WO2017189448A1 - Bivalent immunogenic conjugate for malaria and typhoid - Google Patents

Bivalent immunogenic conjugate for malaria and typhoid Download PDF

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WO2017189448A1
WO2017189448A1 PCT/US2017/029182 US2017029182W WO2017189448A1 WO 2017189448 A1 WO2017189448 A1 WO 2017189448A1 US 2017029182 W US2017029182 W US 2017029182W WO 2017189448 A1 WO2017189448 A1 WO 2017189448A1
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protein
vip
malaria
conjugates
conjugate
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PCT/US2017/029182
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French (fr)
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SoJung AN
Puthupparampil V. Scaria
Patrick E. Duffy
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The United States Of America, As Represented By The Secretary, Department Of Health And Human Services
International Vaccine Institute
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Publication of WO2017189448A1 publication Critical patent/WO2017189448A1/en

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    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K47/00Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/646Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent the entire peptide or protein drug conjugate elicits an immune response, e.g. conjugate vaccines
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K39/00Medicinal preparations containing antigens or antibodies
    • A61K39/002Protozoa antigens
    • A61K39/015Hemosporidia antigens, e.g. Plasmodium antigens
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K39/00Medicinal preparations containing antigens or antibodies
    • A61K39/02Bacterial antigens
    • A61K39/025Enterobacteriales, e.g. Enterobacter
    • A61K39/0275Salmonella
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04Antibacterial agents
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P33/00Antiparasitic agents
    • A61P33/02Antiprotozoals, e.g. for leishmaniasis, trichomoniasis, toxoplasmosis
    • A61P33/06Antimalarials
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K39/00Medicinal preparations containing antigens or antibodies
    • A61K2039/555Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55505Inorganic adjuvants
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K39/00Medicinal preparations containing antigens or antibodies
    • A61K2039/70Multivalent vaccine

Abstract

Disclosed herein are conjugates including at least one malaria protein or portion thereof and at least one capsular Vi polysaccharide (ViP), wherein the at least one malaria protein is linked to the at least one ViP through at least one linking group. In some embodiments, the at least one malaria protein is Pfs25, Pvs25, Pfs48/45, Pvs48/45, Pfs47, Pvs47, Pfs230, Pvs230, PfCSP, PvCSP, or a portion of any one thereof. Also disclosed herein are compositions including one or more of the conjugates including at least one malaria protein and at least one ViP and a pharmaceutically acceptable carrier, an adjuvant, and/or other components. Disclosed herein are methods for eliciting an immune response in a subject to Plasmodium and/or Salmonella typhi, including administering an effective amount of one or more of the disclosed conjugates or a composition including one or more of the disclosed conjugates to the subject.

Description

BIVALENT IMMUNOGENIC CONJUGATE FOR MALARIA AND TYPHOID

CROSS REFERENCE TO RELATED APPLICATION

This application claims priority to U.S. Provisional Application No. 62/327,184, filed April 25, 2016, which is incorporated by reference in its entirety.

FIELD

This disclosure relates to conjugates, particularly immunogenic conjugates containing malaria and typhoid antigens, and methods of their use.

BACKGROUND

Malaria and typhoid fever are co-endemic in large parts of the world. Despite significant efforts to combat these diseases, they remain a major source of morbidity and mortality and a serious threat to public health in developing countries and travelers to the region. Typhoid fever results from bacterial infection by Salmonella enterica serovar typhi (S. typhi) and is transmitted through contaminated water and food, mostly in regions of the world with insufficient water and sewage systems. Malaria is a parasitic infection (Plasmodium) propagated by mosquito vector through bites of female Anopheles mosquito. Though the source and route of these two infections are different, their prevalence have significant regional commonality, particularly in Africa and tropical developing countries. In addition, both diseases disproportionally affect children under 5 years of age. In the co-endemic region, malaria infection may enhance susceptibility to typhoid fever due to a weakened immune system and co-infection can lead to misdiagnosis due to similarity of symptoms such as fever, headaches, muscle aches, nausea and vomiting.

Due to the complexity of the malaria parasite life cycle, development of a malaria vaccine has been a substantial challenge to vaccine research and development. The most advanced malaria vaccine candidate is a pre-erythrocytic vaccine called RTS,S, which in clinical studies has shown modest efficacy against clinical malaria in infants and children over 4 years. In young children but not in infants, RTS,S has also shown modest efficacy against severe malaria, but this benefit for children required a 4th booster dose. Other major vaccine efforts against malaria include whole organism vaccines, blood stage vaccines, and Transmission Blocking Vaccines (TBV). TBV is a distinctive vaccine concept compared to traditional vaccines that protect vaccinated individuals against disease. TBV antigens are expressed in the mosquito stages of the parasite life cycle and vaccination of humans with these antigens is expected to generate antibodies that are transferred to the mosquitos during a blood meal and prevent development of the mosquito stage parasite and block further transmission to humans.

Currently, two typhoid vaccines, Vi capsular polysaccharide vaccine (Vi) and oral live attenuate vaccine (S. typhi Ty21a) are licensed and available in the global vaccine market.

However both typhoid vaccines induce insufficient efficacy in infants and children less than 2 years of age and are not recommended in infant immunization. Two Vi-TT (Tetanus Toxoid) conjugate vaccines have been licensed in China and India respectively. Additional conjugates such as Vi- rEPA (recombinant Exoprotein A from Pseudomonas aeruginosa), Vi-DT (diphtheria toxoid) and Vi-CRMi97 (nontoxic recombinant diphtheria toxin) are being evaluated as typhoid vaccines including for use in children and infants.

SUMMARY

There remains a need for safe and effective vaccines for malaria and typhoid, particularly for infants and young children. Disclosed herein are conjugates including a malaria protein or portion thereof and Vi capsular polysaccharide (such as ViP from S. typhi or Citrobacter). These conjugates exhibit unexpected enhancement of immunogenicity for both antigens and may function as a bivalent vaccine to decrease or inhibit malaria and/or malaria transmission and decrease or inhibit typhoid fever.

Disclosed herein are conjugates (such as immunogenic conjugates) including at least one malaria protein or portion thereof and at least one Vi polysaccharide (ViP), wherein the at least one malaria protein is linked to the at least one ViP through at least one linking group. In some embodiments, the at least one malaria protein is Plasmodium falciparum s25 protein (Pfs25), Plasmodium vivax s25 protein (Pvs25), P. falciparum s230 protein (Pfs230), P. vivax s230 protein (Pvs230), P. falciparum circumsporozoite (CSP) protein (PfCSP), P. vivax CSP protein (PvCSP), P. falciparum s48/45 protein (Pfs48/45), P. vivax s48/45 protein (Pvs48/45), P. falciparum s47 protein (Pfs47), P. vivax s47 protein (Pvs47), or a portion of any one thereof. Exemplary malaria proteins or portions thereof include the amino acid sequences of SEQ ID NOs: 1-18 or amino acid sequences with at least 95% sequence identity to SEQ ID NOs: 1-18. The at least one malaria protein is linked to at least one ViP (such as S. typhi ViP or Citrobacter ViP) through a linking group. In some embodiments, the linking group is a hydrazide, a dihydrazide, an amide group, a thioether group, N-succinimidyl-4-formylbenzoate, N-succinimidyl-3-bromoacetamidopropionate, or N-succinimidyl-3-(2-pyridyldithio)-propionate. In one specific example, the linking group is adipic acid dihydrazide (ADH). Also disclosed herein are compositions including one or more of the conjugates including at least one malaria protein and at least one ViP and a pharmaceutically acceptable carrier, an adjuvant, and/or other components. In some examples, the composition includes an adjuvant, such as alum.

Disclosed herein are methods for eliciting an immune response in a subject to Plasmodium

(such as an immune response to one or more malaria proteins) and/or Salmonella typhi (such as an immune response to ViP). The methods include administering an effective amount of one or more of the disclosed conjugates or a composition including one or more of the disclosed conjugates to the subject. In some examples, the methods elicit a transmission blocking response or a protective response to Plasmodium (such as P. falciparum or P. vivax) or a protective response to S. typhi. In some examples, two or more doses of the immunogenic conjugates or compositions are administered to a subject.

The foregoing and other features of the disclosure will become more apparent from the following detailed description, which proceeds with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and IB are schematic diagrams of exemplary methods of conjugating a malaria protein (e.g. , Pfs25) and Vi polysaccharide (ViP). FIG. 1A shows an exemplary method including derivatization of ViP with adipic acid dihydrazide (ADH) followed by conjugation to Pfs25. FIG. IB shows an exemplary method including derivatization of Pfs25 with ADH followed by conjugation to ViP.

FIG. 2 is an image of a Western blot of ViP, Pfs25, and Pfs25-ViP conjugates (Lots 21-25) with anti-Pfs25 monoclonal antibody 4B7.

FIGS. 3A and 3B are graphs showing antibody titer against Pfs25 (FIG. 3A) or immune response against ViP (FIG. 3B) by ELISA two weeks after immunization with the indicated formulation (day 42).

FIGS. 4A and 4B are graphs showing antibody titer against Pfs25 about five weeks (day 83, PBS) or six weeks (day 91, ALHYDROGEL®) after immunization of mice with the indicated formulations (FIG. 4A) or anti-Vi titer 2-6 weeks after immunization mice with the indicated formulations (FIG. 4B; solid symbols, day 42; open symbols, day 70-90). FIG. 4C shows serum bactericidal activity (SB A) titer as the geometric mean (GM) with the percent confidence interval (% CI) against Salmonella typhi 90 days after immunization of mice with the indicated formulations in either PBS (closed symbols) or ALHYDROGEL® (AH, open symbols). FIG. 5 is a graph showing percent reduction in oocysts in mosquitos fed with immune sera from mice immunized with the indicated formulations. Results are shown as percentage reduction in oocyst count relative to oocyst count from mosquitos fed on control sera. SEQUENCES

The nucleic acid and amino acid sequences provided herein are shown using standard letter abbreviations for nucleotide bases and amino acids, as defined in 37 C.F.R. 1.822. Only one strand of each nucleic acid sequence is shown, but the complementary strand is understood as included by any reference to the displayed strand.

SEQ ID NO: 1 is the amino acid sequence of an exemplary Pfs25 protein.

SEQ ID NO: 2 is the amino acid sequence of a portion of a Pfs25 protein used in some conjugates.

SEQ ID NO: 3 is the amino acid sequence of an exemplary Pvs25 protein.

SEQ ID NO: 4 is the amino acid sequence of a portion of a Pvs25 protein used in some conjugates.

SEQ ID NO: 5 is the amino acid sequence of an exemplary Pfs230 protein.

SEQ ID NO: 6 is the amino acid sequence of a portion of a Pfs230 protein used in some conjugates.

SEQ ID NO: 7 is the amino acid sequence of an exemplary Pvs230 protein.

SEQ ID NO: 8 is the amino acid sequence of a portion of a Pvs230 protein used in some conjugates.

SEQ ID NO: 9 is the amino acid sequence of an exemplary PfCSP protein.

SEQ ID NO: 10 is the amino acid sequence of a portion of a PfCSP protein used in some conjugates.

SEQ ID NO: 1 1 is the amino acid sequence of an exemplary PvCSP protein.

SEQ ID NO: 12 is the amino acid sequence of a portion of a PvCSP protein used in some conjugates.

SEQ ID NO: 13 is the amino acid sequence of an exemplary Pfs48/45 protein.

SEQ ID NO: 14 is the amino acid sequence of a portion of a Pfs48/45 protein used in some conjugates.

SEQ ID NO: 15 is the amino acid sequence of an exemplary Pvs48/45 protein.

SEQ ID NO: 16 is the amino acid sequence of a portion of a Pvs48/45 protein used in some conjugates.

SEQ ID NO: 17 is the amino acid sequence of an exemplary Pfs47 protein. SEQ ID NO: 18 is the amino acid sequence of an exemplary Pvs47 protein.

DETAILED DESCRIPTION

The disclosed conjugates simultaneously target two infections - typhoid fever and malaria. The conjugates include a malaria protein (or portion thereof) and Vi polysaccharide (ViP) and in at least some examples, provide unexpectedly enhanced immune response to both antigens compared to the response induced by the antigens when used individually. Inclusion of ViP in the conjugates provides a direct benefit to subjects, such as an immune response that reduces, inhibits, or even prevents infection with S. typhi and/or symptoms of typhoid fever. In some examples, the conjugates include a malaria transmission blocking vaccine (TBV) antigen (such as P25 protein, P230 protein, P48/45 protein, P47 protein, or a portion thereof). TBV antigens are expressed in the mosquito stages of the parasite life cycle and are expected to generate antibodies that are transferred to mosquitos during a blood meal and prevent development of the mosquito stage parasite and thus further transmission. TBV antigen-containing conjugates thus provide an indirect benefit to the immunized subject, namely reduced malaria transmission in the community. In other examples, the disclosed conjugates include a CSP protein or portion thereof, which provides a direct benefit to subjects, such as an immune response that reduces, inhibits, or even prevents infection with Plasmodium and/or symptoms of malaria. I. Abbreviations

ADH: adipic acid dihydrazide

CSP: circumsporozoite protein

EDC: l-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride

ELISA: enzyme-linked immunosorbent assay

MES: 2-(N-morpholino) ethanesulfonic acid

Pfs25: Plasmodium falciparum s25 protein

Pvs25: Plasmodium vivax s25 protein

Pfs47: Plasmodium falciparum s47 protein

Pvs47: Plasmodium vivax s47 protein

Pfs48/45: Plasmodium falciparum s48/45 protein

Pvs48/45: Plasmodium vivax s48/45 protein

Pfs230: Plasmodium falciparum s230 protein

Pvs230: Plasmodium vivax s230 protein

TBV: transmission blocking vaccine ViP: Vi capsular polysaccharide

II. Terms

Unless otherwise noted, technical terms are used according to conventional usage.

Definitions of common terms in molecular biology may be found in Benjamin Lewin, Genes VII, published by Oxford University Press, 2000 (ISBN 019879276X); Kendrew et al. (eds.), The Encyclopedia of Molecular Biology, published by Blackwell Publishers, 1994 (ISBN 0632021829); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by Wiley, John & Sons, Inc., 1995 (ISBN 0471186341); and other similar references.

