ ID NO : 112 ), or VEQAKI- 25 (2000 ); Sambrook et al., Molecular Cloning : A Laboratory
AEEAGAIGVMVLENIPSELR (SEQ ID NO : 113 ). When Manual, 3rd ed ., Cold Spring Harbor Press, Cold Spring
the composition comprises an isolated polypeptide compris- Harbor, N .Y . (2001 ); and Ausubel et al., Current Protocols
ing at least 20 contiguous amino acid residues (e.g., 20 , 21, in Molecular Biology , Greene Publishing Associates and
22, or 23 contiguous amino acid residues ) of SEQ ID NO : John Wiley & Sons, New York ( 1994 )).
106 , the isolated polypeptide can comprise, for example, 30 In another embodiment of the invention , the composition
SEQ ID NO : 106 . When the composition comprises an can comprise a pharmaceutically acceptable carrier and one
isolated polypeptide comprising at least 27 contiguous or more isolated nucleic acid sequences, each of which
amino acid residues ( e. g., 27 , 28 , 29 , 30 , 31 , 32 , 33 , 34 , 35 , encodes any of the aforementioned isolated polypeptides. In
36 , 37 , 38 , 39 , 40 , or 41 contiguous amino acid residues ) of this respect, the composition can comprise one or more
SEQ ID NO : 107, X can be any suitable amino acid residue , 35 isolated nucleic acid sequences , wherein each of the one or
but preferably is an isoleucine (I) residue or a methionine more nucleic acid sequences encodes a polypeptide com
(M ) residue. In this respect, the isolated polypeptide can prising the amino acid sequence of any one of SEQ ID NO :
comprise, for example , at least 27 contiguous amino acid 1 -SEQ ID NO : 16 , SEQ ID NO : 20 -SEQ ID NO : 26 , SEQ
residues of KHKFKTPFVCGCTNLGEALRRISEGAS - ID NO : 30 - SEO ID NO : 36 , SEO ID NO : 40 - SEO ID NO :
MIRTKGEAGTGNII (SEQ ID NO : 207) , or KHKFKTP - 40 51 , SEQ ID NO : 55 - SEQ ID NO : 65 , SEQ ID NO : 69 -SEQ
FVCGCTNLGEALRRMSEGASMIRTKGEAGTGNII ID NO : 76 , SEQ ID NO : 80 -SEQ ID NO : 86 , SEQ ID NO :
(SEQ ID NO : 208). In particular, the isolated polypeptide 90 -SEQ ID NO : 93 , SEQ ID NO : 98 -SEQ ID NO : 100 , SEQ
can comprise , for example , the amino acid sequence of ID NO : 104 -SEQ ID NO : 118 , SEQ ID NO : 202 , SEQ ID
KHKFKTPFVCGCTNLGEALRRISEGASMIRTK NO : 205 , SEQ ID NO : 207 -SEQ ID NO : 209, SEQ ID NO :
GEAGTGNII (SEQ ID NO : 207 ), or KHKFKTP - 45 211 , or SEQ ID NO : 212 .
FVCGCTNLGEALRRMSEGASMIRTKGEAGTGNII In one embodiment, the one or more isolated nucleic acid
(SEQ ID NO : 208 ). When the composition comprises an sequences comprise codons expressed more frequently (and
isolated polypeptide comprising at least 20 contiguous preferably , most frequently ) in humans than in Plasmodium .
amino acid residues ( e.g., 20 , 21, 22 , 23, 24 , 25 , 26 , 27 , 28 , While the genetic code is generally universal across species ,
or 29 contiguous amino acid residues ) of SEQ ID NO : 108, 50 the choice among synonymous codons is often species
the isolated polypeptide can comprise , for example SEQ ID dependent. One of ordinary skill in the art would appreciate
NO : 108 . When the composition comprises an isolated that, to achieve maximum protection against Plasmodium
polypeptide comprising least 20 contiguous amino acid infection , high levels of Plasmodium antigens must be
residues ( e.g., 20 , 21, 22 , 23 , 24 , 25 , 26 , 27 , 28 , 29 , 30 , 31, expressed in a mammalian ,preferably a human , host. In this
32, 33 , 34 , 35 , 36 , 37 , 38 , 39, or 40 contiguous amino acid 55 respect, the nucleic acid sequence preferably encodes the
residues ) of SEQ ID NO : 109, X can be any suitable amino native amino acid sequence of a Plasmodium antigen , but
acid residue , but preferably is an isoleucine (1) residue or a comprises codons that are expressed more frequently in
valine ( V ) residue . In this respect, the isolated polypeptide mammals ( e . g ., humans ) than in Plasmodium . Changing
can comprise , for example , at least 20 contiguous amino native Plasmodium codons to the most frequently used in
acid residues of LPVVNFAAGGIATPADAAMCMQLGM - 60 mammals will increase expression of the Plasmodium anti
DGVFVGSGIFESENP (SEQ ID NO : 114 ), or LPVVN gen in a mammal (e .g., a human ). Such modified nucleic
FAAGGVATPADAAMCMQLGMDGVFVGSGIFESENP acid sequences are commonly described in the art as
(SEQ ID NO : 115 ). In particular , the isolated polypeptide " humanized ," as " codon -optimized ,” or as utilizing “mam
can comprise, for example , LPVVNFAAGGIATPADAAM - malian -preferred " or " human -preferred ” codons
CMQLGMDGVFVGSGIFESENP (SEQ ID NO : 114 ), or 65 In the context of the invention, a Plasmodium nucleic acid
LPVVNFAAGGVATPADAAMCMQLGMDGVFVGS sequence is said to be " codon - optimized ” if at least about
GIFESENP (SEQ ID NO : 115 ). 60 % ( e .g ., at least about 70 % , at least about 80 % , or at least
 US 10 ,314 , 901 B2
15 16.
about 90 % ) of the wild - type codons in the nucleic acid sequences that are non -native with respect to the adenoviral
sequence are encoded by mammalian -preferred codons . genome. Typically , an adenoviral vector is generated by
That is , a Plasmodium nucleic acid sequence is codon - introducing one or more mutations ( e . g ., a deletion , inser
optimized if at least about 60 % of the codons in the nucleic tion , or substitution ) into the adenoviral genome of the
acid sequence are mammalian -preferred codons. 5 adenovirus so as to accommodate the insertion of a non
Alternatively , the one or more isolated nucleic acid native nucleic acid sequence, for example , for gene transfer,
sequences can utilize particular codons at frequencies that into the adenovirus .
best approximate codon usage frequencies of native Plas - Non -human adenovirus (e .g., ape , simian , avian , canine ,
modium species . Methods and algorithms have been devel- ovine , or bovine adenoviruses ) can be used to generate the
oped in order to facilitate and adjust codon usage in this 10 adenoviral vector ( i. e ., as a source of the adenoviral genome
manner when expressing genes in heterologous systems, and for the adenoviral vector ). For example , the adenoviral
these methods are referred to as " codon harmonization ” ( see , vector can be based on a simian adenovirus, including both
e .g., U . S . Patent Application Publication 2004/0209323; new world and old world monkeys ( see , e .g ., Virus Tax
Angov et al., PLoS ONE, 3 (5 ): e2189 ( 2008 ); and Angov et onomy: VIIIth Report of the International Committee on
al., Methods Mol. Biol., 705 : 1 - 13 (2011 )). Adjusting codons 15 Taxonomy of Viruses (2005 )). A phylogeny analysis of
of the isolated nucleic acid sequence so as to mimic the adenoviruses that infect primates is disclosed in , e . g ., Roy et
codon usage of a native Plasmodium gene may improve al., PLoS Pathog ., 5 (7 ) : e100050 . doi: 10 .1371/ journal.p
gene and /or protein expression by, for example , improving pat. 1000503 ( 2009). For instance , a simian adenovirus can
protein folding or protein solubility. Such modified nucleic be of genotype 1, 3 , 6 , 7, 11, 16 , 18 , 19, 20 , 27 , 33, 38 , 39 ,
acid sequences are commonly described in the art as “ har- 20 48, 49 , or 50 , or any other simian adenoviral genotype . Other
monized ” or “ codon -harmonized .” non -human adenoviruses which can be used in the invention
Additionally and alternatively, the codon - optimized or include non -human primate adenoviruses that are geneti
codon -harmonized nucleic acid sequence encoding a Plas- cally and / or phenotypically similar to or distinct from group
modium antigen can be any sequence that hybridizes to an C human adenoviruses .
