Attenuated plasmodium sporozoite vectors for expression of heterologous antigens and host immunity modifiers

Abstract

The present disclosure is directed to compositions and methods of use of Plasmodium-species sporozoites (SPZ) which are attenuated to arrest the Plasmodium-species sporozoite's developmental progression in liver stage, thereby preventing reentry into the bloodstream and subsequent red blood cell infection, and wherein the Plasmodium-species sporozoite comprises heterologous antigens and / or host immunity modifiers, and is therefore useful as a liver-specific vector for the expression of heterologous antigens and / or immunoregulatory elements. Disclosed is the use of this technology for the enhancement of the immunogenicity of the SPZ per se, as well as treatment of liver-specific pathologies. In some aspects, the Plasmodium-species sporozoite comprises a heterologous transgene encoding: a) antigens of Hepatitis B virus; b) an immune modulator; or c) both (a) and (b). The disclosure is also directed to vaccines comprising the same and methods of use.

Description

Attenuated Plasmodium Sporozoite Vectors for Expression of Heterologous Antigens and Host Immunity ModifiersCROSS-REFERENCES TO RELATED APPLICATIONS

[0001] This application claims the benefit of U. S. Provisional Application No. 63 / 719426 filed November 12, 2024, and U. S. Provisional Application No. 63 / 719426 filed April 8, 2025, the disclosure of which is incorporated herein by reference in its entirety.STATEMENT REGARDING SEQUENCE LISTING

[0002] The Sequence Listing XML associated with this application is provided in XML format and is hereby incorporated by reference into the specification. The name of the XML file containing the sequence listing is 3399-P48WO_Seq Listing_20251112.xml. The XML file is 200,439 bytes; was created on November 12, 2025; and is being submitted electronically via Patent Center with the filing of the specification.STATEMENT OF GOVERNMENT LICENSE RIGHTS

[0003] This invention was made with government support under Al 170777 awarded by the National Institutes of Health. The government has certain rights in the invention.BACKGROUND

[0004] The liver is immunologically unique: it is continuously exposed to gut-derived antigens and microbial products delivered by the portal circulation, and it balances active immune surveillance with pronounced tolerogenic mechanisms that limit collateral damage to parenchyma. This distinctive immune milieu — characterized by specialized resident immune and non-immune cell populations (e.g., Kupffer cells, liver sinusoidal endothelial cells, hepatic stellate cells, tolerogenic dendritic cells), rapid antigen clearance, and active immunoregulatory signaling — creates both therapeutic opportunities and substantial obstacles for treating infectious, inflammatory, fibrotic, metabolic, and neoplastic diseases of the liver.

[0005] Clinically important liver diseases (for example, chronic viral hepatitis, immune-mediated liver disease, malaria, and primary or metastatic hepatic malignancies) remain major medical problems. Despite advances in systemic immunotherapy and antivirals, outcomes are frequently limited by the liver’s tolerogenic bias and by physiological features of hepatic biology that impede effective, durable, and safe therapeutic responses.

[0006] Accordingly, there is a need for compositions, delivery systems, and methods that overcome the liver's innate tolerogenic mechanisms, counteract local immune suppressionand exhaustion, achieve selective intrahepatic targeting and retention of therapeutic agents, and minimize hepatotoxicity. Solutions that accomplish one or more of these goals — preferably in a scalable, manufacturable, and clinically translatable format — would represent a meaningful advance over current therapeutic approaches for liver-associated infectious, inflammatory', metabolic, and neoplastic diseases.

[0007] The present disclosure addresses one or more of the needs and problems set forth above.SUMMARY

[0008] The present disclosure relates generally to therapeutic and prophylactic approaches to diseases of the liver, and more particularly to compositions and methods that address challenges arising from the liver’s distinctive immune environment. In particular, the present disclosure relates to the generation of transgenic Plasmodium parasites that have been adapted to act as liver-specific vectors, encoding and delivering non-PtomorfzMm / heterologous proteins, for example, modifiers of the host immune system and / or one or more antigens associated with pathogens, into hepatocytes to enhance the immune response to parasites, viruses, and other hepatotropic infections, or hepatic maladies. A vaccine platform and methods of treatment that combine naturally encoded anti-parasite immunity and / or the inclusion of known modifiers of host immunity are also disclosed.

[0009] Certain aspects of the disclosure pertain to the creation and use of attenuated Plasmodium-si eciss sporozoites as hepatotropic, liver-specific vectors for the expression of heterologous antigens and / or immunomodulators, useful for generating and / or enhancing liverspecific antigen expression and immune responses. In some aspects, attenuated Plasmodium-species sporozoites comprise a nucleic acid sequence encoding a heterologous antigen and / or an immunomodulator inserted into an attenuated Plasmodium genome.