Unless otherwise explained, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. The singular terms "a," "an," and "the" include plural referents unless context clearly indicates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly indicates otherwise. Hence "comprising A or B" means including A, or B, or A and B. It is further to be understood that all base sizes or amino acid sizes, and all molecular weight or molecular mass values, given for nucleic acids or polypeptides are approximate, and are provided for description. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. Sequences associated with GenBank Accession Numbers are herein incorporated by reference as present in GenBank on April 25, 2016, unless otherwise noted. In case of conflict, the present specification, including explanations of terms, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

In order to facilitate review of the various embodiments of the disclosure, the following explanations of specific terms are provided:

Adjuvant: A substance or vehicle that non-specifically enhances the immune response to an antigen. Adjuvants can include a suspension of minerals (such as alum, aluminum hydroxide, or aluminum phosphate) on which antigen is adsorbed; or water-in-oil emulsion in which antigen solution is emulsified in oil (for example, Freund's incomplete adjuvant), sometimes with the inclusion of killed mycobacteria (Freund's complete adjuvant) to further enhance antigenicity. Immunostimulatory oligonucleotides (such as those including a CpG motif) can also be used as adjuvants (for example, see U.S. Patent Nos. 6,194,388; 6,207,646; 6,214,806; 6,218,371 ; 6,239,116; 6,339,068; 6,406,705; and 6,429,199). Adjuvants also include biological molecules, such as costimulatory molecules. Exemplary biological adjuvants include IL-2, RANTES, GM- CSF, TNF-a, IFN-γ, G-CSF, LFA-3, CD72, B7-1, B7-2, OX-40L and 41 BBL.

Antibody: An immunoglobulin molecule produced by B lymphoid cells with a specific amino acid sequence. Antibodies are evoked in humans or other animals by a specific antigen (immunogen). Antibodies are characterized by reacting specifically with the antigen in some demonstrable way, antibody and antigen each being defined in terms of the other. "Eliciting an antibody response" refers to the ability of an antigen or other molecule to induce the production of antibodies.

Antigen: A compound, composition, or substance that can stimulate the production of antibodies or a T-cell response in a subject, including compositions that are injected or absorbed into a subject. An antigen reacts with the products of specific humoral or cellular immunity, including those induced by heterologous immunogens. In one embodiment, an antigen is a Plasmodium antigen (such as a Plasmodium sexual stage surface protein, CSP, or an immunogenic portion thereof). In other examples, an antigen is a bacterial capsular polysaccharide (such as ViP).

Circumsporozoite protein (CSP): The circumsporozoite protein is a major malaria parasite surface protein during the sporogonic cycle. CSP covers the surface of Plasmodium sporozoites, which are transmitted from the mosquito salivary gland to host hepatocytes. It is highly immunogenic, and in endemic areas high antibody titers against this protein are observed in circulating blood. See, e.g. , Dame et al, Science 225:593-599, 1985; Zavala et al, Science 228: 1436-1440, 1985; International Pat. Publ. No. WO 2008/107370.

CSP sequences are publicly available. For example, GenBank accession numbers

XM_001351086 and XP_001351122 disclose P. falciparum CSP nucleic acid and amino acid sequences, respectively, both of which are incorporated herein by reference as present in GenBank on April 25, 2016. GenBank accession numbers XM_001616843and JX461259 disclose P. vivax CSP nucleic acid sequences and XP_001616893 and AGN05248 disclose P. vivax CSP amino acid sequences, all of which are incorporated herein by reference as present in GenBank on April 25, 2016. In some examples, CSP (or a portion thereof) includes the amino acid sequences provided in SEQ ID NOs: 9-12. Additional P. falciparum and P. vivax CSP nucleic acid and amino acid sequences can be identified by one of ordinary skill in the art. Orthologs of CSP are present in other Plasmodium species (such as P. ovale, P. malariae, and P. knowlesi) and can also be identified.

Conjugate: A compound formed by joining two or more compounds (such as two or more proteins or fragments of proteins, two or more polysaccharides, one or more proteins and one or more polysaccharides, or combinations thereof). The conjugates described herein are formed by co valently joining one or more proteins or a portion thereof and one or more polysaccharides. As used herein, the terms "linked," "joined," conjugated," or "attached" refer to covalent bond linkage of a polysaccharide to a protein or portion thereof. The covalent bond linkage can be direct or can be indirect, e.g., linked though a spacer molecule (a "linker"). In some examples, the components of a conjugate are joined by a non-peptide linker, such as a hydrazone linker, an amide linker, a thioether linker, or combinations of two or more thereof. In other examples, the components of a conjugate are joined by a peptide linker, such as a linker including about one to twelve peptide bonds.

Effective amount: A quantity of a specified agent sufficient to achieve a desired effect, such as an amount of an agent sufficient to inhibit or treat a disease without causing a substantial cytotoxic effect in a subject. For example, this may be the amount of a conjugate useful for eliciting an immune response in a subject, for example, for reducing infection by, symptoms of, or transmission of malaria and/or for reducing infection by or symptoms of typhoid fever.

Host: A cell or organism which harbors another organism or biological entity, usually a parasite (such as a malaria parasite). In one example, a host is a human or non-human primate that can be or is infected by the malaria parasite Plasmodium (such as P. falciparum, P. vivax, P. ovale, P. malariae, or P. knowlesi). The term "host" is used interchangeably with the term "subject" herein.

Immune response: A response of a cell of the immune system, such as a B-cell, T-cell, macrophage or polymorphonucleocyte, to a stimulus such as an antigen. An immune response can include any cell of the body involved in a host defense response, including for example, an epithelial cell that secretes an interferon or a cytokine. An immune response includes, but is not limited to, an innate immune response or inflammation. As used herein, a protective immune response refers to an immune response that protects a subject from infection (inhibits or prevents infection or inhibits or prevents the development of disease associated with infection).

Immunogen: A compound, composition, or substance which is capable, under appropriate conditions, of stimulating an immune response, such as the production of antibodies or a T-cell response in a subject, including compositions that are injected or absorbed into a subject. As used herein, an "immunogenic composition" is a composition comprising an immunogen.

Immunogenic conjugate or composition: A composition useful for stimulating or eliciting a specific immune response (or immunogenic response) in a subject. In some

embodiments, the immunogenic response is protective or provides protective immunity, in that it enables the subject to better resist infection or disease progression from the organism against which the immunogenic composition is directed. In other embodiments, the immunogenic response reduces or inhibits transmission of a disease-causing agents (such as Plasmodium parasites)

between subjects (such as between a mosquito and a host). One specific example of a type of immunogenic composition is a vaccine.

Inhibiting or treating a disease: "Inhibiting" refers to reducing or delaying (or even preventing) the full development of a disease, disorder or condition, for example, in a subject who is at risk for a disease. "Treatment" refers to a therapeutic intervention that ameliorates a sign or symptom of a disease or pathological condition after it has begun to develop. As used herein, the term "ameliorating," with reference to a disease, pathological condition or symptom, refers to any observable beneficial effect of the treatment. The beneficial effect can be evidenced, for example, by a delayed onset of clinical symptoms of the disease in a susceptible subject, a reduction in severity of some or all clinical symptoms of the disease, a slower progression of the disease, a reduction in the number of relapses of the disease, an improvement in the overall health or well- being of the subject, or by other parameters known to one of ordinary skill in the art that are

specific to the particular disease.

Isolated: An "isolated" biological component (such as a nucleic acid, protein or pathogen) has been substantially separated or purified away from other biological components (such as cell debris, or other proteins or nucleic acids). Biological components that have been "isolated" include those components purified by standard purification methods. The term also embraces recombinant nucleic acids, proteins or pathogens, as well as chemically synthesized nucleic acids or peptides.

Malaria: Malaria is a parasitic infection of humans and non-human primates by the

Plasmodium species P. falciparum, P. vivax, P. ovale, P. malariae, and P. knowlesi. Humans become infected following the bite of an infected anopheline mosquito, the host of the malarial parasite. Malaria also occasionally occurs in humans following a blood transfusion or subsequent to needle-sharing. Clinical manifestations of malarial infection which may occur include blackwater fever, cerebral malaria, respiratory failure, hepatic necrosis, and occlusion of myocardial capillaries. Additional Plasmodium species infect other hosts, such as rodents (P. berghei, P. chabaudi, P. vinckei, and P. yoelii), other mammals, birds, and reptiles.

P25: A family of cysteine-rich 25 kDa antigens that includes Plasmodium falciparum mosquito stage antigen Pfs25 and its ortholog in Plasmodium vivax, Pvs25 (see Kaslow et al, Nature 333:74-76, 1988; Malkin et al, Vaccine 23:3131-3138, 2005). P25 proteins are composed of four tandem epidermal growth factor-like domains and are expressed on zygotes and mature ookinete stages of parasites within mosquitoes. Because P25 is only expressed in the mosquito midgut and not in the vertebrate host, these proteins have not been under selective pressure by the host immune system and antigenic variation of P25 appears to be more limited than most vaccine candidates present in asexual blood stages. See, e.g. , U.S. Pat. No. 5,853,739; International Pat. Publ. No. WO 2006/124712.

P25 sequences are publicly available. For example, GenBank accession numbers

XM_001347551 and XP_001347587 disclose P. falciparum Pfs25 nucleic acid and amino acid sequences, respectively, both of which are incorporated herein by reference as present in GenBank on April 25, 2016. GenBank accession numbers XM_001608410 and XP_001608460 disclose P. vivax Pvs25 nucleic acid and amino acid sequences, respectively, both of which are incorporated herein by reference as present in GenBank on April 25, 2016. In particular examples, P25 proteins (or a portion thereof) include the amino acid sequences of SEQ ID NOs: 1-4. Additional P.

falciparum and P. vivax P25 nucleic acid and amino acid sequences can be identified by one of ordinary skill in the art. Orthologs of P25 are present in other Plasmodium species (such as P.

ovale, P. malariae, and P. knowlesi) and can also be identified.

P47: A sexual stage surface protein expressed by female Plasmodium gametes (van Schaijk et al, Mol. Biochem. Parasitol. 149:216-222, 2006). P47 sequence are publicly available. For example, GenBank accession numbers XM_001350146 and XP_001350182 disclose P. falciparum Pfs47 nucleic acid and amino acid sequences, respectively, both of which are incorporated herein by reference as present in GenBank on April 25, 2016. GenBank accession numbers

XM_001614197 and XP_001614247 disclose P. vivax Pvs47 nucleic acid and amino acid sequences, respectively, both of which are incorporated herein by reference as present in GenBank on April 25, 2016. In particular examples, a P47 protein (or a portion thereof) includes the amino acid sequences of SEQ ID NOs: 17 and 18. Additional P. falciparum and P. vivax P47 nucleic acid and amino acid sequences can be identified by one of ordinary skill in the art. Orthologs of P47 are present in other Plasmodium species (such as P. ovale, P. malariae, and P. knowlesi) and can also be identified.

P48/45: A sexual stage surface protein expressed by male and female Plasmodium gametes and containing two six cysteine (6-Cys) domains (van Dijk et al., Cell 104: 153-164, 2001).

Antibodies against Pfs48/45 have been shown to block or reduce transmission of the P. falciparum parasite (see, e.g., Outchkourov et al, Proc. Natl. Acad. Sci. USA 105:4301-4305, 2008).

P48/45 sequence are publicly available. For example, GenBank accession numbers

XM_001350145 and XP_001350181 disclose P. falciparum Pfs48/45 nucleic acid and amino acid sequences, respectively, both of which are incorporated herein by reference as present in GenBank on April 25, 2016. GenBank accession numbers XM_001614196 and XP_001614246 disclose P. vivax Pvs48/45 nucleic acid and amino acid sequences, respectively, both of which are incorporated herein by reference as present in GenBank on April 25, 2016. In particular examples, a P48/45 protein (or a portion thereof) includes the amino acid sequences of SEQ ID NOs: 13-16. Additional P. falciparum and P. vivax P48/45 nucleic acid and amino acid sequences can be identified by one of ordinary skill in the art. Orthologs of P48/45 are present in other Plasmodium species (such as P. ovale, P. malariae, and P. knowlesi) and can also be identified.

P230: A Plasmodium gametocyte surface antigen that is retained on the surface of gametes following emergence (see, e.g. , U.S. Pat. No. 5,733,772). Antibodies against Pfs230 have been shown to block or reduce transmission of the malaria parasite, (see, e.g. , Healer et al, Infect.

Immun. 65:3017-3023, 1997). P230 sequences are publicly available. For example, GenBank accession numbers XM_001349564 and XP_001349600 disclose P. falciparum Pfs230 nucleic acid and amino acid sequences, respectively, both of which are incorporated herein by reference as present in GenBank on April 25, 2016. GenBank accession numbers XM_001612970 and

XP_001613020 disclose P. vivax Pvs230 nucleic acid and amino acid sequences, respectively, both of which are incorporated herein by reference as present in GenBank on April 25, 2016. In

particular examples, P230 proteins (or a portion thereof) include the amino acid sequences of SEQ ID NOs: 5-8. Additional P. falciparum and P. vivax P230 nucleic acid and amino acid sequences can be identified by one of ordinary skill in the art. Orthologs of P230 are present in other

Plasmodium species (such as P. ovale, P. malariae, and P. knowlesi) and can also be identified.

Pharmaceutically acceptable carrier: The pharmaceutically acceptable carriers (vehicles) useful in this disclosure are conventional. Remington: The Science and Practice of Pharmacy, The University of the Sciences in Philadelphia, Editor, Lippincott, Williams, & Wilkins, Philadelphia, PA, 21st Edition (2005), describes compositions and formulations suitable for pharmaceutical delivery of one or more therapeutic compounds or molecules, such as one or more peptide conjugate, and additional pharmaceutical agents.