above - described sequence under at least moderate , prefer - 25 A gorilla adenovirus can be used as the source of the
ably high , stringency conditions , such as described in , e . g ., adenoviral genome for the adenoviral vector. There are four
Sambrook et al., Molecular Cloning, a Laboratory Manual, widely recognized gorilla subspecies within the two species
3rd edition , Cold Spring Harbor Press , Cold Spring Harbor, of Eastern Gorilla (Gorilla beringei ) and Western Gorilla
N . Y . ( 2001). Determining the degree of homology can be (Gorilla gorilla ). The Western Gorilla species includes the
accomplished using any suitable method known in the art, 30 subspecies Western Lowland Gorilla (Gorilla gorilla
such as those described herein . gorilla ) and Cross River Gorilla (Gorilla gorilla diehli ). The
In a preferred embodiment, the composition can comprise Eastern Gorilla species includes the subspecies Mountain
any one , or combination of, the following nucleic acid Gorilla (Gorilla beringei beringei) and Eastern Lowland
sequences : SEQ ID NO : 17 , SEQ ID NO : 18 , SEQ ID NO : Gorilla (Gorilla beringei graueri) ( see , e . g ., Wilson and
19. SEO ID NO : 27 , SEO ID NO : 28 , SEQ ID NO : 29 , SEQ 35 Reeder, eds., Mammalian Species of the World , 3rd ed .,
ID NO : 37 , SEQ ID NO : 38, SEQ ID NO : 39 , SEQ ID NO : Johns Hopkins University Press , Baltimore, Md. ( 2005 )).
52 , SEO ID NO : 53, SEO ID NO : 54 , SEQ ID NO : 66 , SEQ The adenoviral vector can be based on an adenovirus
ID NO : 67, SEQ ID NO : 68 , SEQ ID NO : 77 , SEQ ID NO : isolated from any of the aforementioned subspecies . Pref
78 , SEQ ID NO : 79 , SEQ ID NO : 87 , SEQ ID NO : 88, SEQ erably, the adenoviral vector is based on an adenovirus
ID NO : 89, SEO ID NO : 94 , SEO ID NO : 95 , SEQ ID NO : 40 isolated from Mountain Gorilla (Gorilla beringei beringei)
96 , SEQ ID NO : 97, SEQ ID NO : 101, SEQ ID NO : 102, or Eastern Lowland Gorilla (Gorilla beringei graueri).
SEO ID NO : 103 , SEQ ID NO : 119 , SEQ ID NO : 120 , SEQ Gorilla adenoviruses and adenoviral vectors are described
ID NO : 121, SEQ ID NO : 203, SEQ ID NO : 204, SEQ ID in , e .g ., International Patent Application Nos. PCT/US2012/
NO : 206 , or SEO ID NO : 210 . 058956 , PCT/US2012 /058978, and PCT/US2012 / 059006 .
In one embodiment of the invention , the one or more 45 A human adenovirus can be used as the source of the
isolated nucleic acid sequences which encode the Plasmo- adenoviral genome for the adenoviral vector. For instance ,
dium antigens are present in a vector. Any vector can be an adenovirus can be of subgroup A (e . g ., serotypes 12 , 18 ,
employed in the context of the invention , including viral and and 31 ), subgroup B ( e .g ., serotypes 3 , 7 , 11 , 14 , 16 , 21 , 34 ,
non -viral vectors . Examples of suitable viral vectors include , 35 , and 50 ), subgroup C ( e. g ., serotypes 1 , 2 , 5 , and 6 ),
but are not limited to , retroviral vectors, adeno - associated 50 subgroup D (e. g ., serotypes 8 , 9 , 10 , 13 , 15 , 17 , 19 , 20 ,
virus vectors , poxviral vectors (e . g ., vaccinia virus vectors ), 22 - 30 , 32 , 33 , 36 - 39 , and 42 -48 ), subgroup E ( e . g., serotype
herpesvirus vectors, parainfluenza -RSV chimeric vectors 4 ), subgroup F ( e .g ., serotypes 40 and 41 ) , an unclassified
( PIV -RSV ) , adenoviral vectors , poliovirus, alphavirus, serogroup ( e. g ., serotypes 49 and 51), or any other adeno
baculovirus, and Sindbis virus . Examples of suitable non viral serogroup or serotype. Adenoviral serotypes 1 through
viral vectors include , but are not limited to , plasmids (e . g ., 55 51 (i. e ., Adl through Ad51) are available from the American
DNA plasmids ), yeast (e.g., Saccharomyces ), liposomes , Type Culture Collection (ATCC , Manassas, Va .). Non - group
nanoparticles, and molecular conjugates (e.g., transferrin ). Cadenoviral vectors , methods of producing non -group C
When the vector is a plasmid ( e . g ., DNA plasmid ), the adenoviral vectors , and methods of using non - group C
plasmid can be administered with adjuvants , such as CPG or a denoviral vectors are disclosed in , for example , U . S . Pat.
polymeric adjuvants. The vector also can be a virus - like 60 Nos . 5 , 801, 030 , 5 ,837,511, and 5 , 849,561, and International
particle (VLP ) (see , e.g., Petry et al., Curr. Opin . Molecular Patent Application Publications WO 1997 /012986 and WO
Therapeutics, 5 (5 ) : 524 -528 ( 2003 ); and U . S . Pat. No . 1998 /053087 .
5 ,298 , 244 ). The adenoviral vector can comprise a combination of
In a preferred embodiment, the vector is an adenoviral subtypes and thereby be a “ chimeric ” adenoviral vector. A
vector. The term “ adenoviral vector," as used herein , refers 65 chimeric adenoviral vector can comprise an adenoviral
to an adenovirus in which the adenoviral genome has been genome that is derived from two or more ( e .g ., 2 , 3 , 4 , etc .)
manipulated to accommodate one or more nucleic acid different adenovirus serotypes . In the context of the inven
 US 10 ,314 , 901 B2
17 18
tion , a chimeric adenoviral vector can comprise approxi Whether the adenoviral vector is replication -competent or
mately different or equal amounts of the genome of each of replication -deficient, the adenoviral vector retains at least a
the two or more different adenovirus serotypes . For portion of the adenoviral genome. The adenoviral vector can
example, when the chimeric adenoviral vector genome is comprise any portion of the adenoviral genome, including
comprised of the genomes of two different adenovirus 5 protein coding and non -protein coding regions. Desirably ,
serotypes, the chimeric adenoviral vector genome preferably the adenoviral vector comprises at least one nucleic acid
comprises no more than about 99 . 9 % ( e . g ., no more than sequence that encodes an adenovirus protein . The adenoviral
about 99 % , no more than about 98 % , no more than about vector can comprise a nucleic acid sequence that encodes
95 % , no more than about 85 % , no more than about 80 % , no any suitable adenovirus protein , such as, for example , a
more than about 75 % , no more than about 60 % , no more 10 protein encoded by any one of the early region genes ( i. e .,
than about65 % , or no more than about 50 % ) of the genome E1A , E1B , E2A , E2B , E3 , and /or E4 regions ), or a protein
of one of the adenovirus serotypes , with the remainder of the encoded by any one of the late region genes , which encode
chimeric adenovirus genomebeing derived from the genome the virus structural proteins (i. e ., L1, L2 , L3 , L4, and L5
of the other adenovirus serotype. regions ).