[0010] Other aspects of the present disclosure provide for compositions and methods of generating HBV-specific T-cell responses within the liver by targeted expression and presentation of HBV antigens intracellularly in hepatocytes.

[0011] Other aspects of the present disclosure provide for liver-directed immune modulation to overcome poor trafficking of protective T-cells to the liver and immune exhaustion / dysregulation in the liver that occurs when administered peripherally. These strategies both utilize the immunogenic potential and large payload capacity of hepatocyte-targeted eukaryotic Plasmodium-s ec es sporozoites (SPZ) (e.g., the full genome is about 23 mb on 14 chromosomes) as the vector, particularly P. falciparum (Pf), to generate therapeutic adaptive and innate immune responses against hepatic maladies such as HBV, CHB, and / or HCC cancer.

[0012] In some aspects, the Plasmodium-species sporozoites of human host range comprise one or more heterologous transgenes encoding one or more antigens of hepatitis B virus (HBV) or antigenic fragments thereof. In some aspects, the human hepatic pathogen comprises a hepatitis virus or an encoded antigen thereof. In some aspects, the hepatitis virus is hepatitis B virus (HBV). In some aspects, the encoded antigen of the human hepatic pathogen (e.g., HBV) comprises HBxAg, HBpolAg, HBsAg, HBcAg, an antigenic fragment thereof, or any combination thereof.

[0013] In some aspects, the encoded antigen of the human hepatic pathogen comprises hepatitis B virus (HBV) HBxAg or an antigenic fragment thereof, wherein the HBV HBxAg or antigenic fragment thereof is expressible during liver stage sporozoite infection.

[0014] In some aspects, the encoded antigen of the human hepatic pathogen comprises HBV HBpolAg or an antigenic fragment thereof, wherein the HBV HBpolAg or antigenic fragment thereof is expressible during liver stage sporozoite infection.

[0015] In some aspects, the encoded antigen of the human hepatic pathogen comprises HBV HBsAg or an antigenic fragment thereof, wherein the HBV HBsAg or antigenic fragment thereof is expressible during liver stage sporozoite infection.

[0016] In some aspects, the encoded antigen of the human hepatic pathogen comprises HBV HBcAg or an antigenic fragment thereof, wherein the HBV HBcAg or antigenic fragment thereof is expressible during liver stage sporozoite infection.

[0017] In some aspects, the Plasmodium-species sporozoite comprises a heterologous transgene encoding a human immunomodulatory' molecule. In some aspects, the Plasmodiumspecies sporozoite comprises a gene encoding a human cytokine (e g., human interleukin 2 (hlL- 2), engineered analogues of hIL-2. or a functional fragment thereof.

[0018] In some aspects, the encoded human cytokine comprises an engineered IL-2 fusion protein such as cIL-2eRa.

[0019] In some aspects, the Plasmodium-species sporozoite further comprises a promoter operably linked to a transgene disclosed herein (e.g., a heterologous trans gene encoding a human immunomodulatory' and / or a heterologous transgene encoding an antigen of hepatitis B virus (HBV) or antigenic fragments thereof). In some aspects the promoter is a Plasmodium falciparum (Pf) promoter. In some aspects, the Plasmodium-species sporozoite comprises a Plasmodium falciparum (Pf) CSP. PfEXPl, PfLSAl, or PfHSP70 promoter, wherein said promoter is configured to drive expression of the heterologous transgene. In some aspects, the promoter is PfCSP.

[0020] In some aspects, the Plasmodium-species sporozoite further comprises an export signal. In some aspects, the export signal facilitates secretion of one or more proteins to the cytosol of the liver cells.

[0021] Certain aspects of the disclosure are directed to a Plasmodium-species sporozoite of human host range which is attenuated to arrest the Plasmodium-species sporozoite developmental progression at late liver stage and prior to red blood cell infection (e.g.. PfSPZ-LARC2), wherein said Plasmodium-species sporozoite comprises a polynucleotide comprising (i) a heterologous transgene encoding a human immunomodulator, a functional fragment thereof, or an engineered analogue thereof (e.g., hIL-2 or cIL-2eRa), which is operably linked to a promoter and optionally an export signal; (ii) a heterologous transgene encoding one or more antigens of hepatitis B virus (HBV) or antigenic fragments thereof, which is operably linked to a promoter and optionally an export signal. In some aspects, the polynucleotide further comprises a 3’ UTR and / or a 5’ UTR

[0022] In some aspects, the infectious hepatotropic pathogen is hepatitis B virus (HBV), and the one or more antigens of HBV comprise HBxAg, HBpolAg, HBsAg, HBcAg, an antigenic fragment thereof, or any combination thereof.