In general, the nature of the carrier will depend on the particular mode of administration being employed. For instance, parenteral formulations usually comprise injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a carrier. For solid compositions (for example, powder, pill, tablet, or capsule forms), conventional non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate. In addition to biologically-neutral carriers, pharmaceutical compositions to be administered can contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan

monolaurate. Purified: The term "purified" does not require absolute purity; rather, it is intended as a relative term. Thus, for example, a purified peptide, protein, nucleic acid, or polysaccharide is one that is isolated in whole or in part from naturally associated proteins and other contaminants. In certain embodiments, the term "substantially purified" refers to a peptide, protein, nucleic acid, or polysaccharide that has been isolated from a cell, cell culture medium, or other crude preparation and subjected to fractionation to remove various components of the initial preparation, such as proteins, cellular debris, and other components.

Recombinant: A recombinant nucleic acid, protein or parasite is one that has a sequence that is not naturally occurring or has a sequence that is made by an artificial combination of two otherwise separated segments of sequence. This artificial combination is often accomplished by chemical synthesis or by the artificial manipulation of isolated segments of nucleic acids, for example, by genetic engineering techniques.

Subject: Living multi-cellular vertebrate organisms, a category that includes both human and non-human mammals. Subjects include veterinary subjects, including livestock such as cows and sheep, rodents (such as mice and rats), and non-human primates, as well as humans.

Vi polysaccharide (ViP): A capsular polysaccharide of Salmonella enterica serovar typhi (Salmonella typhi) or Citrobacter (such as C. freundii). ViP is a linear homopolymer of poly- a(l→4)-GalpA, N-acetylated at position C-2 and O-acetylated at position C-3. ViP is a virulence factor of S. typhi that prevents antibody binding to the S. typhi O antigen and inhibits binding of C3 component of complement from binding to the surface of S. typhi.

III. Immunogenic Conjugates

Disclosed herein are conjugates including at least one Plasmodium protein (such as at least one of SEQ ID NOs: 1-18) or a portion thereof and at least one Vi capsular polysaccharide. In some embodiments, the conjugate includes a P25 protein or portion thereof (such as a Pfs25 protein or Pvs25 protein or portion thereof) conjugated to ViP. In some examples, the conjugate includes a Pfs25 protein including or consisting of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2 conjugated to ViP or a Pvs25 protein including or consisting of the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 4 (or amino acids 7-179 of SEQ ID NO: 4) conjugated to ViP. In other embodiments, the conjugate includes a P230 protein or portion thereof (such as a Pfs230 protein or Pvs230 protein or portion thereof) conjugated to ViP. In some examples, the conjugate includes a Pfs230 protein including or consisting of the amino acid sequence of SEQ ID NO: 5 or SEQ ID

NO: 6 conjugated to ViP or a Pvs230 protein including or consisting of the amino acid sequence of SEQ ID NO: 7 or SEQ ID NO: 8 (or amino acids 1-201 of SEQ ID NO: 8) conjugated to ViP. In still further embodiments, the conjugate includes a CSP protein or portion thereof (such as a PfCSP protein or PvCSP protein or portion thereof) conjugated to ViP. In some examples, the conjugate includes a PfCSP protein including or consisting of SEQ ID NO: 9 or SEQ ID NO: 10 conjugated to ViP or a PvCSP protein including or consisting of SEQ ID NO: 11 or SEQ ID NO: 12 conjugated to ViP. In additional embodiments, the conjugate includes a P48/45 protein (such as Pfs48/45 or Pvs48/45) or a portion thereof conjugated to ViP. In some examples, the conjugate includes a Pfs48/45 protein including or consisting of the amino acid sequence of SEQ ID NO: 13 or SEQ ID NO: 14 or a Pvs48/45 protein including or consisting of the amino acid sequence of SEQ ID NO: 15 or SEQ ID NO: 16. In further embodiments, the conjugate includes a P47 protein (such as Pfs47 or Pvs47) or a portion thereof conjugated to ViP. In some examples, the conjugate includes a Pfs47 protein including or consisting of the amino acid sequence of SEQ ID NO: 17 or a Pvs47 protein including or consisting of the amino acid sequence of SEQ ID NO: 18. In particular examples, linkage of the malaria protein to ViP results in enhancement in immunogenicity against the malaria protein, ViP, or both, for example, compared to the unconjugated malaria protein or ViP or compared to conjugates of the malaria protein or ViP to a different protein or

polysaccharide.

In some embodiments, the at least one Plasmodium protein or portion thereof and the ViP are conjugated using carbodiimide-mediated conjugation with a dihydrazide as the linker. In one non-limiting example, the Plasmodium protein and ViP conjugate has the structure ViP- CONHNHCO(CH2)nCONHNHOC-Protein, where n is 1, 2, 3, 4, or 5. In one specific example, n=4 (adipic acid dihydrazide linker). However, as discussed below, additional conjugation methods, which produce different linkages are also contemplated herein.

Both the protein and polysaccharide components of the disclosed conjugates contain multiple reactive groups per molecule. Thus, an activated polysaccharide molecule can react with and form more than one linkage to more than one protein molecule. Likewise, an activated protein molecule can react with and form more than one linkage to more than one polysaccharide molecule. Therefore, the conjugate product is a mixture of various structures. For example, a single linkage can be present, or 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 75, 100, or more linkages can be present. The average number of linkages between a polysaccharide and a protein can be adjusted, for example, by adjusting the molar ratio of EDC to carboxyl group such that the desired number of protein or ViP carboxylic acid groups are modified (for example, with ADH). Generally, an average of 1-30 linkages (such as about 5-25, 10-20, or 15-30 linkages) is present, for example, to reduce interfering with the ability of the protein and/or polysaccharide to stimulate the immune system. However, in certain embodiments more than 30 linkages can be tolerated or even desirable. Thus, in some examples, the disclosed conjugates include one or more (such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, or more) proteins per ViP in the conjugate. In non-limiting examples, the conjugates may have one of the following structures:

P - PS or P - PS - P or P - PS - P

I

P wherein P is a malaria protein or portion thereof; and PS is ViP.

In some embodiments, the ratio of the protein component of the conjugate to the total conjugate (w/w) is less than 1 (such as less than 0.9, less than 0.8, less than 0.7, less than 0.6, less than 0.5, less than 0.4, less than 0.3, less than 0.2, or less than 0.1). For example, the ratio of protein to total conjugate (w/w) is in some examples, about 0.1-0.8, about 0.2-0.8, about 0.1-0.5, about 0.2-0.4, about 0.3-0.6, about 0.4-0.5, or about 0.5-0.7. In particular examples, some conjugates have a ratio of Pfs25 (such as SEQ ID NO: 2) to total conjugate (w/w) of about 0.3-0.6. In some embodiments, the conjugate size is about 0.2-20 mDa, such as about 0.2-0.9, 0.4-10, 0.5- 15, 0.8-5, 1-8, 5-12, 2-20, or 10-15 mDa.

A. Plasmodium Proteins

The disclosed conjugates include at least one Plasmodium protein or a potion thereof, such as a P25 protein, a P230 protein, a CSP protein, a P48/45 protein, a P47 protein, or a portion of any one thereof.

In some embodiments, the disclosed conjugates include a malaria P25 protein or portion thereof. In some examples, the P25 antigen utilized in the conjugates is a portion of the P25 protein such as a processed form of the P25 protein (for example, a P25 protein lacking the signal sequence) and/or a form of the P25 protein lacking the hydrophobic C-terminal domain or a portion thereof. Exemplary P25 proteins or portions thereof included in the disclosed conjugates include or consist of the amino acid sequences set forth below.

Pfs25 full length (SEQ ID NO: 1 ; XP_001347587)

MNKLYSLFLFLFIQLSIKYNNAKVTVDTVCKRGFLIQMSGHLECKCENDLVLVNEETCEEK VLKCDEKTVNKPCGDFSKCIKIDGNPVSYACKCNLGYDMVNNVCIPNECKNVTCGNGKCI LDTSNPVKTGVCSCNIGKVPNVQDQNKCSKDGETKCSLKCLKENETCKAVDGIYKCDCKD GFIIDNESSICTAFSAYNILNLSIMFILFSVCFFIM

Pfs25 partial construct (SEQ ID NO: 2)

KVTVDTVCKRGFLIQMSGHLECKCENDLVLVNEETCEEKVLKCDEKTVNKPCGDFSKCIKI DGNPVSYACKCNLGYDMVNNVCIPNECKQVTCGNGKCILDTSNPVKTGVCSCNIGKVPN VQDQNKCSKDGETKCSLKCLKEQETCKAVDGIYKCDCKDGFIIDQESSICT

Pvs25 full length (SEQ ID NO: 3; XP_001608460)

MNSYYSLFVFFLVQIALKYSKAAVTVDTICKNGQLVQMSNHFKCMCNEGLVHLSENTCEE KNECKKETLGKACGEFGQCIENPDPAQVNMYKCGCIEGYTLKEDTCVLDVCQYKNCGES GECIVEYLSEIQSAGCSCAIGKVPNPEDEKKCTKTGETACQLKCNTDNEVCKNVEGVYKC QCMEGFTFDKEKNVCLSYSVFNILNYSLFFIILLVLSYVI Pvs25 partial construct (SEQ ID NO: 4)

EAEAYVAVTVDTICKNGQLVQMSNHFKCMCNEGLVHLSENTCEEKNECKKETLGKACGE FGQCIENPDPAQVNMYKCGCIEGYTLKEDTCVLDVCQYKNCGESGECIVEYLSEIQSAGCS CAIGKVPNPEDEKKCTKTGETACQLKCNTDNEVCKNVEGVYKCQCMEGFTFDKEKNVCL GPHHHHHH

In other embodiments, the disclosed conjugates include a malaria P230 protein or portion thereof. In some examples, the P230 antigen utilized in the conjugates includes one or more domains of the P230 protein, such as all or a portion of domain I of the P230 protein (see e.g., Gerloff et al, Proc. Natl. Acad. Set USA 102:13598-13603, 2005). Exemplary P230 proteins or portions thereof included in the disclosed conjugates include or consist of the amino acid sequences set forth below.

Pfs230 full-length (SEQ ID NO: 5; XP_001349600)

MKKIITLKNLFLIILVYIFSEKKDLRCNVIKGNNIKDDEDKRFHLFYYSHNLFKTPETKEKKN KKECFYKNGGIYNLSKEIRMRKDTSVKIKQRTCPFHKEGSSFEMGSKNITCFYPIVGKKERK TLDTIIIKKN VTNDH V VS SDMHS NVQEKNMILIRNID KENKNDIQN VEEKIQRDT YENKD Y ESDDTLIEWFDDNTNEENFLLTFLKRCLMKIFSSPKRKKTVVQKKHKSNFFINSSLKYIYMY LTPSDSFNLVRRNRNLDEEDMSPRDNFVIDDEEEEEEEEEEEEEEEEEEEEEEEEEYDDYVY EESGDETEEQLQEEHQEEVGAESSEESFNDEDEDSVEARDGDMIRVDEYYEDQDGDTYDS TIKNEDVDEEVGEEVGEEVGEEVGEEVGEEVGEEVGEEVGEEVGEEEGEEVGEGVGEEVG EEEGEEVGEEEGEYVDEKERQGEIYPFGDEEEKDEGGESFTYEKSEVDKTDLFKFIEGGEG DDVYKVDGSKVLLDDDTISRVSKKHTARDGEYGEYGEAVEDGENVIKIIRSVLQSGALPSV GVDELDKIDLSYETTESGDTAVSEDSYDKYASNNTNKEYVCDFTDQLKPTESGPKVKKCE VKVNEPLIKVKIICPLKGSVEKLYDNIEYVPKKSPYVVLTKEETKLKEKLLSKLIYGLLISPT VNEKENNFKEGVIEFTLPPVVHKATVFYFICDNSKTEDDNKKGNRGIVEVYVEPYGNKING CAFLDEDEEEEKYGNQIEEDEHNEKIKMKTFFTQNIYKKNNIYPCYMKLYSGDIGGILFPKN IKSTTCFEEMIPYNKEIKWNKENKSLGNLVNNSVVYNKEMNAKYFNVQYVHIPTSYKDTL NLFCSIILKEEESNLISTSYLVYVSINEELNFSLFDFYESFVPIKKTIQVAQKNVNNKEHDYTC DFTDKLDKTVPSTANGKKLFICRKHLKEFDTFTLKCNVNKTQYPNIEIFPKTLKDKKEVLK LDLDIQYQMFSKFFKFNTQNAKYLNLYPYYLIFPFNHIGKKELKNNPTYKNHKDVKYFEQS SVLSPLSSADSLGKLLNFLDTQETVCLTEKIRYLNLSINELGSDNNTFSVTFQVPPYIDIKEPF YFMFGCNNNKGEGNIGI VELLIS KQEEKIKGCNFHES KLD YFNENIS SDTHECTLH A YENDII GFNCLETTHPNEVEVEVEDAEIYLQPENCFNNVYKGLNSVDITTILKNAQTYNINNKKTPTF LKIPPYNLLEDVEISCQCTIKQVVKKIKVIITKNDTVLLKREVQSESTLDDKIYKCEHENFINP RVNKTFDENVEYTCNIKIENFFNYIQIFCPAKDLGIYKNIQMYYDIVKPTRVPQFKKFNNEE LHKLIPNSEMLHKTKEMLILYNEEKVDLLHFYVFLPIYIKDIYEFNIVCDNSKTMWKNQLG GKVIYHITVSKREQKVKGCSFDNEHAHMFSYNKTNVKNCIIDAKPKDLIGFVCPSGTLKLT NCFKDAIVHTNLTNINGILYLKNNLANFTYKHQFNYMEIPALMDNDISFKCICVDLKKKKY NVKSPLGPKVLRALYKKLNIKFDNYVTGTDQNKYLMTYMDLHLSHKRNYLKELFHDLGK KKPADTDANPESIIESLSINESNESGPFPTGDVDAEHLILEGYDTWESLYDEQLEEVIYNDIES LELKDIEQYVLQVNLKAPKLMMSAQIHNNRHVCDFSKNNLIVPESLKKKEELGGNPVNIH CYALLKPLDTLYVKCPTSKDNYEAAKVNISENDNEYELQVISLIEKRFHNFETLESKKPGN GDVVVHNGVVDTGPVLDNSTFEKYFKNIKIKPDKFFEKVINEYDDTEEEKDLESILPGAIVS PMKVLKKKDPFTSYAAFVVPPIVPKDLHFKVECNNTEYKDENQYISGYNGIIHIDISNSNRK INGCDFSTNNSSILTSSVKLVNGETKNCEININNNEVFGIICDNETNLDPEKCFHEIYSKDNK TVKKFREVIPNIDIFSLHNSNKKKVAYAKVPLDYINKLLFSCSCKTSHTNTIGTMKVTLNKD EKEEEDFKTAQGIKHNNVHLCNFFDNPELTFDNNKIVLCKIDAELFSEVIIQLPIFGTKNVEE GVQNEEYKKFSLKPSLVFDDNNNDIKVIGKEKNEVSISLALKGVYGNRIFTFDKNGKKGEG ISFFIPPIKQDTDLKFIINETIDNSNIKQRGLIYIFVRKNVSENSFKLCDFTTGSTSLMELNSQV KEKKCTVKIKKGDIFGLKCPKGFAIFPQACFSNVLLEYYKSDYEDSEHINYYIHKDKKYNL KPKDVIELMDENFRELQNIQQYTGISNITDVLHFKNFNLGNLPLNFKNHYSTAYAKVPDTF NSIINFSCNCYNPEKHVYGTMQVESDNRNFDNIKKNENVIKNFLLPNIEKYALLLDDEERQ KKIKQQQEEEQQEQILKDQDDRLSRHDDYNKNHTYILYDSNEHICDYEKNESLISTLPNDT KKIQKSICKINAKALDVVTIKCPHTKNFTPKDYFPNSSLITNDKKIVITFDKKNFVTYIDPTK KTFSLKDIYIQSFYGVSLDHLNQIKKIHEEWDDVHLFYPPHNVLHNVVLNNHIVNLSSALE GVLFMKSKVTGDETATKKNTTLPTDGVSSILIPPYVKEDITFHLFCGKSTTKKPNKKNTSLA LIHIHISSNRNIIHGCDFLYLENQTNDAISNNNNNSYSIFTHNKNTENNLICDISLIPKTVIGIK CPNKKLNPQTCFDEVYYVKQEDVPSKTITADKYNTFSKDKIGNILKNAISINNPDEKDNTYT YLILPEKFEEELIDTKKVLACTCDNKYIIHMKIEKSTMDKIKIDEKKTIGKDICKYDVTTKVA TCEIIDTIDSSVLKEHHTVHYSITLSRWDKLIIKYPTNEKTHFENFFVNPFNLKDKVLYNYNK PINIEHILPGAITTDIYDTRTKIKQYILRIPPYVHKDIHFSLEFNNSLSLTKQNQNIIYGNVAKIF IHINQGYKEIHGCDFTGKYSHLFTYSKKPLPNDDDICNVTIGNNTFSGFACLSHFELKPNNC FSSVYDYNEANKVKKLFDLSTKVELDHIKQNTSGYTLSYIIFNKESTKLKFSCTCSSNYSNY TIRITFDPNYIIPEPQSRAIIKYVDLQDKNFAKYLRKL