The adenoviral vector can be replication -competent, con - 15 Preferably , the adenoviral vector is replication deficient,
ditionally replication -competent, or replication - deficient. such that the replication -deficient adenoviral vector requires
A replication -competent adenoviral vector can replicate in complementation of at least one replication -essential gene
typical host cells , i. e ., cells typically capable of being function of one or more regions of the adenoviral genome
infected by an adenovirus . A replication -competent adeno for propagation (e .g ., to form adenoviral vector particles ).
viral vector can have one or more mutations as compared to 20 The replication -deficient adenoviral vector can be modi
a wild -type adenovirus ( e . g ., one or more deletions, inser - fied in any suitable manner to cause the deficiencies in the
tions, and/or substitutions ) in the adenoviral genomethat do one or more replication -essential gene functions in one or
not inhibit viral replication in host cells . For example , the more regions of the adenoviral genome for propagation . The
adenoviral vector can have a partial or entire deletion of the complementation of the deficiencies in the one or more
adenoviral early region known as the E3 region , which is not 25 replication -essential gene functions of one or more regions
essential for propagation of the adenoviral vector genome. of the adenoviral genome refers to the use of exogenous
Aconditionally -replicating adenoviral vector is an adeno - means to provide the deficient replication - essential gene
viral vector that has been engineered to replicate under functions . Such complementation can be effected in any
pre - determined conditions . For example , replication - essen - suitable manner, for example, by using complementing cells
tial gene functions, e . g ., gene functions encoded by the 30 and / or exogenous DNA ( e . g ., helper adenovirus) encoding
adenoviral early regions, can be operably linked to an the disrupted replication -essential gene functions.
inducible , repressible , or tissue -specific transcription control The adenoviral vector can be deficient in one or more
sequence , e .g ., a promoter. In such an embodiment, repli- replication -essential gene functions of only the early regions
cation requires the presence or absence of specific factors (i.e ., E1- E4 regions ) of the adenoviral genome, only the late
that interact with the transcription control sequence. Con - 35 regions (i. e ., L1-L5 regions of the adenoviral genome, both
ditionally -replicating adenoviral vectors are further the early and late regions of the adenoviral genome, or all
described in U . S . Pat. No. 5 ,998 , 205 . adenoviral genes (i. e., a high capacity adenovector (HC
A replication- deficient adenoviral vector is an adenoviral Ad)). See Morsy et al., Proc. Natl. Acad. Sci. USA , 95 :
vector that requires complementation of one or more gene 965 - 976 ( 1998 ) ; Chen et al., Proc. Natl. Acad. Sci. USA , 94 :
functions or regions of the adenoviral genome that are 40 1645- 1650 ( 1997 ); and Kochanek et al., Hum . Gene Ther.,
required for replication , as a result of, for example , a 10: 2451- 2459 ( 1999 ). Examples of replication -deficient
deficiency in one or more replication - essential gene function adenoviral vectors are disclosed in U . S . Pat. Nos . 5 ,837 ,511;
or regions, such that the adenoviral vector does not replicate 5 ,851 ,806 ; 5 , 994 , 106 ; 6 , 127, 175 ; 6 ,482,616 ; and 7 , 195 ,896 ,
in typical host cells , especially those in a human to be and International Patent Application Publications WO 1994 /
infected by the adenoviral vector. 45 028152 , WO 1995 /002697 , WO 1995 /016772 , WO 1995 /
A deficiency in a gene function or genomic region , as used 034671, WO 1996 /022378 , WO 1997 /012986 , WO 1997 /
herein , is defined as a disruption (e.g ., deletion ) of sufficient 021826 , and WO 2003 / 022311 .
genetic material of the adenoviral genome to obliterate or The early regions of the adenoviral genome include the
impair the function of the gene ( e .g ., such that the function E1, E2, E3, and E4 regions. The El region comprises the
of the gene product is reduced by at least about 2 - fold , 50 EIA and E1B subregions, and one or more deficiencies in
5 - fold , 10 - fold , 20 - fold , 30 - fold , or 50 - fold ) whose nucleic replication -essential gene functions in the El region can
acid sequence was disrupted (e . g ., deleted ) in whole or in include one or more deficiencies in replication -essential
part. Deletion of an entire gene region often is not required gene functions in either or both of the E1A and E1B
for disruption of a replication -essential gene function. How subregions , thereby requiring complementation of the E1A
ever , for the purpose of providing sufficient space in the 55 subregion and/or the E1B subregion of the adenoviral
adenoviral genome for one or more transgenes, removal of genome for the adenoviral vector to propagate (e . g ., to form
a majority of one or more gene regions may be desirable . adenoviral vector particles ). The E2 region comprises the
While deletion of genetic material is preferred , mutation of E2A and E2B subregions, and one or more deficiencies in
genetic material by addition or substitution also is appro - replication -essential gene functions in the E2 region can
priate for disrupting gene function . Replication - essential 60 include one or more deficiencies in replication -essential
gene functions are those gene functions that are required for gene functions in either or both of the E2A and E2B
adenovirus replication (e.g ., propagation ) and are encoded subregions, thereby requiring complementation of the E2A
by , for example, the adenoviral early regions (e .g ., the E1, subregion and /or the E2B subregion of the adenoviral
E2, and E4 regions), late regions (e .g ., the L1, L2, L3 , L4, genome for the adenoviral vector to propagate (e .g ., to form
and L5 regions), genes involved in viral packaging (e .g ., the 65 adenoviral vector particles ).
IVa2 gene), and virus -associated RNAs (e .g., VA -RNA -1 The E3 region does not include any replication -essential
and/or VA -RNA - 2 ). gene functions, such that a deletion of the E3 region in part
 US 10 ,314 , 901 B2
19 20
or in whole does not require complementation of any gene ficient adenoviral vector) for propagation (e . g ., to form
functions in the E3 region for the adenoviral vector to adenoviral vector particles ). The adenoviral vector can be
propagate (e . g ., to form adenoviral vector particles ). In the deficient in at least one replication - essential gene function
context of the invention , the E3 region is defined as the (desirably all replication -essential gene functions ) of the E1
region that initiates with the open reading frame that 5 region of the adenoviral genome and at least one gene
encodes a protein with high homology to the 12 .5K protein function of the nonessential E3 region of the adenoviral
from the E3 region of human adenovirus 5 (NCBIreference genome (denoted an E1/E3 - deficient adenoviral vector ). The
sequence AP _ 000218 ) and ends with the open reading frame adenoviral vector can be deficient in at least one replication
that encodes a protein with high homology to the 14 .7K essential gene function (desirably all replication -essential
protein from the E3 region of human adenovirus 5 (NCBI 10 gene functions ) of the E4 region of the adenoviral genome
reference sequence AP _ 000224 . 1 ) . The E3 region may be and at least one gene function of the nonessential E3 region
deleted in whole or in part, or retained in whole or in part of the adenoviral genome ( denoted an E3 /E4 -deficient
The size of the deletion may be tailored so as to retain an adenoviral vector ).
adenoviral vector whose genome closely matches the opti- In one embodiment, the adenoviral vector is replication
mum genome packaging size . A larger deletion will accom - 15 deficient and requires , at most, complementation of the E2
modate the insertion of larger heterologous nucleic acid region, preferably the E2A subregion , of the adenoviral
sequences in the adenoviral genome. In one embodiment of genome, for propagation ( e. g ., to form adenoviral vector
the invention , the L4 polyadenylation signal sequences, particles ). Thus , the replication deficient adenoviral vector
which reside in the E3 region , are retained . requires complementation of at least one replication - essen
The E4 region comprises multiple open reading frames 20 tial gene function of the E2A subregion of the adenoviral
(ORFs ). An adenoviral vector with a deletion of all of the genome (denoted an E2A -deficient adenoviral vector) for
open reading frames of the E4 region except ORF6 , and in propagation ( e . g ., to form adenoviral vector particles ). The
some cases ORF3 , does not require complementation of any adenoviral vector can be deficient in at least one replication
gene functions in the E4 region for the adenoviral vector to essential gene function ( desirably all replication -essential
propagate ( e . g ., to form adenoviral vector particles ). Con - 25 gene functions ) of the E2 A region of the adenoviral genome
versely , an adenoviral vector with a disruption or deletion of and at least one gene function of the nonessential E3 region
ORF6 , and in some cases ORF3, of the E4 region ( e . g ., with of the adenoviral genome (denoted an E2A / E3 -deficient
a deficiency in a replication - essential gene function based in adenoviral vector) .