[0023] In some aspects, the at least one heterologous expressible transgene encodes a human immunomodulator or a functional fragment thereof that is an antagonist of at least one of an interferon regulatory factor (1RF) or an interferon signaling protein.

[0024] In some aspects, the immunomodulator is an antagonist of at least one of IRF3, IRF1, and IRF7. In some aspects, the immunomodulator is an antagonist of IRF3 and the antagonist is selected from the group consisting of Flavivirus Nonstructural Protein 1 (NS1), Human Immunodeficiency Virus Viral Protein R (HIV VPR), West Nile Virus (WNV) NS1, Severe Acute Respiratory Syndrome Coronavirus 2 Nonstructural Protein 3 (S ARS Cov-2 NSP3), SARS Cov-2 NSP13, SARS Cov-2 NSP15, SARS Cov-2 Open Reading Fram 6 (ORF6), SARS Cov-2 ORF3b, SARS Cov-2 NSP1, Ebolavirus VP35, and Ebola virus Nucleoprotein (NP).

[0025] In certain aspects, the immunomodulator is an antagonist of the interferon signaling protein t pe 1 interferon (IFN-1).

[0026] In some aspects, the immunomodulator is human interleukin 2 (hIL-2). In certain aspects, the heterologous transgene encodes the engineered hIL-2 analogue cIL-2eRa (SEQ ID NO: 38).

[0027] In some aspects, the heterologous transgene encoding the immunomodulator is inserted into a P230p locus within the Plasmodium genome. In certain aspects, the heterologous transgene encoding the immunomodulator is operably linked to a promoter sequence of the Plasmodium liver stage protein UIS4. In some aspects, the heterologous transgene encoding theimmunomodulator is operably linked to a promoter sequence of the Plasmodium circumsporozoite protein (CSP) PEXEL. In some aspects, the Plasmodium-species sporozoite comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 40 or 41.

[0028] In certain aspects, the Plasmodium-species sporozoite, further comprises a Plasmodium falciparum (PI) PfCSP (SEQ ID NO: 25), PIEXP1(SEQ ID NO: 31), PILSA1 (SEQ ID NO: 32), or PIHSP70 (SEQ ID NO: 29) promoter. In some aspects, the promoter is configured to drive expression of the heterologous transgene.

[0029] Aspects of the present disclosure provide a live Plasmodium organism that is genetically modified to encode and express an antagonist of at least one of an interferon regulatory factor (IRF) or an interferon signaling protein. In certain aspects, the antagonist is directed to IRF3, IRF1, and IRF7. In certain aspects, the antagonist is directed to ty pe 1 interferon (IFN-1). In certain aspects, the antagonist comprises a viral antagonist of IRF3. In certain aspects, the antagonist is selected from the group consisting of Flavivirus Nonstructural Protein 1 (NS1), Human Immunodeficiency Virus Viral Protein R (HIV VPR), West Nile Virus (WNV) NS1, Severe Acute Respiratory Syndrome Coronavirus 2 Nonstructural Protein 3 (S ARS Cov-2 NSP3), SARS Cov-2 NSP13, SARS Cov-2 NSP15, SARS Cov-2 Open Reading Frame 6 (ORF6), SARS Cov-2 ORF3b, SARS Cov-2 NSP1, Ebolavirus VP35, and Ebola virus Nucleoprotein (NP).

[0030] In certain aspects, the antagonist is inserted into a P230p locus within the Plasmodium genome.

[0031] In other aspects, the hepatotropic vector further comprises a promoter, and optionally an export signal. In some aspects, the promoter is configured to drive expression of the heterologous trans genes.

[0032] In certain aspects, the antagonist is operably linked to a promoter sequence of the Plasmodium liver stage protein UIS4.

[0033] In certain aspects, the antagonist is operably linked to a sequence of the Plasmodium circumsporozoite protein (CSP) PEXEL.

[0034] In certain aspects, the Plasmodium organism comprises a nucleotide coding sequence having at least 80% sequence identity to SEQ ID NO:40 or 41. In certain aspects, the Plasmodium organism comprises a nucleotide coding sequence having at least 85%, at least 90%, or at least 95% sequence identity’ to SEQ ID NO:40 or 41.

[0035] In certain aspects, the Plasmodium-species sporozoite comprises both (i) a heterologous transgene encoding a human immunomodulator, a functional fragment thereof, or an engineered analogue thereof and (ii) one or more heterologous transgenes encoding one or more antigens of an infectious hepatotropic pathogen or antigenic fragments thereof.