Pfs230 domain I construct (SEQ ID NO: 6)

SVLQSGALPSVGVDELDKIDLSYETTESGDTAVSEDSYDKYASQNTNKEYVCDFTDQLKPT ESGPKVKKCEVKVNEPLIKVKIICPLKGSVEKLYDNIEYVPKKSPYVVLTKEETKLKEKLLS KLIYGLLISPTVNEKENNFKEGVIEFTLPPVVHKATVFYFICDNSKTEDDNKKGNRGIVEVY VEPYGNKING

Pvs230 full-length (SEQ ID NO: 7; XP_001613020)

MGKLAKLKRVLLLLPLVRIALEQSHGAQAGGTSGVLGGGRGGSGAHRGSYEGIHQVIHQV IHRGRCEGIHQVIHRGRCDGGHHVIHRGRCDGGHHVIHRGMCEEIHRVVHRVIHREIYEGT HFSRSGRPYSGRSDSTGGRYNRGDAISMGRYPLHGQAKIGDSDMVITPRRVARHLAEEDD GDDEDGDVDDDDGNDDGEGTHTQPQVKGMDDEDLEGPPGEDDCFVLPEAGASDGVFDK VDEAFETTIKGDGNVLQASDPEVETFASSNTNKEYVCDFVKHITMKEASKKVVICEMKIQE PLVKVKILCPTKYADVIKYGSMEFFPKKAPYVTLVNANEGSEEKKESLKEKRLAALIHGVII TPEVNEKENNFEKGVIEFVLPPVLKEKKKLYYICDNGKSADGVSNHGDRGVAAITIEPYGQ SVKGCNYTGKGKHFFSYDYEEGADMKHPCVFQLNAGEIGGIMFPEGTKSTSCFDRMVPHS ANEKWDKKKKSLSQVINGAVVYNNDFKAKYFTVKYFSIPKSFREATHLMCHIQLTDGDK KHMVYISINQELNLSVFDLYEAFANVKKVIQIAKQTDDKKEYTCDFTDQLDKKAGESEKK VIICRKRLTEFDSFNMKCAINKAKYANLEMIPKMLKEEKKKKKNVLKVQLDVQYELFKKY FHFHDKYGRYLPGYPSFFSFPLNRVAKLELKKNSLFKNHKDSSYFDEVASPSDGFVKLLSF LDAQDTVILNEQVSDLTISTEQSTEDLFTLQLQIPPYITTNEPLYFLIGCNNTKDGGNLGIVEL VISKNEHLVKGCNFSDGKMEHFTNNVGSTGEKCNITAFPNDVVGFNCSKTVLPPSDEAEDS ATSNVVIEPEGCFTQVYSSSQKTDIVSILPGVLLYTSRAKSSKAFIKIPPVLLKDKFTFSCKCK IESVQNEMEVNVRRERESKEAVIAELKQEATTTGKEKDEGGMIYSCANKTLIEPKLEKLDD RYVKNTCHVDAKEMHSYVELFCPSRDLSIHQNINLYFNTVKPTKVESPKVLNQVELEKLIP HAEMLHKTRSILPLQSCTSGKEMVDISHVYLFFPYYVKEEQEFNILCDNSNTVFEGKRGGL LTYQVKVPKRSKKIKGCDFSASKSEAFLNESTANDCLVDAMPKDVIGFVCPPGTVKITSCF KEAVVGDNLINISEALELTDNMANYTHSHRFSYLEIPSVVSKDLSFKCICVQLKKDYALTSP PSAKLLEVIYQKMSIKKGEGYNKGEPKNKFLMSSLHLHLSHKKRQIVSIFQKVDAKNPSEE DANPEQLFEEVAGQEVYDDVTGATCSMSQIRDNHISGDDYSVFTSVSDSLLEETITRDISSL NASDFKVYTLKVNLKAPKLLKPKKLSDNEYLCDFSKKSLIVPEPLTEESPPDIHCYSALKPL DTLYVKCPTEKAAYEVAKGKTGEEDEEEAKGIIALLEGDPADAGDPSAPPLDDASFVKYFE KVSMKPSGFFKNVLNEDGDKEEEIERVLPGAANTSMIVLKKKDPFTSYAAVIIPPSVSRNTF FKVQCNNDEYKEGAKNGGYKGVIHLDISRSEKKTIGCDFTAETSSIFTKGVLLPSGQSKECE VEATKNDIFGVRCSNESTFDPTNCFHEIYDKAGGKKKIKELIPDVKVFTLPNSKTKVAYGKI PLDYVNKINFSCSCTKADDSTKGTIKVIVNKEEASLEDMKLTDAVKHGQVNFCNFLDDEA LMFEKNPEKIVQCKVDADLFSEVVVILPKMGSTSGEAEHKNLSVTPPLTEGEDIKVLTDEK TQVSLTTALKGVYGSRVFSFEKNSKKGIGLSFFIPPTFENKNFKLLINQSGLLSSNKQRGIVFI IVRKNSEPGSVKLCDFTSPENTLAELDHTNNEKVCSVRIAKGDLFGITCPKGFFLYPEACFS NVTLDYYKGELQTVGGEAEVEGYDADINYAERTQTNMRIKDLLHLLGDEDTEMEDLQKF SEFSNLPEMLNFETFHLGSMHLDFKKAYTSAYARVPTKFRSAIKFSCSCYNPQKKVFGTMD VETEYASSEEEPPIRADPFVRNKLLPLRSKISIDADEVEPIVEPDAVDDLEATGSEGCKEEGP CGNMLSQGISYTCDFSQESLFTLIEGKLQRNTCTVDARALDVVTVKCPAIANHVHAEGIAD EAVETEKVIITIDDEQFVTYQEEKTKKKTFSLKEIYDKEYYGMTSEEVVKLRKVDAGWEK HQVYYPKRVLNDVVVANKVTKLEEALPGVLLLQSKVNDELKTTELIADGVVRFIVPPYVR NDFQFHLFCGKSSETKPQGKNTSLGVIRVNVSANRKELQGCDFVNEKEDPHLVIFTNKRET ASNTVCPISLIPNTVVGINCPSGKLLPDSCFHETYYVAESEAVTAANQVGSLPKEKITNVIKG AIPLSNFANKKNTYTYLILPKDMKSVATLNKHFYCTCNEGIIKMKVNTIYLKREKDNRVDK ESCSYDSVKNVTTCNVVEFMESLAQKDNKTNHYSVQLAKWDKLILKYPTNEKENYEKVY INPINIKDKVLFRKVPTHIEDLLPGAITTNKLDSRTKIIEYTLRVPPYVSKEVHFSIEFNNSLTS KMVNYNVAYGGVVEIFIHVREGYTEISGCDFTGQYNHLFSHNFAPNLKESKTCSVVFGNN SYAGFACPTKFEIVPHNCFASVYDRNDGEKVKKLVDLSEEAEYDFVQYNSRGVSLSYLTF KRDTKTHSVSCKCVSPQVTHTINVTFEPDADHSLPKTRIRIRYFDLSRASFSSHLRGG

Pvs230 domain I construct (SEQ ID NO: 8)

VLPEAGASDGVFDKVDEAFETTIKGDGNVLQASDPEVETFASSNTNKEYVCDFVKHITMK EASKKVVICEMKIQEPLVKVKILCPTKYADVIKYGSMEFFPKKAPYVTLVNANEGSEEKKE SLKEKRLAALIHGVIITPEVNEKENNFEKGVIEFVLPPVLKEKKKLYYIDNGKSADGVSNHG DRGVAAITIEPYGQSVKGAGHHHHHH

In additional embodiments, the disclosed conjugates include a malaria CSP protein or portion thereof. In some examples, the CSP antigen utilized in the conjugates is a portion of the CSP protein, for example, a portion including all or part of the central repeat region. Exemplary CSP proteins or portions thereof included in the disclosed conjugates include or consist of the amino acid sequences set forth below. PfCSP full-length (SEQ ID NO: 9; XP_001351122)

MMRKLAILSVSSFLFVEALFQEYQCYGSSSNTRVLNELNYDNAGTNLYNELEMNYYGKQE NWYSLKKNSRSLGENDDGNNEDNEKLRKPKHKKLKQPADGNPDPNANPNVDPNANPNV DPNANPNVDPNANPNANPNANPNANPNANPNANPNANPNANPNANPNANPNANPNANP NANPNANPNANPNANPNANPNVDPNANPNANPNANPNANPNANPNANPNANPNANPNA NPN ANPN ANPN ANPN ANPN ANPN ANPN ANPN ANPN ANPNKNNQGNGQGHNMPNDPNRN VDENANANSAVKNNNNEEPSDKHIKEYLNKIQNSLSTEWSPCSVTCGNGIQVRIKPGSANK PKDELDYANDIEKKICKMEKCSSVFNVVNSSIGLIMVLSFLFLN

PfCSP partial construct (SEQ ID NO: 10)

ENDDGNNEDNEKLRKPKHKKLKQPADGNPDPNANPNVDPNANPNVDPNANPNVDPNAN PNANPNANPNANPNANPNANPNANPNANPNANPNANPNANPNANPNANPNANPNANPN ANPNANPNVDPNANPNANPNANPNANPNANPNANPNANPNANPNANPNANPNANPNAN PNANPNANPNANPNANPNANPNANPNKNNQGNGQGHNMPNDPNRNVDENANANSAVK NNNNEEPSDKHIKEYLNKIQNSLSTEWSPCSVTCGNGIQVRIKPGSANKPKDELDYANDIEK KICKMEK

PvCSP full-length (SEQ ID NO: 11; AGN05248)

MKNFILLAVSSILLVDLFPTHCGHNVDLSKAINLNGVGFNNVDASSLGAAHVGQSASRGR GLGENPDDEEGDAKKKKDGKKAEPKNPRENKLKQPEDGAGNQPGANGAGNQPGANGAG NQPGANGAGNQPGANGAGNQPGANGAGNQPGANGAGNQPGANGAGNQPGANGADDQP GANGAGNQPGANGAGNQPGANGAGNQPGANGAGDQPGANGAGNQPGANGAGDQPGA NGAGNQPGANGAGNQPGANGAGNQPGANGAGNQPGANGAGNQPGANGAGGQAAGGN AANKKAGDAGAGQGQNNEGANAPNEKSVKEYLDKVRATVGTEWTPCSVTCGVGVRVR RRVNAANKKPEDLTLNDLETDVCTMDKCAGIFNVVSNSLGLVILLVLALFN PvCSP partial construct (SEQ ID NO: 12)

DAKKKKDGKKAEPKNPRENKLKQPEDGAGNQPGANGAGNQPGANGAGNQPGANGAGN QPGANGAGNQPGANGAGNQPGANGAGNQPGANGAGNQPGANGADDQPGANGAGNQPG ANGAGNQPGANGAGNQPGANGAGDQPGANGAGNQPGANGAGDQPGANGAGNQPGAN GAGNQPGANGAGNQPGANGAGNQPGANGAGNQPGANGAGGQAAGGNAANKKAGDAG AGQGQNNEGANAPNEKSVKEYLDKVRATVGTEWTPCSVTCGVGVRVRRRVNAANKKPE DLTLNDLETDVCTMDK In additional embodiments, the disclosed conjugates include a malaria P48/45 protein or portion thereof. In some examples, the P48/45 antigen utilized in the conjugates is a portion of the P48/45 protein, such as a 6-Cys domain of the P48/45 protein. Exemplary P48/45 proteins or portions thereof included in the disclosed conjugates include or consist of the amino acid sequences set forth below.