ORF6 and / or ORF3 of the E4 region ), with or without a In one embodiment , the adenoviral vector is replication
disruption or deletion of any of the other open reading 30 deficient and requires , atmost, complementation of the El
frames of the E4 region or the native E4 promoter , poly - and E4 regions of the adenoviral genome for propagation
adenylation sequence , and / or the right- side inverted terminal ( e . g ., to form adenoviral vector particles ). Thus, the repli
repeat ( ITR ), requires complementation of the E4 region cation - deficient adenoviral vector requires complementation
(specifically , ofORF6 and/or ORF3 of the E4 region ) for the of at least one replication -essential gene function of both the
adenoviral vector to propagate ( e . g ., to form adenoviral 35 El and E4 regions of the adenoviral genome (denoted an
vector particles ). The late regions of the adenoviral genome E1/E4-deficient adenoviral vector ) for propagation (e. g., to
include the L1, L2, L3 , L4 , and L5 regions. The adenoviral form adenoviral vector particles ). The adenoviral vector can
vector also can have a mutation in the major late promoter be deficient in at least one replication - essential gene func
(MLP ), as discussed in International Patent Application tion (desirably all replication - essential gene functions ) of the
Publication WO 2000 /000628 , which can render the adeno - 40 El region of the adenoviral genome, at least one replication
viral vector replication - deficient if desired . essential gene function of the E4 region of the adenoviral
The one or more regions of the adenoviral genome that genome, and at least one gene function of the nonessential
contain one or more deficiencies in replication - essential E3 region of the adenoviral genome ( denoted an E1 /E3 /E4
gene functions desirably are one or more early regions of the deficient adenoviral vector ). The adenoviral vector prefer
adenoviral genome, i.e ., the E1, E2, and / or E4 regions, 45 ably requires , atmost , complementation of the El region of
optionally with the deletion in part or in whole of the E3 the adenoviral genome for propagation , and does not require
region . complementation of any other deficiency of the adenoviral
The replication deficient adenoviral vector also can have genome for propagation . More preferably, the adenoviral
one or more mutations as compared to a wild -type adeno - vector requires, at most, complementation of the El and E4
virus ( e . g ., one or more deletions, insertions, and / or substi - 50 regions of the adenoviral genome for propagation , and does
tutions ) in the adenoviral genome that do not inhibit viral not require complementation of any other deficiency of the
replication in host cells. Thus, in addition to one or more adenoviral genome for propagation .
deficiencies in replication - essential gene functions, the The adenoviral vector, when deficient in multiple repli
adenoviral vector can be deficient in other respects that are cation -essential gene functions of the adenoviral genome
not replication -essential. For example , the adenoviral vector 55 ( e . g ., an E1/E4 -deficient adenoviral vector ), can include a
can have a partial or entire deletion of the adenoviral early spacer sequence to provide viral growth in a complementing
region known as the E3 region , which , as discussed herein , cell line similar to that achieved by adenoviruses or adeno
is not essential for propagation of the adenoviral genome. viral vectors deficient in a single replication - essential gene
In one embodiment, the adenoviral vector is replication - function (e. g., an El - deficient adenoviral vector). The spacer
deficient and requires , at most, complementation of the El 60 sequence can contain any nucleotide sequence or sequences
region or the E4 region of the adenoviral genome, for which are of a desired length , such as sequences at least
propagation ( e. g., to form adenoviral vector particles ). Thus , about 15 base pairs ( e. g., between about 15 nucleotides and
the replication - deficient adenoviral vector requires comple about 12 ,000 nucleotides ), preferably about 100 nucleotides
mentation of at least one replication - essential gene function to about 10 ,000 nucleotides , more preferably about 500
of the E1 A subregion and/or the E1B region of the adeno - 65 nucleotides to about 8 ,000 nucleotides, even more prefer
viral genome (denoted an El- deficient adenoviral vector ) or ably about 1 ,500 nucleotides to about 6 ,000 nucleotides, and
the E4 region of the adenoviral genome (denoted an E4 - de most preferably about 2 ,000 to about 3 ,000 nucleotides in
 US 10 ,314 , 901 B2
21 22
length , or a range defined by any two of the foregoing terminators , internal ribosome entry sites ( IRES ), and the
values . The spacer sequence can be coding or non -coding like , that provide for the expression of the nucleic acid
and native or non- native with respect to the adenoviral sequence in a host cell . Exemplary expression control
genome, but does not restore the replication - essential func sequences are known in the art and are described in , for
tion to the deficient region . The spacer also can contain an 5 example , Goeddel, Gene Expression Technology : Methods
expression cassette .More preferably , the spacer comprises a in Enzymology , Vol. 185, Academic Press, San Diego , Calif.
polyadenylation sequence and/ or a gene that is non - native (1990 ) . Ideally , the Plasmodium antigen - encoding nucleic
with respect to the adenoviral vector. The use of a spacer in acid sequence is operably linked to a promoter and a
an adenoviral vector is further described in , for example , polyadenylation sequence . The promoter desirably is a con
U .S . Pat. No. 5 ,851,806 and International Patent Application 10 stitutive or inducible promoter , preferably a constitutive
Publication WO 1997 /021826 . promoter. A large number of promoters, including constitu
By removing all or part of the adenoviral genome, for tive , inducible , and repressible promoters, from a variety of
example , the E1, E3 , and E4 regions of the adenoviral different sources are well known in the art. Representative
genome, the resulting adenoviral vector is able to accept sources of promoters include for example, virus, mammal,
inserts of exogenous nucleic acid sequences while retaining 15 insect, plant, yeast, and bacteria , and suitable promoters
the ability to be packaged into adenoviral capsids. An from these sources are readily available , or can be made
exogenous nucleic acid sequence can be inserted at any synthetically , based on sequences publicly available , for
position in the adenoviral genome so long as insertion in the example , from depositories such as the ATCC as well as
position allows for the formation of the adenoviral vector other commercial or individual sources . Promoters can be
particle . The exogenous nucleic acid sequence ( i.e ., a 20 unidirectional (i. e ., initiate transcription in one direction ) or
nucleic acid sequence encoding a Plasmodium antigen ) bi-directional (i.e., initiate transcription in either a 3 ' or 5 '
preferably is positioned in the El region , the E3 region , or direction ). Non - limiting examples of promoters include, for
the E4 region of the adenoviral genome. example , the T7 bacterial expression system , pBAD (araA )
The replication -deficient adenoviral vector of the inven bacterial expression system , the cytomegalovirus (CMV)
tion can be produced in complementing cell lines that 25 promoter (human ormouse ), the ß -actin promoter (human or
provide gene functions not present in the replication - defi - chicken ), the EF1 - a promoter, the ubiquitin promoter, the
cient adenoviral vector, but required for viral propagation , at SV40 promoter, and the Rous Sarcoma Virus promoter.
appropriate levels in order to generate high titers of viral Inducible promoters include, for example , the Tet system
vector stock . Such complementing cell lines are known and (U . S . Pat . Nos . 5 , 464,758 and 5 ,814 ,618 ), the Ecdysone
include , but are not limited to , 293 cells (described in , e . g ., 30 inducible system (No et al., Proc . Natl. Acad. Sci., 93 :
Graham et al., J. Gen . Virol., 36 : 59 - 72 (1977 )), PER .C6 3346 -3351 ( 1996 )), the T-REXTM system ( Invitrogen , Carls
cells ( described in , e .g ., International Patent Application bad , Calif .), LACSWITCHTM system ( Stratagene, San
Publication WO 1997 /000326 , and U . S . Pat. Nos. 5, 994 , 128 Diego, Calif.), and the Cre - ERT tamoxifen inducible recom
and 6 ,033 , 908 ), and 293 -ORF6 cells (described in , e . g., binase system (Indra et al., Nuc. Acid . Res., 27 : 4324 -4327
International Patent Application Publication WO 1995 / 35 ( 1999 ) ; Nuc. Acid . Res., 28 : e99 (2000 ) ; U . S . Pat. No .
034671 and Brough et al., J. Virol., 71: 9206 - 9213 ( 1997 )). 7 ,112 ,715 ; and Kramer & Fussenegger,Methods Mol. Biol.,
Other suitable complementing cell lines to produce the 308 : 123 - 144 ( 2005 )).
replication - deficient adenoviral vector of the invention A promoter can be selected by matching its particular
include complementing cells that have been generated to pattern of activity with the desired pattern and level of
propagate adenoviral vectors encoding transgenes whose 40 expression of an antigen ( s ). For example , the adenoviral
expression inhibits viral growth in host cells ( see , e .g ., U .S . vector can comprise two or more nucleic acid sequences that
Patent Application Publication 2008 /0233650 ). Additional encode the same or different antigens and are operably
suitable complementing cells are described in , for example , linked to different promoters displaying distinct expression
U . S . Pat. Nos. 6 ,677 , 156 and 6 ,682 ,929, and International profiles . In this regard , a first promoter can be selected to
Patent Application Publication WO 2003 /020879 . In some 45 mediate an initial peak of antigen production , thereby prim
instances, the cellular genome need not comprise nucleic ing the immune system against an encoded antigen . A
acid sequences , the gene products of which complement for second promoter can be selected to drive production of the
all of the deficiencies of a replication -deficient adenoviral same or different antigen such that expression peaks several
vector . One or more replication -essential gene functions days after that of the first promoter, thereby " boosting ” the
lacking in a replication -deficient adenoviral vector can be 50 immune system against the antigen . Alternatively , a hybrid
supplied by a helper virus, e . g ., an adenoviral vector that promoter can be constructed which combines the desirable
supplies in trans one or more essential gene functions aspects of multiple promoters . For example , a CMV- Rous
required for replication of the replication -deficient adenovi- Sarcoma Virus hybrid promoter combining the CMV pro
ral vector. Alternatively, the adenoviral vector can comprise moter 's initial rush of activity with the Rous Sarcoma Virus
a non - native replication - essential gene that complements for 55 promoter ' s high maintenance level of activity can be
the one or more replication - essential gene functions lacking employed . In that antigens can be toxic to eukaryotic cells ,
in the inventive replication -deficient adenoviral vector. For it may be advantageous to modify the promoter to decrease
example , an E1/E4 -deficient adenoviral vector can be engi- activity in complementing cell lines used to propagate the
neered to contain a nucleic acid sequence encoding E4 adenoviral vector.
ORF6 that is obtained or derived from a different adenovirus 60 To optimize protein production , preferably the Plasmo
( e. g ., an adenovirus of a different serotype than the inventive dium antigen - encoding nucleic acid sequence further com
adenoviral vector, or an adenovirus of a different species prises a polyadenylation site following the coding sequence .
than the inventive adenoviral vector) . Any suitable polyadenylation sequence can be used , includ
In addition to the one or more nucleic acid sequences ing a synthetic optimized sequence , as well as the polyade
encoding Plasmodium antigens, the adenoviral vector pref- 65 nylation sequence of BGH (Bovine Growth Hormone ),
erably comprises expression control sequences, such as polyoma virus , TK ( Thymidine Kinase ), EBV (Epstein Barr
promoters , enhancers, polyadenylation signals , transcription Virus), mouse globin D protein , and the papillomaviruses,
 US 10 ,314 , 901 B2
23 24
including human papillomaviruses and BPV (Bovine Pap - to administration . For example , the composition can be
illoma Virus). A preferred polyadenylation sequence is the formulated to reduce loss of the adenoviral vector on devices
SV40 (Simian Virus -40 ) polyadenylation sequence . Also used to prepare , store , or administer the adenoviral vector,
preferably all the proper transcription signals (and transla such as glassware, syringes , or needles. The composition can
tion signals , where appropriate ) are correctly arranged such 5 be formulated to decrease the light sensitivity and /or tem
that the nucleic acid sequence is properly expressed in the perature sensitivity of the adenoviral vector. To this end, the
cells into which it is introduced . If desired , the nucleic acid composition preferably comprises a pharmaceutically
sequence also can incorporate splice sites (i.e ., splice accep acceptable liquid carrier, such as , for example , those
tor and splice donor sites ) to facilitate mRNA production . described above , and a stabilizing agent selected from the
If the nucleic acid sequence encodes a processed or 10 group consisting of polysorbate 80 , L -arginine, polyvi
secreted protein or peptide , or a protein that acts intracel- nylpyrrolidone, trehalose , and combinations thereof. Use of
lularly , preferably the nucleic acid sequence further com such a composition will extend the shelf life of the adeno
prises the appropriate sequences for processing, secretion , viral vector, and facilitate its administration . Formulations
intracellular localization , and the like. The nucleic acid for adenoviral vector -containing compositions are further
sequence can be operably linked to a signal sequence , which 15 described in , for example , U . S . Pat. Nos. 6 ,225 ,289, 6 , 514 ,
targets a protein to cellular machinery for secretion . Appro - 943 , and International Patent Application Publication WO
priate signal sequences include, but are not limited to, leader 2000 /034444.
sequences for immunoglobulin heavy chains and cytokines The composition also can be formulated to enhance
( see , for example , Ladunga et al., Current Opinions in transduction efficiency of the composition . In addition , one
Biotechnology, 11 : 13 - 18 (2000 )). Other protein modifica - 20 of ordinary skill in the art will appreciate that the isolated
tions can be required to secrete a protein from a host cell, Plasmodium polypeptides and/or nucleic acid sequences can
which can be determined using routine laboratory tech - be present in a composition with other therapeutic or bio
niques . Preparing expression constructs encoding antigens logically -active agents . For example , factors that control
and signal sequences is further described in , for example, inflammation, such as ibuprofen or steroids, can be part of
U . S . Pat. No . 6 ,500 ,641 . Methods of secreting non - secre - 25 the composition to reduce swelling and inflammation asso
table proteins are further described in , for example, U . S . Pat. ciated with in vivo administration of the composition. Anti
No. 6 , 472 , 176 , and International Patent Application Publi - biotics , i.e ., microbicides and fungicides , can be present to
cation WO 2002/048377 . treat existing infection and/or reduce the risk of future
Plasmodium antigens encoded by the one or more nucleic infection , such as infection associated with gene transfer
acid sequences can be modified to attach or incorporate the 30 procedures.
antigen on a host cell surface. In this respect, the antigen can The invention further provides a method of inducing a
comprise a membrane anchor, such as a gpi- anchor, for protective immune response against a Plasmodium parasite
conjugation onto a cell surface . A transmembrane domain in a mammal. The method comprises administering to the
can be fused to the antigen to incorporate a terminus of the mammal the inventive composition comprising isolated
antigen protein into the cell membrane . Other strategies for 35 Plasmosdium polypeptides or nucleic acid sequences ,
displaying peptides on a cell surface are known in the art and whereupon a protective immune response against a Plasmo
are appropriate for use in the context of the invention . dium parasite in the mammal is induced . When the compo
The composition is a physiologically acceptable ( e .g ., sition comprises one or more Plasmodium nucleic acid
pharmaceutically acceptable ) composition , which comprises sequences, the one or more nucleic acid sequences encoding
a carrier, preferably a physiologically ( e . g ., pharmaceuti- 40 the Plasmodium polypeptides are expressed in the mammal
cally ) acceptable carrier. Any suitable carrier can be used to produce the Plasmodium polypeptide. In accordance with
within the context of the invention , and such carriers are the invention , the composition is administered to a mammal,
well known in the art. The choice of carrier will be deter- most preferably a human . The human preferably is in a
mined, in part, by the particular use of the composition ( e. g., population that has a high risk of acquiring Plasmodium
administration to an animal) and the particular method used 45 parasites . Such high - risk populations include residents of,
to administer the composition . The composition optionally and travelers to , parts of Africa, Asia , the Middle East,
can be sterile . Central and South America , Hispaniola , and Oceania , as
Suitable compositions include aqueous and non -aqueous well as military personnel deployed to these areas.
isotonic sterile solutions, which can contain anti-oxidants, The immune response can be directed against any Plas
buffers , and bacteriostats , and aqueous and non -aqueous 50 modium species that infects a particular mammal, such as
sterile suspensions that can include suspending agents , solu - those described herein . Preferably , the mammal is a human
bilizers , thickening agents , stabilizers, and preservatives. and the immune response is directed against a human
The composition can be presented in unit -dose or multi - dose infecting Plasmodium species.Most preferably , the immune
sealed containers , such as ampules and vials , and can be response is directed against Plasmodium falciparum and /or
stored in a freeze -dried (lyophilized ) condition requiring 55 Plasmodium vivax. The immune response can be a humoral
only the addition of the sterile liquid carrier, for example , immune response, a cell-mediated immune response , or,
water, immediately prior to use . Extemporaneous solutions desirably, a combination of humoral and cell-mediated
and suspensions can be prepared from sterile powders , immunity . Ideally , the immune response provides protection
granules , and tablets. Preferably, the carrier is a buffered to the animal, typically a mammal such as a human , upon
saline solution . The composition can be generated in accor- 60 subsequent challenge with Plasmodium . The inventive
dance with conventional techniques described in , e. g ., Rem - method also can be used for antibody production and
ington : The Science and Practice of Pharmacy, 21st Edition , harvesting .