Pfs48/45 full-length (SEQ ID NO: 13; XP_001350181)

MMLYISAKKAQVAFILYIVLVLRIISGNNDFCKPSSLNSEISGFIGYKCNFSNEGVHNLKPD MRERRSIFCTIHSYFIYDKIRLIIPKKSSSPEFKILPEKCFQKVYTDYENRVETDISELGLIEYEI EENDTNPNYNERTITISPFSPKDIEFFCFCDNTEKVISSIEGRSAMVHVRVLKYPHNILFTNLT NDLFTYLPKTYNESNFVSNVLEVELNDGELFVLACELINKKCFQEGKEKALYKSNKIIYHK NLTIFKAPFYVTS KD VNTECTCKFKNNN YKI VLKPKYEKKVIHGCNFS SN VS S KHTFTDS L DISLVDDSAHISCNVHLSEPKYNHLVGLNCPGDIIPDCFFQVYQPESEELEPSNIVYLDSQINI GDIEYYEDAEGDDKIKLFGIVGSIPKTTSFTCICKKDKKSAYMTVTIDSAYYGFLAKTFIFLI VAILLYI

Pfs48/45 partial construct (SEQ ID NO: 14)

KPKYEKKVIHGCNFSSNVSSKHTFTDSLDISLVDDSAHISCNVHLSEPKYNHLVGLNCPGDI IPDCFFQVYQPESEELEPSNIVYLDSQINIGDIEYYEDAEGDDKIKLFGIVGSIPKTTSFTCICK KDKKSAYMTVTIDSA

Pvs48/45 full-length (SEQ ID NO: 15; XP_001614246)

MLKRQLANLLLVLSLLRGITHTQMAKGEVKYVPPEELNKDVSGFFGFKCNFSSKGVHNLE

PILTEKRSLVCSIYSYFIYDKIKLTIPKKIPGSKFKMLPEKCFQTVYTNYEKRTEEKIENMGLV

EYEVKEDDSNSEYTEKILTISPFNTKDVEFFCICDNSENVISNVKGRVALVQVNVLKYPHKI TSINLTKEPYSYLPNQVDKTSFKSHKLDLELQDGELVVLACEKVDDKCFKKGKDTSPLSLY KSKKIVYHKNLSIFKAPVYVKSADVTAECSCNVDSTIYTLSLKPVYTKKLIHGCNFSSDKST HNFTNHVDMAELGENAQITCSIELVDTSYNHLIGMSCPGEVLPECFFQVYQRESPELEPSKI VYLDAQLNIGNVEYFEDSKGENIVKIFGLVGSIPKTTSFTCICRKGKKIGYMSVKIAAGYFG FLAKIFILLIVLLLLYF

Pvs48/45 partial construct (SEQ ID NO: 16)

KPVYTKKLIHGCNFSSDKSTHNFTNHVDMAELGENAQITCSIELVDTSYNHLIGMSCPGEV LPECFFQVYQRESPELEPSKIVYLDAQLNIGNVEYFEDSKGENIVKIFGLVGSIPKTTSFTCIC RKGKKIGYMSVKIAAG

In additional embodiments, the disclosed conjugates include a malaria P47 protein or portion thereof. In some examples, the P47 antigen utilized in the conjugates is a portion of the P47 protein. Exemplary P47 proteins included in the disclosed conjugates include or consist of the amino acid sequences set forth below.

Pfs47 full-length (SEQ ID NO: 17; XP_001350182)

MCMGRMISIINIILFYFFLWVKKSISELLSSTQYVCDFYFNPLTNVKPTVVGSSEIYEEVGCTI NNPTLGDHIVLICPKKNNGDFSNIEIVPTNCFESHLYSAYKNDSSAYHLEKLDIDKKYAINSS FSDFYLKILVIPNEYKSHKTIYCRCDNSKTEKNIPGQDKILKGKLGLVKIILRNQYNNIIELEK TKPIIHNKKDTYKYDIKLKESDILMFYMKEETIVESGNCEEILNTKINLLSNNNVVIKMPSIFI NNINCMLSSQDQNNEKNYINLKADKTKHIDGCDFTKPKGKGIYKNGFIINDIPNEEERICTV HLWNKKNQTIAGIKCPYKLIPPYCFKHVLYEKEIDSQKTYKTFLLSDVLDTPNIEYYGNNK EGMYMLALPTKPEKTNKIRCICEQGGKKAVMELHIASTSTKYISMFLIFFLIVIFYMYVSI

Pvs47 full-length (SEQ ID NO: 18; XP_001614247)

MKLLTFAAATYGFLLKECLNSFIFPTKHLCDFALNPHSSIKPVLKEASGKDEEVWCSVHNP SLTDYVAMVCPKKKGGDYTELETVPANCFTKHLYSPYDSEENEKDMELLELDPKLSFNRT FNDFVLKVLVIPGYYKHNKTIYCRCDNRKTKKGEDQEKIEEGKVGLVKIVLNKKEKKPRGI DFTETDELEQTDIVQNGNDKLVKVKENETIHFKFNSNQKLEIKECENVINMKYGFLQEHVL NFRFPAVFLSSENCTITVIESAKTPVRIIIKTQKTENIDGCDFTKPSGEGDYQDGFALEELKSN EKICTIHIGSSKKKISAGIKCPYKLTPTYCFRHVLYEKDVNGVKSYHPFLLTDVLGTLDVEF YSNAQEGSYIIGLPTNPQKYSVVRCVCEHNGKAGIMELRIASSSGWAFLSLTLLLLLIALLS AC In some embodiments, the Plasmodium proteins (or portions thereof) of use in the disclosed conjugates have a sequence at least 95%, 96%, 97%, 98%, or 99%, such as 100% identical to the amino acid sequence set forth in one of SEQ ID NOs: 1-18. In other examples, the conjugates include a Plasmodium protein with an amino acid sequence at least 95% identical to (such as at least 96%, 97%, 98%, 99%, or 100% identical to) amino acids 7-179 of SEQ ID NO: 4 or amino acids 1-201 of SEQ ID NO: 8. Exemplary sequences with the indicated level of sequence identity can be obtained using computer programs that are readily available on the internet and the amino acid sequences set forth herein. In one example, the polypeptide retains a function of the

Plasmodium protein, such as generating an immune response and/or reducing or inhibiting

Plasmodium transmission (for example, from human to mosquito or from mosquito to human).

Minor modifications of a malaria protein primary amino acid sequence may result in peptides which have substantially equivalent activity (such as immunogenic activity) as compared to the unmodified counterpart polypeptide described herein. Such modifications may be deliberate, as by site-directed mutagenesis, or may be spontaneous. All of the polypeptides produced by these modifications are included herein. In some non-limiting examples, the malaria proteins disclosed herein include one or more conservative amino acid substitutions, for example 1-10 conservative substitutions, 2-5 conservative substitutions, 4-9 conservative substitutions, such as 1, 2, 5 or 10 conservative substitutions). A table of exemplary conservative substitutions is provided as Table 1. Substitutions of the amino acids sequence shown in SEQ ID NOs: 1-18 can be made based on this table. One of ordinary skill in the art can identify additional conservative amino acid substitutions that can be utilized.

Table 1. Exemplary conservative amino acid substitutions

Original Residue Conservative Substitution(s)

Ala Ser

Arg Lys

Asn Gin, His

Asp Glu

Cys Ser

Gin Asn

Glu Asp

His Asn; Gin

He Leu, Val Original Residue Conservative Substitution(s)

Leu He; Val

Lys Arg; Gin; Glu

Met Leu; lie

Phe Met; Leu; Tyr

Ser Thr

Thr Ser

Trp Tyr

Tyr Trp; Phe

Val lie; Leu

In some examples, the malaria protein or portion thereof does not include an N-terminal methionine; however, an N-terminal methionine can be present, for example as a result of expression in a bacterial, yeast, or mammalian system. In additional examples, the malaria protein or portion thereof includes a tag, for example an N-terminal or C-terminal sequence or component that facilitates protein production and/or purification. Examples of such tags include the His6-tag (e.g., Roche Applied Science, Mannheim, Germany) or streptavidin binding peptide (e.g., Sigma- Aldrich, St. Louis, MO). In some examples, the tag is removed from the malaria protein prior to conjugation; however, in other examples, the tag is included in the conjugate.

The malaria proteins or portions thereof (such as SEQ ID NOs: 1-18) disclosed herein can be chemically synthesized by standard methods, or can be produced recombinantly. They can also be isolated by methods including preparative chromatography and immunological separations. Polypeptides can also be produced using molecular genetic techniques, such as by inserting a nucleic acid encoding a malaria protein or portion thereof into an expression vector, introducing the expression vector into a host cell, and isolating the polypeptide. In particular examples, the disclosed proteins (such as SEQ ID NOs: 1-18) are produced by expression in E. coli, Pischia pastoris, or insect cell lines.

Suitable vectors for expression of a protein in a host cell include bacterial, viral, insect, yeast, and mammalian expression vectors. In some examples, a nucleic acid encoding a malaria protein or portion thereof (such as SEQ ID NOs: 1-18) is incorporated into a vector, such as an autonomously replicating plasmid or virus, or into the genomic DNA of a prokaryote or eukaryote, or exists as a separate molecule (such as a cDNA) in a cell independent of other sequences. One of ordinary skill in the art can identify suitable bacterial, viral, insect, yeast, or mammalian vectors for expression of the disclosed proteins. In some examples, the vector includes one or more expression control sequences operatively linked to the malaria protein-encoding nucleic acid. An expression control sequence operatively linked to a malaria protein-encoding nucleic acid is linked (for example, ligated) such that expression of the coding sequence is achieved under conditions compatible with the expression control sequences. Expression control sequences include, but are not limited to, appropriate promoters, enhancers, transcription terminators, a start codon in front of a protein-encoding gene, splicing signal for introns, maintenance of the correct reading frame to permit proper translation of mRNA, and stop codons.

DNA sequences encoding a malaria protein or portion thereof (such as those described herein) can be expressed in vitro by DNA transfer into a suitable host cell. The cell may be prokaryotic or eukaryotic. Methods of stable transfer, meaning that the foreign DNA is continuously maintained in the host, are known in the art. Host cells can include microbial, yeast, insect, and mammalian host cells. Methods of expressing DNA sequences having eukaryotic or viral sequences in prokaryotes are well known in the art. Non-limiting examples of suitable host cells include bacteria, archea, insect, fungi (for example, yeast), mycobacterium (such as M.

smegmatis), plant, and animal cells (for example, mammalian cells, such as human). Exemplary cells of use include E. coli, Bacillus subtilis, Saccharomyces cerevisiae, Salmonella typhimurium, Pichia pastoris, Sf9 cells, C129 cells, 293 cells, Neurospora, and immortalized mammalian cell lines. Examples of commonly used mammalian host cell lines are VERO cells, HeLa cells, CHO cells, WI38 cells, BHK cells, and COS cells, although other cell lines may be used, such as cells designed to provide higher expression, desirable glycosylation patterns, or other features.

Transformation of a host cell with recombinant DNA can be carried out by conventional techniques as are well known to those skilled in the art. Where the host cell is prokaryotic, such as, but not limited to, E. coli, competent cells which are capable of DNA uptake can be prepared from cells harvested after exponential growth phase and subsequently treated by the CaCh method using procedures well known in the art. Alternatively, MgCh or RbCl can be used. Transformation can also be performed after forming a protoplast of the host cell if desired, or by electroporation. When the host cell is eukaryotic, such methods of transfection of DNA as calcium phosphate

coprecipitates, conventional mechanical procedures such as microinjection, electroporation, insertion of a plasmid encased in liposomes, or virus vectors can be used.

B. Vi Polysaccharide

The disclosed conjugates include Vi capsular polysaccharide, for example from S. typhi or Citrobacter. S. typhi ViP is a linear homopolymer of poly-a(l→4)-GalpA, N-acetylated at position C-2 and O-acetylated at position C-3. Citrobacter produces a Vi capsular polysaccharide that is indistinguishable from S. typhi ViP. In additional examples, a polysaccharide that is antigenically similar to ViP can be prepared from fruit pectin (e.g. , Szu et al. , Vaccine 32:2618-2622, 2014) and is utilized in the disclosed conjugates.

Methods of purifying bacterial capsular polysaccharides, such as ViP include purification from cultures of S. typhi or Citrobacter (such as C. freundii). ViP for use in the disclosed conjugates can be purified from cultures of S. typhi or Citrobacter, for example using methods including precipitation of the ViP and subsequent purification (for example concentrating the ViP, diafiltering or dialyzing to change buffer(s), and ultrafiltration). An exemplary method of purifying ViP is provided in Example 1, below; however, additional methods for purifying ViP known in the art and can also be used (e.g. , Jang et al., J. Biotechnol. 135:71-77, 2008; Kothari et al , Vaccine 31:4714-4719, 2013).

C. Production of Conjugates

The proteins and polysaccharides described herein can be conjugated (or linked) directly or indirectly to one another by suitable covalent linkages. In some examples, the linkage is directly between the protein and polysaccharide (for example, direct conjugation between carboxyl groups of the polysaccharide and amino groups of the protein). In other examples, the conjugate includes a linking group between the protein and polysaccharide, such as a non-peptide linker (for example, a linker including a hydrazone group, an amide group, or a thioether group) or a peptide linker. Non- limiting examples of linking groups include adipic acid dihydrazide (ADH), N-succinimidyl-4- formylbenzoate (SFB), and N-succinimidyl-3-(2-pyridyldithio)-propionate (SPDP) or diamines separated by -(CH2)n-, wherein n is 1-8 (for example, 1,3-diaminopropane, 1 ,4-diaminobutane, 1,5- diaminopentane, or 1,6-diaminohexane).