Lippincott Williams & Wilkins, Philadelphia , Pa. (2001 ). To enhance the immune response generated against a
When the composition comprises an adenoviral vector, Plasmodium antigen , the composition also can comprise an
the composition preferably is free of replication -competent 65 immune stimulator , or a nucleic acid sequence that encodes
adenovirus. In addition , the composition preferably is for an immune stimulator. Immune stimulators also are referred
mulated to protect the adenoviral vector from damage prior to in the art as “ adjuvants,” and include , for example ,
 US 10 ,314 , 901 B2
25 26
cytokines, chemokines , or chaperones. Cytokines include, ticles, 1x107-1x1012 particles, 1x108 -1x1011 particles,
for example , Macrophage Colony Stimulating Factor (e.g., 3x108 -3x1011 particles , 1x109 -1x1012 particles , 1x109 -1x
GM -CSF), Interferon Alpha (IFN - a ), Interferon Beta ( IFN 1011 particles , 1x10°-1x1010 particles, or 1x1010 -1x1012
B ), Interferon Gamma ( IFN -Y ), interleukins (IL - 1, IL -2 , particles, of the adenoviral vector. In other words , a single
IL -4 , IL -5, IL -6 , IL - 8, IL -10 , IL - 12 , IL - 13, IL - 15, IL - 16 , and 5 dose of adenoviral vector can comprise, for example , about
IL - 18 ), the TNF family of proteins, Intercellular Adhesion 1x10 pu , 2x10 pu , 4x10 pu , 1x10 pu , 2x10 pu , 4x107
Molecule - 1 ( ICAM - 1 ), Lymphocyte Function - Associated pu, 1x108 pu , 2x108 pu , 3x108 pu , 4x108 pu, 1x109 pu ,
antigen -3 (LFA -3 ),B7-1, B7 -2 , FMS-related tyrosine kinase 2x10° pu , 3x10 pu , 4x10° pu , 1x1010 pu , 2x101° pu3x1010
3 ligand , (Flt3L ) , vasoactive intestinal peptide (VIP ), and pu , 4x1010 pu, 1x1011 pu , 2x1011 pu , 3x1011 pu , 4x1011 pu ,
CD40 ligand . Chemokines include , for example , B Cell - 10 1x1012 pu, 2x1012 pu , 3x1012 pu , or 4x1012 pu of the
Attracting chemokine - 1 (BCA - 1), Fractalkine, Melanoma adenoviral vector.
Growth Stimulatory Activity (MGSA ) protein , Hemofiltrate Administration of the inventive composition can be one
CC chemokine 1 (HCC - 1 ), Interleukin 8 (IL - 8 ), Interferon - component of a multistep regimen for inducing a protective
stimulated T- cell alpha chemoattractant (I- TAC ), Lymphot immune response against Plasmodium parasites (e .g ., Plas
actin , Monocyte Chemotactic Protein 1 (MCP - 1 ), Monocyte 15 modium falciparum and/ or Plasmodium vivax ) in a mammal.
Chemotactic Protein 3 (MCP - 3 ), Monocyte Chemotactic In this respect , themethod of inducing a protective immune
Protein 4 (CP -4 ), Macrophage -Derived Chemokine (MDC ), response can further comprise administering to the mammal
a macrophage inflammatory protein (MIP ), Platelet Factor 4 a boosting composition after administering the inventive
( PF4 ), RANTES , BRAK , eotaxin , exodus 1 - 3 , and the like . composition to the mammal. In this embodiment, therefore ,
Chaperones include, for example , the heat shock proteins 20 the immune response is “ primed” upon administration of the
Hsp170 , Hsc70 , and Hsp40 . inventive composition , and is “ boosted ” upon administration
The composition ideally comprises a " therapeutically of the boosting composition . Alternatively , the inventive
effective amount of the isolated Plasmodium polypeptide or method further comprises administering to the mammal a
polypeptides. A “ therapeutically effective amount” refers to priming composition to the mammal prior to administering
an amount effective, at dosages and for periods of time 25 the inventive composition to the mammal. In this embodi
necessary, to achieve a desired therapeutic result . The thera - ment, therefore , the immune response is " primed ” upon
peutically effective amount may vary according to factors administration of the priming composition , and is “ boosted ”
such as the disease state , age, sex , and weight of the upon administration of the inventive composition .
individual, and the ability of the Plasmodium polypeptide to In one embodiment, the Plasmodium polypeptide(s ) pres
elicit a desired response in the individual. For example , a 30 ent in , or encoded by a vector present in , the priming
therapeutically effective amount of a Plasmodium polypep - composition and the boosting composition are desirably the
tide of the invention is an amount which ameliorates a same. For example , if the priming composition comprises a
malaria infection in a human . hypothetical Plasmodium " protein A ," then the boosting
Alternatively , the pharmacologic and /or physiologic composition also comprises “ protein A ," or comprises a
effect may be prophylactic , i.e ., the effect completely or 35 vector which encodes “ protein A .” At least one of the
partially prevents a disease or symptom thereof. In this priming composition or the boosting composition desirably
respect, the inventive method comprises administering a comprises an adenoviral vector that comprises a nucleic acid
" prophylactically effective amount” of the composition . A sequence encoding a Plasmodium antigen , while the other of
" prophylactically effective amount” refers to an amount the priming composition and the boosting composition can
effective, at dosages and for periods of time necessary, to 40 comprise the inventive composition or a different effective
achieve a desired prophylactic result (e . g ., prevention of agent, though desirably a gene transfer vector that comprises
Plasmodium infection or onset of malaria ). For example, a a nucleic acid sequence encoding a Plasmodium antigen .
prophylactically effective amount of a Plasmodium poly - Any gene transfer vector can be employed , including viral
peptide of the invention is an amount which protects a and non -viral gene transfer vectors. Examples of suitable
human upon subsequent challenge with Plasmodium . 45 viral gene transfer vectors include , but are not limited to ,
In embodiments where the composition comprises an retroviral vectors , adeno -associated virus vectors, vaccinia
adenoviral vector comprising one or more nucleic acid virus vectors , herpesvirus vectors , parainfluenza -RSV chi
sequences that encode a Plasmodium polypeptide, the com - meric vectors ( PIV -RSV ) , and adenoviral vectors. Examples
position comprises a “ therapeutically effective amount" of of suitable non - viral vectors include , but are not limited to ,
the adenoviral vector , i.e ., a dose of adenoviral vector which 50 plasmids, liposomes , and molecular conjugates ( e . g ., trans
provokes a desired immune response in the mammal. Desir- ferrin ). Preferably , the priming composition or the boosting
ably , a single dose of adenoviral vector comprises about composition comprises a plasmid or an adenoviral vector .
1x10 % or more particles (which also are referred to as Alternatively , an immune response can be primed or boosted
particle units (pu )) of the adenoviral vector, e . g ., about by administration of a Plasmodium protein itself (e . g ., any
1x100 or more particles, about 1x10 or more particles, 55 of the Plasmodium proteins described herein ) with or with
about 1x108 or more particles , about 1x10° or more par out a suitable adjuvant (e .g., alum , QS -21, insulin - derived
ticles, or about 3x10° or more particles of the adenoviral adjuvant, etc .), a live-attenuated Plasmodium parasite , and
vector. Alternatively, or in addition , a single dose of adeno - the like. When the priming composition or the boosting
viral vector comprises about 3x1014 particles or less of the composition comprises an adenoviral vector , the adenoviral
adenoviral vector, e .g ., about 1x1013 particles or less , about 60 vector can be, or can be derived from , any adenovirus that
1x1012 particles or less , about 3x1011 particles or less , about infects a human or non - human animal, such as those
1x1011 particles or less, about 1x1010 particles or less, or described herein . In this respect, the priming composition or
about 1x10° particles or less of the adenoviral vector. Thus , the boosting composition can comprise a human adenoviral
a single dose of adenoviral vector can comprise a quantity of vector (e . g ., serotype 5 , 28 , or 35 ), a simian adenoviral
particles of the adenoviral vector in a range defined by any 65 vector (such as described in , e .g ., International Patent Appli
two of the aforementioned values. For example , a single cation Publication WO 2011 /057254 ) , or a gorilla adenoviral
dose of adenoviral vector can comprise 1x10 ^ -1x1014 par- vector. For example, a priming composition containing a
 US 10 ,314 , 901 B2
27 28
human serotype 5 adenoviral vector can be administered to on expression abundance data from microarray analysis and
a human , followed by administration of a boosting compo- protein mass spectrometry analysis by several research
sition containing the inventive composition comprising one groups, 300 sporozoite stage and liver stage candidate P .
or more isolated Plasmodium polypeptides described herein yoelii genes with identifiable P. falciparum orthologs were
(i.e ., a “ heterologous ” prime- boost regimen ). Alternatively, 5 prioritized for generation of an adeno - array.
a priming composition containing the inventive composition The candidate P. yoelii genes were cloned into a high
comprising one or more isolated Plasmodium polypeptides level adenovirus vector-based expression cassette using
described herein can be administered to a human , followed high -throughput methodologies (such as those described in ,
by administration of a boosting composition containing a e .g ., U . S . Patent Application Publication 2010 / 0222234 ) to
human serotype 5 adenoviral vector . In another embodiment, 10 produce an adeno -array. Specifically , PCR -amplified candi
the priming composition contains an adenoviral vector of a date antigens genes were first cloned into a pCR8/GW / Topo
first serotype comprising a nucleic acid sequence encoding cloning vector (Life Technologies , Carlsbad , Calif.). The P.
a Plasmodium antigen , and the boosting composition con - yoelii genes were then individually subcloned into a CMV
tains an adenoviral vector of a different serotype comprising expression cassette which resides in a plasmid containing
a nucleic acid sequence encoding the same Plasmodium 15 the human adenovirus 5 genome in which the El and E3
antigen as the first adenoviral vector. Alternatively , the regions were deleted . The CMV expression cassette resides
priming composition can contain a plasmid that comprises a in the El region of this plasmid . Cloning was carried out
nucleic acid sequence encoding a Plasmodium antigen , and using the attR1-CmR -ccB - attR2 lambda recombination
the boosting composition can contain the inventive compo technology (Life Technologies, Carlsbad , Calif.). To con
sition comprising an adenoviral vector. In yet another 20 struct the adenoviral vectors , the adenoviral genomes were
embodiment, the inventive composition comprising one or liberated from the plasmid backbone and used to transfect
more isolated Plasmodium polypeptides can be administered 293 -ORF6 complementing cells (Brough et al., J. Virol., 71:
to a human , followed by a second administration of the same 9206 - 9213 ( 1997 )).
composition (i.e ., a “homologous ” prime-boost regimen ). A20 /2J antigen presenting cells (APCs) were infected
One of ordinary skill in the art will appreciate that any 25 with adenoviral vectors expressing Py antigens from the
combination of vectors encoding one or more Plasmodium adeno - array and used as targets in an IFNy assay to recall
antigens and/or Plasmodium polypeptides themselves can be T-cell responses from mice immunized with Plasmodium
employed as the priming and /or boosting compositions in yoelii- irradiated sporozoites . The infected APCs were then
conjunction with the inventive composition described incubated with splenocytes from mice immunized with
herein . 30 known protective regimens of Radiation Attenuated Sporo
Administration of the priming composition and the boost - zoites (RAS ), and antigen -specific CD8 + T cell responses
ing composition can be separated by any suitable timeframe were measured by Intercellular Cytokine Staining (ICS).
( e. g ., at least any of about 1 week , 2 weeks , 4 weeks, 8 Adenoviral vectors encoding the Py circumsporozoite pro
weeks, 12 weeks, 16 weeks, 24 weeks, 52 weeks, 2 years , tein (PyCSP ) and lacking a transgene (AdNull) were used as
and 5 years, or any range defined by any two of the 35 controls .New antigens that recalled relatively frequent IFNY
foregoing values ). The boosting composition preferably is responses in RAS- immunized , but not in naïve mice , were
administered to a mammal ( e . g ., a human ) at least about 2 identified . The putative gene product encoded by each of the
weeks ( e .g ., at least any of about 3 weeks, 4 weeks , 5 weeks , P . yoelii antigens and their corresponding amino acid SEQ
6 weeks, 7 weeks, 8 weeks , 9 weeks, 10 weeks , 11 weeks, ID NOs, as well as the SEQ ID NOs for the corresponding
12 weeks, 13 weeks, 14 weeks, 15 weeks, 16 weeks, 20 40 P. falciparum and P. vivax orthologs, are set forth in Table 1 .
weeks, 24 weeks, 28 weeks, 35 weeks, 40 weeks, 50 weeks,
52 weeks, 2 years , and 5 years , or any range defined by any TABLE 1
two of the foregoing values ) following administration of the
P . yoelii P . falciparum P. vivax
priming composition . More than one dose of priming com ( 17XNL ) (307) (Sal- 1)
position and / or boosting composition can be provided in any 45 SEQ Ortholog Ortholog
suitable timeframe. The dose of the priming composition Putative Gene Product ID NO SEQ ID NO SEQ ID NO
and boosting composition administered to the mammal putative WD -40 repeat 122 149 176
depends on a number of factors , including the extent of any protein
side- effects , the particular route of administration, and the serine
50 hydroxymethyltransferase ,
123 150 177
like.
The following examples further illustrate the invention mitochondrial precursor
49 kDa zinc finger protein 124 151 178
but, of course, should not be construed as in any way hypothetical protein 98 99 100
limiting its scope. 60S ribosomal protein L6 , 51
putative
EXAMPLE 1 55 Arabidopsis thaliana
Atlg20220 / T20H2_ 3 -related
translation elongation factor 34
This example demonstrates a method for screening and EF - 1 , subunit alpha
isolating Plasmodium antigen sequences that provide pro adenosine deaminase 14 15 16
tection against malaria challenge. translation initiation factor
eIF -5A
125 152 179
Adenovirus-based microarray ( adeno - array ” ) technol - 60
ogy ( see , e. g ., U . S . Patent Application Publication 2010 / stress -induced protein stil 126 153 180
like protein
0222234) was used for high - throughput discovery of pre ethylene-inducible protein 116 117 118
erythrocytic Plasmodium falciparum antigens using hever replication licensing
orthologs identified in a P . yoelii (Py) mouse model. Spe DNA factor mis5
127 154 181
cifically , bioinformatics data mining using publicly available 65 pyruvate dehydrogenase E1 128 155 182
genomic and proteomic databases was performed to identify alpha subunit
highly expressed P. yoelii pre -erythrocytic antigens. Based
 US 10 ,314 ,901 B2
29 30
TABLE 1 - continued Antigens that recalled the most robust T cell responses
P. yoelii P. falciparum P. vivax
were selected and their capacity to protect mice from a P.
(17XNL ) (3D7) (Sal- 1 ) yoelii sporozoite challenge was tested . In this regard , 100 ug
SEQ Ortholog of plasmid vector expressing each of the above Py antigens
Ortholog
Putative Gene Product ID NO SEQ ID NO SEQ ID NO 5 were injected into mice at the start (Day 0 ) of the experi
ribosomal protein varl , 129 156 183 ment, and 6 weeks later, animals were boosted by intramus
putative cular (IM ) injection of adenoviral vectors expressing the
asparagine-rich protein , 130 157 184 same antigens at 1x101° particles per mouse . Immunized
putative
succinyl- coa ligase beta 131 158 185 10
mice were challenged with 300 or 600 live sporozoites two
chain , hydrogenosomal weeks after boost. Tail vein bleeds were performed from 6
precursor to 14 days after live sporozoite challenge and blood films
Drosophila melanogaster 132 159 186 were prepared for analysis of blood - stage parasitemia. Out
RE21692p , putative
cyclophilin 133 160 187 bred CD1mice were immunized with a DNA prime-adeno
hypothetical protein 134 161 188 15 virus boost regimen and sterile protection was measured
Protein of unknown function ,
putative
135 162 189 following sporozoite challenge as set forth in Table 2 .