In some examples, at least one protein or polysaccharide is reacted with hydrazine, carbohydrazide, hydrazine chloride, a dihydrazide, or a mixture thereof under conditions sufficient to produce a hydrazide-activated protein or hydrazide-activated polysaccharide. The hydrazide- activated protein is reacted with a polysaccharide or the hydrazide-activated polysaccharide is reacted with a protein to produce a protein-polysaccharide conjugate. In some examples, the conjugation reaction is catalyzed by a carbodiimide, such as l-ethyl-3-(3- dimethylaminopropyl)carbodiimide (EDC), l-Cyclohexyl-3-(2-morpholinoethyl) carbodiimide (CMC), NN'-dicyclohexyl carbodiimide (DCC), diisopropyl carbodiimide (DIC), or Ν,Ν'- carbonyldiimidazole (CDC). In one non-limiting example, the carbodiimide is EDC.

In particular examples, a dihydrazide or a suitable analog, such as a member of the group of compounds of the formula:

Figure imgf000027_0001

where n is one, two, three, four, or five, is used to produce the conjugates described herein. In particular examples, the dihydrazide is adipic acid dihydrazide (ADH).

In some embodiments, a carboxylic acid moiety of a protein or polysaccharide is reacted with a hydrazide group of the linking agent to yield an amide linkage. In some examples, the reaction can be performed in two steps, the first step involving derivatization of the protein or the polysaccharide with the dihydrazide followed by a second step involving further reaction of the derivatized protein or polysaccharide with a protein or polysaccharide (e.g. , FIGS. 1 A and IB). Thus, in one non-limiting example, a malaria protein, such as Pfs25, Pvs25, Pfs230, Pvs230, PfCSP, PvCSP, Pfs48/45, Pvs48/45, Pfs47, Pvs47, or a portion of any one thereof (such as any one of SEQ ID NOs: 1-18 or amino acids 7-179 of SEQ ID NO: 4 or amino acids 1-201 of SEQ ID NO: 8) is derivatized with a dihydrazide (such as ADH) in the presence of a carbodiimide (such as EDC). The protein-dihydrazide derivative is then reacted with ViP in the presence of a

carbodiimide (such as EDC) to produce a protein- ViP conjugate. In an alternative, non-limiting example, ViP is derivatized with a dihydrazide (such as ADH) in the presence of a carbodiimide (such as EDC). The ViP-dihydrazide derivative is then reacted with a malaria protein such as Pfs25, Pvs25, Pfs230, Pvs230, PfCSP, PvCSP, Pfs48/45, Pvs48/45, Pfs47, Pvs47, or a portion of any one thereof (such as any one of SEQ ID NOs: 1-18 or amino acids 7-179 of SEQ ID NO: 4 or amino acids 1-201 of SEQ ID NO: 8) in the presence of a carbodiimide (such as EDC) to produce a protein- ViP conjugate. In examples where the ViP is derivatized with a dihydrazide, reaction conditions may be adjusted to reduce or inhibit ViP- ViP crosslinking, for example, by increasing the ratio of ADH to EDC in the ViP modification step (for example, 2: 1 or more, such as 4: 1, 5: 1, 10: 1, or more).

In other examples, a dihydrazide (such as ADH) and SFB (or a suitable analog) having the formula:

Figure imgf000027_0002

where Z is independently hydrogen, halogen (for example, fluorine, chlorine, bromine, or iodine), or lower alkyl (for example, Ci-C6 alkyl), is used as a linking agent to yield a hydrazone linkage. The reaction is performed in two steps. In the first step, an amine moiety of a protein (such as Pfs25, Pvs25, Pfs230, Pvs230, PfCSP, PvCSP, Pfs48/45, Pvs48/45, Pfs47, Pvs47, or a portion of any one thereof) is reacted with SFB to yield derivatized protein or polypeptide and separately a carboxylic acid moiety of ViP is reacted with ADH to yield derivatized ViP. In the second step, the two derivatives are reacted to yield a conjugate including a hydrazone linking group.

In additional examples, SPDP (or a suitable analog) having the formula:

Figure imgf000028_0001

where q is one, two, three, four, five, six, seven, or eight, is utilized as a linking agent to yield a thioether linkage. The reaction is preferably performed in two steps. The first step involves producing thiolated ViP (for example, by reacting ViP with cystamine) to yield thiolated ViP and separately reacting an amine moiety of a protein (such as Pfs25, Pvs25, Pfs230, Pvs230, PfCSP, PvCSP, Pfs48/45, Pvs48/45, Pfs47, Pvs47, or a portion of any one thereof) with SPDP to yield a derivatized protein. In the second step, the two derivatives are reacted to yield a conjugate including a thioether linking group (see, e.g., Szu et al, J. Exp. Med. 166:1510-1524, 1987).

In additional examples, carboxylic acids on ViP are converted into active esters of succinimide, which can react with amines on the protein. Active esters can be made by reaction of carboxyl group with N-hydroxysuccinimide (NHS) in the presence of a carbodiimide (such as EDC). The NHS ester-modified ViP is reacted with the protein (such as Pfs25, Pvs25, Pfs230, Pvs230, PfCSP, PvCSP, Pfs48/45, Pvs48/45, Pfs47, Pvs47, or a portion of any one thereof) to form amide bonds.

After conjugation, the conjugate can be purified by any suitable method. Purification is employed to remove unreacted polysaccharide, protein, or small molecule reaction byproducts. Purification methods include dialysis, ultrafiltration, size exclusion chromatography, density gradient centrifugation, hydrophobic interaction chromatography, ammonium sulfate fractionation, and the like. The conjugate can be concentrated or diluted, transferred to a different buffer, or processed into any suitable form for use in pharmaceutical compositions, as desired. In some examples, the conjugates are purified by dialysis followed by filtration (such as through a 0.2 μιη membrane).

IV. Methods of Eliciting an Immune Response or Inhibiting or Treating Infection

The conjugates disclosed herein and/or prepared according to the methods disclosed herein are administered to a subject (such as a human subject) in an effective amount (for example, a therapeutically effective amount) to elicit an immune response in the subject (for example, an immune response to a malaria protein and/or ViP). In some embodiments, the disclosed conjugates are capable of treating, inhibiting, or in some examples, even preventing infection or disease (for example malaria infection and/or typhoid infection) in a subject. In other examples, the disclosed conjugates are capable of reducing, inhibiting, or even preventing transmission of a malaria parasite (including, but not limited to P. falciparum and/or P. vivax). The disclosed conjugates in some examples provide one or more advantages over conventional vaccines (such as individual malaria proteins, ViP, or other conjugate vaccines), including enhanced immunogenicity of the antigens, potential reduction in the amount of antigen used, less frequent booster immunizations, improved efficacy, preferential stimulation of immunity, or potential targeting of immune responses.

In some embodiments, disclosed herein are methods of eliciting an immune response in a subject to a malaria protein (or Plasmodium) and/or ViP, including administering to the subject a conjugate including at least one malaria protein (such as Pfs25, Pvs25, Pfs230, Pvs230, PfCSP, PvCSP, Pfs48/45, Pvs48/45, Pfs47, or Pvs47) or a portion of any one thereof linked to ViP (such as any one of SEQ ID NOs: 1-18 linked to ViP). As discussed below, the disclosed conjugates may be administered in the form of a pharmaceutical composition (such as a composition including one or more pharmaceutically acceptable carriers, adjuvants, and/or other components).

In some examples, the methods include administering one or more doses of a single conjugate (including, but not limited to Pfs25-ViP, Pvs25-ViP, Pfs230-ViP, Pvs230-ViP, PfCSP- ViP, PvCSP- ViP, Pfs48/45-ViP, Pvs48/45-ViP, Pfs47-ViP, or Pvs47-ViP) to the subject. In other examples, the methods include administering one or more doses of two or more conjugates (such as 2, 3, 4, 5, or more) selected from Pfs25-ViP, Pvs25-ViP, Pfs230-ViP, Pvs230-ViP, PfCSP- ViP, PvCSP- ViP, Pfs48/45-ViP, Pvs48/45-ViP, Pfs47-ViP, and Pvs47-ViP to the subject. When two or more conjugates are administered to a subject, the conjugates may be administered simultaneously (in the same composition), substantially simultaneously (for example, separate administration of each conjugate within a short period of time, such as within 1 hour or less of one another), or sequentially (for example, separate administration of each conjugate separated by more than 1 hour).

In some examples, the method further includes selecting a subject in need of enhanced immunity to Plasmodium (such as P. falciparum, P. vivax, P. ovale, P. malariae, and/or P.

knowlesi) and/or S. typhi. Hosts in need of enhanced immunity to Plasmodium include subjects who are at risk of malaria infection, subjects who have been exposed to Plasmodium parasites, and subjects who are infected with Plasmodium parasites. Residents of, or travelers to, countries or regions where malaria is endemic (such as Africa, Central and South America, the island of Hispaniola (Haiti and the Dominican Republic), Asia (including the Indian subcontinent, Southeast Asia and the Middle East), Eastern Europe, and the South Pacific) are at risk of contracting malaria, such as malaria caused by infection with Plasmodium. Additional factors that contribute to risk of infection with Plasmodium include the characteristics of the area, time of year, presence of malaria in the area, exposure to mosquito bites, and lack of preventive measures (such as anti-malarial drugs and/or insect repellant). Hosts in need of enhanced immunity to S. typhi include subjects who are residents of, or travelers to, countries or regions where typhoid fever is prevalent, including Central and South America, Africa, and Asia (including the Indian subcontinent, Southeast Asia, and the Middle East).

In some examples, one or more of the immunogenic conjugates disclosed herein are included in a pharmaceutical composition with one or more of adjuvants, diluents, excipients, carriers, preservatives and/or other pharmaceutically acceptable substances. Formulation of the immunogenic conjugates into pharmaceutical compositions can be accomplished using methods known in the art. The term "pharmaceutically acceptable" is used to refer to a non-toxic material that is compatible with a biological system such as a cell, cell culture, tissue, or organism. In some examples, the disclosed compositions are sterile and contain either a therapeutically or

prophylactically effective amount of one or more of the disclosed conjugates in a unit of weight or volume suitable for administration to a subject. The pharmaceutically acceptable carriers (vehicles) useful in this disclosure are conventional. Remington: The Science and Practice of Pharmacy, The University of the Sciences in Philadelphia, Editor, Lippincott, Williams, & Wilkins, Philadelphia, PA, 21st Edition (2005), describes compositions and formulations suitable for pharmaceutical delivery of one or more therapeutic compounds or molecules. The characteristics of the carrier depend on the route of administration. Pharmaceutically acceptable carriers include diluents, fillers, salts, buffers, stabilizers, solubilizers, preservatives, and other materials which are well known in the art.

In some examples, the disclosed compositions contain one or more adjuvants. Suitable adjuvants include, for example, aluminum adjuvants (such as aluminum hydroxide (e.g. , alum) or aluminum phosphate, for example, ALHYDROGEL® adjuvant), Freund's Adjuvant (complete or incomplete), BAY, 3(N,N,-dimethylaminoethane)-carbamyl cholesterol (DC-Chol), poly[di(sodium carboxylatephoneoxy)phosphazene] (PCPP), monophosphoryl lipid A (such as 3 de-O-acylated monophosphoryl lipid A (3D-MPL) or 3 '-O-desacyl-4' -monophosphoryl lipid A (MPL)), CpG, Quillaja saponaria 21 (QS-21), cholera toxin, formyl methionyl peptide, Vibrio cholera 01 proteoliposomes, AS01 (a liquid suspension of liposomes including 3'-0-desacyl-4'- monophosphoryl lipid A and QS-21, GlaxoSmithKline Vaccines), or toll-like receptor 4 agonists (for example, GLA-SE or GLA-LSQ).

The disclosed immunogenic conjugates, conjugates made by the disclosed methods, and/or pharmaceutical compositions including the conjugates are administered to a subject by any suitable route, including but not limited to parenteral, intradermal, transmembranal, transdermal (including topical), intramuscular, intraperitoneal, intravenous, intra-arterial, intralesional, subcutaneous, oral, and intranasal (e.g., inhalation) routes of administration. In some non-limiting examples, the conjugates are administered to a subject intramuscularly, subcutaneously, or intradermally.

Immunogenic conjugates can be administered by bolus injection or by continuous infusion, as well as by localized administration. In some examples, the conjugate is administered in a

pharmaceutically acceptable carrier (e.g. , a vehicle).

The dosage of immunogenic conjugate(s) to be administered to a subject and the regime of administration can be determined in accordance with standard techniques well known to those of ordinary skill in the pharmaceutical and veterinary arts, taking into consideration such factors as the intended use, particular antigen(s), the adjuvant (if present), the age, sex, weight, species, general condition, prior illness and/or treatments of the subject, and the route of administration. Suitable doses and immunization protocols can be determined by one of skill in the art. For example, preliminary doses can be determined according to animal tests, and the scaling of dosages for human administration is performed according to art-accepted practices such as standard dosing trials. For example, the therapeutically effective dose can be estimated initially from serum antibody level testing. The dosage depends on the specific activity of the conjugate and can be readily determined by routine experimentation.

In practicing immunization protocols for inhibition, treatment, and/or prevention of malaria and/or typhoid fever, an effective amount of one or more of the conjugates or a composition including one or more of the conjugates is administered to a subject. In some examples, an effective amount is the total amount of the conjugate(s) or other active component that is sufficient to show a meaningful benefit to the subject, such as immune response, treatment, inhibition, prevention, or amelioration of malaria and/or typhoid fever or an increase in rate of treatment, prevention, or amelioration of such conditions. An effective amount includes the amount of an individual therapeutic agent (such as an individual immunogenic conjugate disclosed herein), a combination of therapeutic agents (such as two or more of the disclosed immunogenic conjugates), or a combination of one or more of the disclosed immunogenic conjugates and other agents. A combination can be administered to a subject simultaneously, substantially simultaneously, or sequentially. In particular examples, a subject is administered an amount of therapeutic agent or composition in an amount and for a time to treat, inhibit, or even prevent an infection, such as an infection with S. typhi or Plasmodium (such as P. falciparum or P. vivax)..

In some examples, the amount of the disclosed conjugate administered to a subject is from about 1 μg/kg to about 100 mg/kg body weight such as from about 0.5 g/kg to 10 mg/kg, about 10 (J-g/kg to about 25 mg/kg, about 1 mg/kg to about 50 mg/kg, about 5 mg/kg to about 75 mg/kg or about 10 mg/kg to 100 mg/kg (for example, 0.5 μg/kg, 1 μg/kg, 5 μg/kg, 10 μg/kg, 25 μg/kg, 50 μg/kg, 75 μg/kg, 100 μg/kg, 250 μg/kg, 500 μg/kg, 1 mg/kg, 5 mg/kg, 10 mg/kg, 25 mg/kg, 50 mg/kg, 75 mg/kg, or 100 mg/kg). In other examples, the amount of the conjugate administered to the subject is from about 1 μg to about 10 g, for example about 10 μg to 1 mg, about 100 μg to 2 mg, about 500 μg to 5 mg, about 1 mg to 25 mg, about 10 mg to about 75 mg, about 50 mg to 100 mg, about 100 mg to 500 mg, about 250 mg to 1 g, about 500 mg to 5 g, or about 1 g to 10 g (such as about 10 μg, 25 μg, 50 μg, 100 μg, 250 μg, 500 μg, 750 μg, 1 mg, 2.5 mg, 5 mg, 10 mg, 25 mg, 50 mg, 75 mg, 100 mg, 250 mg, 500 mg, 750 mg, 1 g, 2.5 g, 5 g, 7.5 g, or 10 g). In some examples, the amount of the conjugate administered to the subject is about 10-500 μg, such as about 20 -200 μg, about 50-100 μg, about 75-250 μg, or about 200-500 μg (for example, about 10 μg, about 20 μg, about 30 μg, about 40 μg, about 50 μg, about 60 μg, about 80 μg, about 100 μg, about 150 μg, about 200 μg, about 250 μg, about 300 μg, about 400 μg, or about 500 μg).

In some embodiments, one or more of the disclosed immunogenic conjugates or a composition including one or more of the conjugates is administered as a single dose or in a series including one or more boosters. In some examples, the subject is administered a single dose. In other examples, the subject is administered a single dose, and at least one booster dose is administered up to 1 week, 2 weeks, 1 month, 2 months, 3 months, 6 months, 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, or more later. More than one booster dose (such as 2, 3, 4, 5, or more booster doses) can be administered if necessary, as determined by one of ordinary skill in the art.

In some examples, the disclosed immunogenic conjugates are formulated for parenteral, subcutaneous, intradermal, intramuscular, intraperitoneal, or intravenous administration, injectable administration, sustained release from implants, or administration by eye drops. Suitable forms for such administration include sterile suspensions and emulsions. Such formulations include admixtures with a suitable carrier, diluent, or excipient such as sterile water, physiological saline, glucose, and the like. The immunogenic conjugates can also be lyophilized. The immunogenic conjugates or formulations thereof can contain auxiliary substances such as wetting or emulsifying agents, pH buffering agents, gelling or viscosity enhancing additives, preservatives, colors, and the like, depending upon the route of administration and the preparation desired. In some examples, such preparations include complexing agents, metal ions, polymeric compounds such as polyacetic acid, polyglycolic acid, hydrogels, dextran, and the like, liposomes, microemulsions, micelles, unilamellar or multilamellar vesicles, erythrocyte ghosts or spheroblasts. Suitable lipids for liposomal formulation include, without limitation, monoglycerides, diglycerides, sulfatides, lysolecithin, phospholipids, saponin, bile acids, and the like. The presence of such additional components can influence the physical state, solubility, stability, rate of in vivo release, and rate of in vivo clearance, and are thus chosen according to the intended application, such that the characteristics of the carrier are tailored to the selected route of administration.

In other examples, the disclosed immunogenic conjugates are formulated into liquid preparations for example, for oral, nasal, anal, rectal, buccal, vaginal, peroral, intragastric, mucosal, perlingual, alveolar, gingival, olfactory, or respiratory mucosa administration. Suitable forms for such administration include suspensions, syrups, and elixirs.

In particular embodiments, the immunogenic conjugates are provided as liquid suspensions or as freeze-dried products. Suitable liquid preparations include, for example, isotonic aqueous solutions, suspensions, emulsions, or viscous compositions that are buffered to a selected pH.

Transdermal preparations include lotions, gels, sprays, ointments or other suitable techniques. If nasal or respiratory (mucosal) administration is desired (e.g., aerosol inhalation or insufflation), compositions can be in a form and dispensed by a squeeze spray dispenser, pump dispenser or aerosol dispenser. Aerosols are usually under pressure by means of a hydrocarbon. Pump dispensers can dispense a metered dose or a dose having a particular particle size.

When in the form of solutions, suspensions, or gels, formulations of the conjugate typically contain a major amount of water (for example, purified water) in addition to the active ingredient. Minor amounts of other ingredients such as pH adjusters, emulsifiers, dispersing agents, buffering agents, preservatives, wetting agents, gelling agents, colors, and the like optionally are present.

In specific examples, the compositions are isotonic with the blood or other body fluid of the subject. The isotonicity of the compositions can be attained using sodium tartrate, propylene glycol, or other inorganic or organic solutes. Sodium chloride is particularly preferred. Buffering agents can be employed, such as acetic acid and salts, citric acid and salts, boric acid and salts, and phosphoric acid and salts. Parenteral vehicles may include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's or fixed oils. Intravenous vehicles may include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like.

Viscosity of the compositions can be maintained at the selected level using a

pharmaceutically acceptable thickening agent. One suitable thickening agent is methylcellulose, because it is readily and economically available and is easy to work with. Other suitable thickening agents include, for example, xanthan gum, carboxymethyl cellulose, hydroxypropyl cellulose, carbomer, and the like. The concentration of the thickener can depend upon the agent selected and is used in an amount that can achieve the selected viscosity. Viscous compositions are normally prepared from solutions by the addition of such thickening agents.

In some examples, a pharmaceutically acceptable preservative is included in the

composition to increase the shelf life of the compositions. Benzyl alcohol is one exemplary preservative, although a variety of preservatives including, for example, parabens, thimerosal, chlorobutanol, or benzalkonium chloride can also be employed. A suitable concentration of the preservative is from 0.02% to 2% based on the total weight, though one of ordinary skill in the art can select additional suitable preservative concentrations based on factors such as the preservative, the particular composition, and the intended use and storage condition.

When the conjugate is administered by intravenous, cutaneous, subcutaneous, or other injection, the composition is in the form of a pyrogen-free, parenterally acceptable aqueous solution. The preparation of parenterally acceptable solutions with suitable pH, isotonicity, stability, and the like, is within the ordinary skill in the art. In one example, a pharmaceutical composition for injection contains an isotonic vehicle such as Sodium Chloride Injection, Ringer's Injection, Dextrose Injection, Dextrose and Sodium Chloride Injection, Lactated Ringer's Injection, or other vehicles as are known in the art. The pharmaceutical compositions can also contain stabilizers, preservatives, buffers, antioxidants, or other additives known to those of ordinary skill in the art.

The duration of the injection can vary depending upon various factors, and can comprise a single injection administered over the course of a few seconds or less, to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours or more of continuous intravenous administration.

The conjugate can be administered topically, systematically, or locally, via a liquid or gel, or as an implant or device.

In some examples, the disclosed conjugates are administered in combination with various vaccines either currently being used or in development, whether intended for human or non-human subjects. Examples of vaccines for human subjects and directed to infectious diseases include

Salmonella paratyphi vaccines, nontyphoidal Salmonella (NTS) vaccines, combined diphtheria and tetanus toxoids vaccine; pertussis whole cell vaccine; inactivated influenza vaccine; 23-valent pneumococcal vaccine; live measles vaccine; live mumps vaccine; live rubella vaccine; Bacille Calmette- Guerin I (BCG) tuberculosis vaccine; hepatitis A vaccine; hepatitis B vaccine; hepatitis C vaccine; rabies vaccine (e.g., human diploid cell vaccine); inactivated polio vaccine; meningococcal polysaccharide vaccine (e.g., Menomune®, Sanofi Pasteur); quadrivalent meningococcal conjugate vaccine (e.g., Menactra® (Sanofi Pasteur) or Menveo® (Novartis)); yellow fever live virus vaccine; typhoid killed whole cell vaccine; cholera vaccine; Japanese B encephalitis killed virus vaccine; adenovirus vaccine; cytomegalovirus vaccine; rotavirus vaccine; varicella vaccine; anthrax vaccine; small pox vaccine; and other commercially available and experimental vaccines.

In some embodiments, the disclosed methods further include administering to the subject one or more agents for treating malaria. In some examples, the therapeutic agent is artemisinin or a derivative thereof (such as artesunate, dihydroartemisinin, or artemether) or an artemisinin-based combination therapy (such as artemether-lumefantrine), atovaquone-proguanil, chloroquine, primaquine, mefloquine, quinine (alone or with doxycycline, tetracycline, or clindamycin), or a combination of two or more thereof. Appropriate treatment options can be selected by a skilled clinician, for example, based on the Plasmodium species prevalent in the geographic area of the subject (or of the subject at the time of exposure), known drug-resistance of the Plasmodium prevalent in the area, age and overall health status of the subject, and other factors. In additional embodiments, the disclosed methods further include administering to the subject one or more agents for treating typhoid fever, for example, one or more antibiotics (such as ciprofloxacin or ceftriaxone).

The conjugates can be provided to an administering physician or other health care professional in the form of a kit. The kit is a package which houses one or more containers which contain the immunogenic conjugate(s) and instructions for administering the composition to a subject. The kit can optionally also contain one or more other therapeutic agents. The kit can optionally contain one or more diagnostic tools and instructions for use. For example, a composition containing two or more of the disclosed immunogenic conjugates or other vaccines can be included, or separate pharmaceutical compositions containing different conjugates, vaccines, or therapeutic agents. The kit can also contain separate doses of the immunogenic conjugate for serial or sequential administration. The kit can contain suitable delivery devices, e.g., syringes, inhalation devices, and the like, along with instructions for administrating the therapeutic agents. The kit can optionally contain instructions for storage, reconstitution (if applicable), and administration of any or all therapeutic agents included. The kits can include a plurality of containers reflecting the number of administrations to be given to a subject. If the kit contains a first and second container, then a plurality of these can be present. The following examples are provided to illustrate certain particular features and/or embodiments. These examples should not be construed to limit the disclosure to the particular features or embodiments described. EXAMPLES

Example 1

Synthesis of Pfs25-Vi Polysaccharide Conjugates

This example describes synthesis of Pfs25-Vi polysaccharide conjugates by two different approaches.

Chemical conjugation of a portion of malaria Pfs25 protein (SEQ ID NO: 2) to ViP was carried out by two different methods, both including a sequential two-step modification and coupling (FIGS. 1A and IB). In one case, following the method by Kossaczka et al. (Inf. Immun. 65:2088-2093, 1997), Pfs25 was modified by reaction with l-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and adipic acid dihydrazide (ADH) to introduce 3-6 molecules of ADH onto Pfs25, which were used for coupling with ViP in the second step. In the second strategy, ViP was modified in the initial step to convert 4-30% of its carboxylic acid groups modified with ADH by varying the molar ratio of EDC and ADH. ADH-modified ViP was then used to couple Pfs25 in the second step. The two methods resulted in Pfs25-ViP conjugates, but with different molecular weights.

ViP was purified from Salmonella enterica typhi isolate number C6524 strain obtained from a patient in Kolkata India by the National Institute of Cholera and Enteric Diseases (NICED). Isolate C6524 was cultivated in a bioreactor to maximize Vi production and inactivated with formalin (Jang et al, J. Biotechnol. 135:71-77, 2008). ViP was released from the cell to the culture supernatant. ViP was clarified from the culture supernatant using a 0.45 μιη Hydrosart® crossflow microfiltration cassette (Sartorius AG, Goettingen, Germany) and concentrated and diafiltered against 1 M NaCl and concentrated and diafiltrated to change buffer to pure water using a

Hydrosart® 30 kDa cutoff ultrafiltration cassette (Sartorius AG). Vi was precipitated by mixing with cetyltrimethlammonium bromide (CTAB, or Cetavlon) at a final concentration of 0.5% for 2 hours. This Cetavlon-treated 1st precipitate was washed with 20% ethanol and ethanol

concentration was increased to 60% to dissolve the ViP precipitate in the 60% ethanol. 5 M NaCl was added to make 1 M NaCl in the dissolved ViP in 60% ethanol and to increase the ethanol concentration to 75%, then the dissolved ViP was precipitated again and settled overnight. The 2nd precipitate in 75% ethanol was washed with absolute ethanol and dissolved in water. Remaining impurities were removed by ammonium sulfate precipitation then filtered through a 0.2 μιη filter. Finally remaining ammonium sulfate was removed by 100 kDa Hydrosart® ultrafiltration cassette (Sartorius) diafiltration and sterilized using 0.2 μιη Sartopore® 2 sterile filter (Sartorius) (Kothari et al, Vaccine 31 :4714-4719, 2013).

Method 1: Modification of ViP with ADH and conjugation to Pfs25

ViP (4 mg/ml) was dissolved in 80 mM 2-(N-morpholino) ethanesulfonic acid (MES) buffer, pH 5.6 and stirred overnight at 4°C. Two different derivatizations of ViP with ADH were prepared, low and high modification. To generate low modification ViP, polysaccharide was reacted with 20 mM ADH and 40 mM EDC at room temperature. For preparing high modification ViP, polysaccharide was reacted with 200 mM ADH and 40 mM EDC. Both reactions were carried out by addition of ADH to ViP solution followed by addition of EDC after 5 minutes with stirring at room temperature. After 1 hour stirring, the mixture was dialyzed using 6-8 kDa dialysis membrane against PBS with 3 buffer changes at 4°C.