Homo sapiens RIKEN DNA 136 163 190
1600015H11 gene-related TABLE 2
translation initiation factor if 63 64 6
2
20
SEQ Sterile protection
Leucine Rich Repeat, putative 137 164 191
hypothetical protein 138 165 192 Antigen ID NO # Protected /# challenged % protected
Dnaj homolog, putative 139 166 193
ADP -ribosylation factor 140 167 194 PyCSP 53 / 112 47 %
GTPase -activating protein GV0032 24 16 /41 39 %
ubiquitin carboxyl-terminal 74 GV0041 10 /42 24 %
hydrolase isozyme 15 25 GV0033 34 6 / 28 21 %
elongation factor 3 related 141 168 195 GV0043 125 1 / 14 7%
protein PFEF3-r1 GV0074 213 4 / 14 29 %
eukaryotic translation 142 169 196 GV0176 7 /28 25 %
initiation factor 3 39 kDa GV0196 7 /28 25 %
subunit
hypothetical protein 90 91
GV0004
GV0013
123
124
3 / 14
3 /14
21 %
21 %
30 GV0199 84 6 / 28 21 %
putative protein 84 85
Rab1 protein 143 170 GV0014 98 8 /42 19 %
peptidyl -prolyl cis-trans 144 171 198 GV0139 216 3 / 14 21 %
isomerase , cyclophilin -type GV0104 271 1 / 14 7%
Ribosomal protein S7e 145 172 199 Naïve control 11 / 112 10 %
similar to RIKEN DNA 35
AdNull 8 / 112 7%
2010107016 gene 146 173 200
neurofilament protein H form 147 174 201
H2 The results of this example demonstrate the identification
60S acidic ribosomal protein 148 175 of new Plasmodium antigens that can induce a protective
P2
putative protein phosphatase 213 214 215 immune response againstmalaria in a mammal.
2C 40 All references, including publications , patent applica
putative small nucleolar 216 217 218 tions, and patents, cited herein are hereby incorporated by
ribonucleoprotein gar1 reference to the same extent as if each reference were
26s protease regulatory 219 220 221 individually and specifically indicated to be incorporated by
subunit 6a (tat-binding protein reference and were set forth in its entirety herein .
homolog 1) (tbp - 1)
Drosophila melanogaster 222 223 224 45 The use of the terms " a " and " an " and " the " and " at least
CG1349 gene product one ” and similar referents in the context of describing the
putative ribosomal protein 225 226 227
S19e invention (especially in the context of the following claims)
glycyl-tRNA synthetase 228 229 230 are to be construed to cover both the singular and the plural,
transketolase 231 232 233 unless otherwise indicated herein or clearly contradicted by
asparaginyl-tRNA synthetase 234 235 236 50 context. The use of the term “ at least one ” followed by a list
putative acyl carrier protein 237 238 239 of one or more items ( for example , " at least one of A and B ” )
putative calcyclin binding 240 241 242
protein is to be construed to mean one item selected from the listed
putative glycerol kinase 243 244 245 items ( A or B ) or any combination of two or more of the
elongation factor 2 246 247 248 listed items ( A and B ), unless otherwise indicated herein or
hexokinase 249 250 251 clearly contradicted by context. The terms " comprising,"
pyruvate dehydrogenase E1 252 253 254 “ having,” “ including ,” and “ containing” are to be construed
beta subunit as open -ended terms (i.e ., meaning " including , but not
U43539 hepatocyte 255 256 257
erythrocyte protein 17 kDa limited to ,” ) unless otherwise noted . Recitation of ranges of
protein kinase domain 258 259 260 values herein are merely intended to serve as a shorthand
merozoite surface protein 1 261 262 263 60 method of referring individually to each separate value
precursor falling within the range , unless otherwise indicated herein ,
merozoite surface protein 7 264 265 , 266, 267 268 and each separate value is incorporated into the specification
precursor
glutathione s -transferase 269 N/ A 270 as if it were individually recited herein . All methods
S -adenosylmethionine 271 272 273
273 described herein can be performed in any suitable order
synthetase 65 unless otherwise indicated herein or otherwise clearly con
tradicted by context. The use of any and all examples, or
exemplary language (e. g., “ such as ” ) provided herein , is
 US 10 ,314 , 901 B2
31 32
intended merely to better illuminate the invention and does inventors expect skilled artisans to employ such variations
not pose a limitation on the scope of the invention unless as appropriate, and the inventors intend for the invention to
otherwise claimed . No language in the specification should be practiced otherwise than as specifically described herein .
be construed as indicating any non - claimed element as Accordingly, this invention includes all modifications and
essential to the practice of the invention . 5 equivalents of the subject matter recited in the claims
Preferred embodiments of this invention are described appended hereto as permitted by applicable law . Moreover,
herein , including the best mode known to the inventors for any combination of the above-described elements in all
carrying out the invention . Variations of those preferred possible variations thereof is encompassed by the invention
embodiments may become apparent to those of ordinary unless otherwise indicated herein or otherwise clearly con
skill in the art upon reading the foregoing description. The tradicted by context.

SEQUENCE LISTING
The patent contains a lengthy “ Sequence Listing” section . A copy of the “ Sequence Listing” is available in
electronic form from the USPTO web site (http :// seqdata .uspto. gov /? pageRequest=docDetail& DocID = US10314901B2 ).
An electronic copy of the “ Sequence Listing” will also be available from the USPTO upon request and payment of the
fee set forth in 37 CFR 1 .19(b )( 3 ).

The invention claimed is : 25 10 . The composition of claim 5 , wherein the adenoviral

1. A composition comprising a pharmaceutically accept- vector requires complementation of a deficiency in the El
able carrier and one ormore isolated nucleic acid sequences, region of the adenoviral genome and a deficiency in the E4
wherein the one or more isolated nucleic acid sequences are region of the adenoviral genome for propagation and does
selected from the group consisting of SEQ ID NOs: 27 -29 . not require complementation of any other deficiency of the
2 . The composition of claim 1 , wherein the one or more 3030 adenoviral genome for propagation .
11 . A method of inducing a protective immune response
isolated nucleic acid sequences are present in a vector. against a Plasmodium parasite in a mammal, which method
3 . The composition of claim 2 , wherein the vector is an comprises administering to the mammal the composition of
adenoviral vector. claim 1, whereupon a protective immune response against
4 . The composition of claim 3 , wherein the adenoviral the Plasmodium parasite in the mammal is induced .
vector is a human adenoviral vector or a gorilla adenoviral 35 12 . The method of claim 11 , wherein the mammal is a
vector. human .
5 . The composition of claim 3 , wherein the adenoviral arasite 13 . The method of claim 11 , wherein the Plasmodiump
vector requires complementation of a deficiency in one or is Plasmodium falciparum or Plasmodium vivax .
more early regions of the adenoviral genome for propagation 14 . The method of claim 11 , wherein the one or more
and does not require complementation of any other defiA - 4040 isolated nucleic acid sequences are present in a vector.
15 . The method of claim 14 , wherein the vector is an
ciency of the adenoviral genome for propagation .
6 . The composition of claim 5 , wherein the one or more adenoviral vector .
early regions are selected from the group consisting of the vector 16 . The method of claim 15 , wherein the adenoviral
El region, the E2 region , and the E4region of the adenovirus 45 vector. is a human adenoviral vector or a gorilla adenoviral
genome.
7. The composition of claim 5 , wherein the adenoviral vector 17 . The method of claim 15 , wherein the adenoviral
vector requires complementation of a deficiency in the E1 more requires complementation of a deficiency in one or
region of the adenoviral genome for propagation and does and does notearly regions of the adenoviral genome for propagation
not require complementation of any other deficiency of the 50 ciency require complementation of any other defi
adenoviral genome for propagation . of tare adenoviral genome for propagation .
18 . The composition of claim 1, comprising a pharma
8 . The composition of claim 5, wherein the adenoviral ceutically
vector requires complementation of a deficiency in the E1A sequence ofacceptable carrier and the isolated nucleic acid
SEQ II) NO : 27 .
region or the E1B region of the adenoviral genome for 19 . The composition of claim 1, comprising a pharma
propagation and does not require complementation of any
other deficiency of the adenoviral genome for propagation 55 ceutically acceptable carrier and the isolated nucleic acid
9 . The composition of claim 5 , wherein the adenoviral sequence of SEQ ID NO : 28 .
20 . The composition of claim 1 , comprising a pharma
vector requires complementation of a deficiency in the ceutically
E4region of the adenoviral genome for propagation and does acceptable carrier and the isolated nucleic acid
not require complementation of any other deficiency of the sequence of SEQ II) NO : 29 .
adenoviral genome for propagation .