Conjugation of Pfs25 to derivatized ViP (VIPAH) was performed by addition of EDC, followed by VIPAH to the Pfs25 with a final concentration of Pfs25, VIPAH, and EDC at 0.85 mg/ml, lmg /ml, and 2 mg/ml, respectively, in 80 mM MES buffer, maintained at pH 5.6-5.8. After 3 hours of stirring at 4°C, unconjugated Pfs25 and residual EDC were removed by 100 KDa dialysis against 2 times of 800 ml 0.5M saline pH 7.4 and then 2 times 800 ml, PBS pH 7.4 at 4°C, changing the buffer at 3 hour intervals. Resulting samples were sterile filtered using 0.2 μιη filtration. Concentration of ViP in the conjugate was determined by Hesterin assay using acetylcholine as the reference and concentration of Pfs25 was determined using A280 using extinction coefficient of 0.312 for Abs 0.1%. Molecular mass was determined by Size Exclusion Chromatography (TSKgel G5000PWXL; 7.8 mm x 30 mm, Tosoh Bioscience, King of Prussia, PA) with Multi- Angle Light Scattering analysis (SEC MALS) with 12 KDa dextran normalization. Method 2: Modification of Pfs25 with ADH and conjugation to ViP

Derivatization of Pfs25 was performed at room temperature in MES buffer at pH 5.6. Pfs25 in MES buffer was mixed with ADH followed by EDC to a final concentration of 0.04 mM, 20 mM, 20 mM, and 80 mM of Pfs25, ADH, EDC, and MES buffer, respectively. After 1 hour of reaction at room temperature, the reaction mixture was dialyzed (using dialysis membrane with 100 KDa MWCO) against PBS, pH 7.4 at 4°C with change of buffer at 3 hour intervals. After dialysis, ADH modified Pfs25 (named Pfs25AH) was concentrated to greater than 2 mg/ml using 5 KDa MWCO membrane spin filter. The hydrazide (ADH) concentration of the derivatized Pfs25 was determined by colorimetric TNBS assay.

ADH modified Pfs25 was conjugated to ViP as follows. ViP at 1 mg/ml concentration in 80 mM MES buffer at pH 5.6 was reacted with 2 mg/ml of EDC for 5 minutes, followed by ADH modified Pfs25 (0.5 mg/ml) for 3 hours at room temperature with continuous stirring. The pH of the reaction mixture was maintained between 5.6 and 5.8 during the reaction. Resulting conjugates were dialyzed using 100 KDa MWCO dialysis membrane against two changes of 0.5 M saline followed by two changes of PBS, pH 7.2. Conjugates were sterile filtered using 0.2 μιη sterile filters. The sterile conjugates were assayed for ViP content, Pfs25 concentration, and molecular mass by Hesterin assay, A280, and SEC MALS, respectively.

Example 2

Characterization of Pfs25-ViP Conjugates

This example describes the characterization of Pfs25-ViP conjugates prepared in Example

1.

Conjugate samples, Pfs25, and ViP were loaded to Tskgel G5000PWXL (7.8 mm X 30 mm) column with IX PBS, 0.02 % sodium azide, pH 7.4 at 0.5 ml /min in an Agilent HPLC system and the molecular weight was analyzed using Multi-angle light scattering (MALS), HELEOS II (Wyatt technology). Molecular weight of the conjugates as determined by SEC-MALS is shown in Table 2.

Table 2. Conjugate characteristics

Figure imgf000038_0001
Western blot analysis was performed on 4-20 % Tris-Glycine gel with Benchmark pre- stained protein ladder. 25 μg of Pfs25 in the conjugate samples were loaded to the gel and run at 30 mA constant current. The gel was transferred to a nitrocellulose membrane using iBlot gel transfer devise (Invitrogen). The blot was incubated with primary antibody (Anti-Pfs25, 4B7) and followed by secondary antibody labelled with alkaline phosphatase (Goat anti-mouse IgG, KPL). The blot was developed with BCIP/NBT phosphatase substrate (KPL) (FIG. 2). Example 3

Immunogenicity of Pfs25-ViP Conjugates

This example describes evaluation of immunogenicity of the Pfs25-ViP conjugates in mice.

Eleven groups of 10 CD-I out-bred mice were immunized with formulations consisting of Pfs25-ViP conjugates and controls (Table 3). Conjugates were tested with or without adsorption to ALHYDROGEL® adjuvant. Animals were immunized by intramuscular injection of 50 of the formulations on days 0 and 28. Bleeds were performed on Day 42, 70, and 90, and the collected sera were analyzed for antibody titer against Pfs25 and ViP by ELISA technique.

Table 3. Immunogenicity study groups of Pfs25-ViP conjugates and controls

Figure imgf000039_0001

Groups 1-6 were formulated in PBS without adjuvant, and contained 50 μg/mL of Pfs25M content. Groups 7-11 were formulated adsorbed on alum, prepared by diluting stock formulations containing 50 μg/mL of Pfs25M content adsorbed on 900 μg/mL of

ALHYDROGEL® (450 μg/mL of aluminum).

Sera from mice immunized with various conjugates and controls were assayed by standard ELISA methods to determine the antibody titer against Pfs25 on 96 well plates coated with Pfs25 antigen. Immune response against ViP was analyzed on ELISA plates coated with Vi

polysaccharide. Immune responses on Day 42 (two weeks post second immunization) are given in FIGS. 3A and 3B. Conjugation of Pfs25 to ViP enhanced the immunogenicity of both antigens on Day 42 (two weeks post second immunization) (FIGS. 3A and 3B). Immunogenicity of Pfs25 increased by 100-fold for two different conjugates. Similarly, immune response against Vi polysaccharide also increased substantially compared to unconjugated Vi polysaccharide, both in the presence and in the absence of ALHYDROGEL® adjuvant. This effect was maintained at Day 70-90, particularly for Pfs25 (FIGS. 4A and 4B). Lot 24 was more heavily modified with ADH, which can lead to internal (ViP-ViP) crosslinking. Without being bound by theory, it is believed that this may be one of the reasons for the lower immunogenicity or Lot 24 compared to the other conjugates tested. Further, serum bactericidal activity (SBA) against Salmonella typhi was tested using sera from Day 90. All three conjugates showed slightly higher SBA titer compared with Vi alone (FIG. 4C). The day 90 titer was considerably lower than that observed on day 42, only moderately higher than the Vi alone.

To evaluate transmission-blocking activity of the Pfs25-ViP conjugate, a set of Anopheles mosquitoes were fed on a mixture of cultured P. falciparum gametocytes mixed with test immune sera or control sera, through a membrane feeding apparatus. Mosquitos were allowed to develop for a week. After a week, mosquitos were dissected and the number of oocysts developed in the midgut of each mosquito was counted. This was compared to the number of oocysts developed in the midgut of mosquitos fed on control sera. A reduction in the number of oocysts in the mosquito midgut fed on the immune sera indicates blockage of development of the parasite in the mosquito. The Pfs25-ViP conjugate blocked transmission of the malaria parasite, as shown by reduction in oocysts (FIG. 5). The data are represented as percentage reduction in oocyst count relative to oocyst count from mosquitos fed on control sera. A 100% reduction in oocyst count indicates complete blockage of parasite development in the mosquito. A greater than 50% reduction in oocyst count is considered to be meaningful transmission blocking activity, and greater than 80% reduction is considered acceptable activity for a Transmission Blocking Vaccine. In PBS formulations, two conjugates (#22 and #25) showed equal to or greater functional activity compared to Pfs25-Exoprotein A (EPA). In an ALHYDROGEL®, one of the conjugates (#25) showed activity comparable to the EPA conjugate, and the remaining samples showed functional activity (>60% reduction in oocyst count).

Example 4

Methods of Eliciting an Immune Response to Plasmodium and/or Salmonella typhi

This example provides exemplary methods for eliciting an immune response to Plasmodium and/or S. typhi in a subject. However, one skilled in the art will appreciate that methods that deviate from these specific methods can also be used to successfully elicit an immune response to these pathogens in a subject. In particular examples, the method includes selecting a subject in need of enhanced immunity to malaria (Plasmodium) and/or typhoid fever (S. typhi). Subjects in need of enhanced immunity include individuals who reside in, have traveled to, or are traveling to, regions where Plasmodium and/or S. typhi are endemic, particularly regions where these pathogens are co- endemic.

Selected subjects are administered an effective amount of one or more of the disclosed immunogenic compositions, including one or more of Pfs25-ViP, Pvs25-ViP, Pfs48/45-ViP, Pvs48/45-ViP, Pfs47-ViP, Pvs47-ViP, Pfs230-ViP, Pvs230-ViP, PfCSP-ViP, and PvCSP-ViP conjugates. In some examples, the one or more conjugates is administered to the subject at doses of about 1 μg to 500 μg of each conjugate. However, the particular dose can be determined by a skilled clinician. The disclosed conjugates can be administered in one or several doses. When administered in several doses, the time separating the administration can be seconds, minutes, hours, days, or even weeks.

The mode of administration can be any used in the art, including but not limited to subcutaneous, intradermal, or intramuscular administration. The amount of agent administered to the subject can be determined by a clinician, and may depend on the particular subject treated. Specific exemplary amounts are provided herein (but the disclosure is not limited to such doses).

The development of immune response (such as development of antibodies, such as transmission blocking antibodies) in a subject is monitored at time points following administration of the immunogenic composition. Methods of detecting antibodies in a sample (such as a blood or serum sample) include those known in the art, for example, ELISA methods. For example, human sera samples from immunized subjects are tested for antibody titer against the antigens (Pfs25 and Vi) using ELISA. Sera also is tested for functional activities using transmission blocking activity of malaria by standard membrane feed assay (such as that described in Example 3) and anti-Vi functional activity is evaluated by bactericidal activity of the immune sera.

In view of the many possible embodiments to which the principles of the disclosure may be applied, it should be recognized that the illustrated embodiments are only examples and should not be taken as limiting the scope of the invention. Rather, the scope of the invention is defined by the following claims. We therefore claim as our invention all that comes within the scope and spirit of these claims.

Claims

We claim:
1. An immunogenic conjugate comprising at least one malaria protein or portion thereof and at least one Vi polysaccharide (ViP), wherein the at least one malaria protein is linked to the at least one ViP through at least one linking group.
2. The immunogenic conjugate of claim 1, wherein the at least one malaria protein comprises Plasmodium falciparum s25 protein (Pfs25), Plasmodium vivax s25 protein (Pvs25), P. falciparum s230 protein (Pfs230), P. vivax s230 protein (Pvs230), P. falciparum circumsporozoite (CSP) protein (PfCSP), P. vivax CSP protein (PvCSP), Plasmodium falciparum s48/45 protein (Pfs48/45), Plasmodium vivax s48/45 protein (Pvs48/45), Plasmodium falciparum s47 protein (Pfs47), Plasmodium vivax s47 protein (Pvs47), or a portion of any one thereof.
3. The immunogenic conjugate of claim 2, wherein the at least one malaria protein comprises an amino acid sequence with at least 95% sequence identity to any one of SEQ ID NOs: 1-18, amino acids 7-179 of SEQ ID NO: 4, or amino acids 1-201 of SEQ ID NO: 8.
4. The immunogenic conjugate of claim 3, wherein the at least one malaria protein comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 1-18, amino acids 7-179 of SEQ ID NO: 4, or amino acids 1-201 of SEQ ID NO: 8.
5. The immunogenic conjugate of any one of claims 1 to 4, wherein the ViP comprises Salmonella enterica serovar typhi ViP or Citrobacter ViP.
6. The immunogenic conjugate of any one of claims 1 to 5, wherein the at least one malaria protein and at least one ViP are linked by a linking group selected from the group consisting of a hydrazide, a dihydrazide, an amide group, a thioether group, N-succinimidyl-4-formylbenzoate, N- succinimidyl-3-bromoacetamidopropionate, and N-succinimidyl-3-(2-pyridyldithio)-propionate.
7. The immunogenic conjugate of any one of claims 1 to 6, wherein the linking group is adipic acid dihydrazide.
8. The immunogenic conjugate of any one of claims 1 to 7, wherein the ratio of the at least one protein to the total conjugate (w/w) is 0.2-0.8.
9. The immunogenic conjugate of any one of claims 1 to 8, wherein immunogenicity against the malaria protein, ViP, or both, is enhanced compared to the unconjugated malaria protein or ViP.
10. A composition comprising one or more of the immunogenic conjugates of any one of claims 1 to 9 and a pharmaceutically acceptable carrier and/or an adjuvant.
11. The composition of claim 10, wherein the adjuvant is alum.
12. A method of eliciting an immune response to Plasmodium and/or Salmonella typhi in a subject, comprising administering an effective amount of one or more of the immunogenic conjugates of any one of claims 1 to 9 or the composition of claim 10 or claim 11 to the subject.
13. The method of claim 12, wherein the immune response to Plasmodium comprises an immune response to P. falciparum or P. vivax.
14. The method of claim 12 or claim 13, wherein the immune response to Plasmodium comprises a transmission blocking immune response and/or a protective immune response.
15. The method of any one of claims 12 to 14, wherein the immune response to S. typhi comprises a protective immune response.
16. The method of any one of claims 12 to 15, wherein administering the conjugate or the composition to the subject comprises administering two or more doses of the conjugate or the composition to the subject.
17. The method of any one of claims 12 to 16, wherein the conjugate or the composition is administered to the subject intramuscularly, subcutaneously, or intradermally.
18. The method of any one of claims 12 to 17, wherein the subject is a human.
19. The method of any one of claims 12 to 18, further comprising selecting a subject in need of enhanced immunity to Plasmodium and/or S. typhi.
20. The method of any one of claims 12 to 19, further comprising administering one or more anti-malarial treatments.
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