[0036] Aspects of the present disclosure provide a Plasmodium-species sporozoite which is attenuated to arrest the P / asmodium-species sporozoite developmental progression during liver stage and prevent subsequent red blood cell infection, the sporozoite comprising one or more heterologous expressible transgenes encoding one or more antigens of an infectious hepatotropic pathogen or antigenic fragments thereof, thereby providing a liver-specific vector for introduction of the heterologous expressible transgene into hepatocytes.

[0037] In some aspects, the attenuation is achieved by radiation attenuation.

[0038] In some aspects, the Plasmodium-species sporozoite is genetically attenuated. In some aspects, the genetically attenuated Plasmodium-species sporozoite lacks: (i) a functional LINUP gene; (ii) a functional Mei2 gene (at times referred to as " SPZ LAR ’); or (iii) both, a functional LINUP gene and a functional Mei2 gene (at time referred to as “SPZ LARC2’’).

[0039] In some aspects, the genetically attenuated Plasmodium-species sporozoite lacks a functional LINUP gene (SEQ ID NO: 19), a functional LINUP protein (SEQ ID NO: 37), or both. In some aspects, the genetically attenuated Plasmodium-species sporozoite lacks a functional Mei2 gene (SEQ ID NO: 18) or a functional Mei2 protein (SEQ ID NO: 20). In other aspects, the genetically attenuated Plasmodium-species sporozoite lacks a functional LINUP gene (SEQ ID NO: 19) and afunctional Mei2 gene (e.g., SEQ ID NO: 18).

[0040] In aspects of the disclosure, the attenuated Plasmodium-species sporozoites are of human host range, comprising a species selected from the group consisting of P. falciparum, P. vivax, P. knowlesi, P. ovcde, P. malarias, or any combination thereof. In certain aspects of the disclosure, the attenuated Plasmodium-species sporozoite of human host range comprises or is the species P. falciparum.

[0041] Certain aspects provide a vaccine composition comprising an attenuated live Plasmodium organism. In some aspects, the vaccine composition comprises a Plasmodiumspecies sporozoite disclosed herein and a carrier. In some aspects, the carrier is selected from the group consisting of phosphate buffered saline (PBS), saline, and human serum albumin, or any combination thereof.

[0042] In certain aspects the vaccine composition further comprises an adjuvant. In certain aspects, the adjuvant is 7DW8-5.

[0043] In certain embodiments, the vaccine composition is administered parenterally, e.g., intravenously, intradermally, subcutaneously, intramuscularly, or intraperitoneally. In some embodiments, the parenteral route is intravenous, in some embodiments the parenteral route is intramuscular.

[0044] Other aspects of the present disclosure provide methods of expressing one or more heterologous transgenes intra-hepatically in a human subject. In some aspects, the methodcomprises administering an effective amount of the tomo zwm-species sporozoite vaccine composition to a subject.

[0045] Certain aspects provide a method of generating an immune reaction to a human hepatic pathogen intra-hepatically in a human subject, the method comprising administering to the subject an effective amount of the Plasmodium-species sporozoite vaccine composition disclosed herein.

[0046] Certain aspects provide methods of preventing or reducing the likelihood of an infection of a human pathogen in a human subject comprising administering to the subject an effective amount of the Plasmodium-species sporozoite vaccine composition disclosed herein.

[0047] Certain aspects provide methods of inhibiting interferon production within a hepatocyte comprising administering the Plasmodium organism modified to express antagonists of an IRF or an interferon signaling protein, inhibiting interferon production thereby.

[0048] In some aspects, methods are provided for HBsAg loss, with seroconversion and undetectable HBV DNA in serum of human subjects with CHB by administration of vaccines comprising attenuated Plasmodium-species SPZ comprising heterologous transgenes.

[0049] Other aspects of the present disclosure provide methods of treating a hepatic infection or hepatic tumor in a human subject in need thereof, the method comprising administering to the subject an effective amount of the Plasmodium-species sporozoite vaccine composition disclosed herein.

[0050] Certain aspects provide a method of preventing or treating malaria in a host subject comprising administering an effective amount of the vaccine compositions disclosed herein.

[0051] Certain aspects of the disclosure are directed to a vaccine composition (e.g., an attenuated Plasmodium SPZ construct comprising functional heterologous sequences, i.e. encoding a vaccine immunogen) comprising a Plasmodium-species sporozoite disclosed herein and a carrier. In some aspects, the carrier is selected from the group consisting of water, PBS, saline, and human serum albumin or any combination thereof.

[0052] Certain aspects of the disclosure are directed to a method of inducing an innate and adaptive immune response in the liver of a human subject with CHB, the method comprising administration of one or more doses of an attenuated vaccine comprising a Plasmodium-species SPZ of human host range disclosed herein.

[0053] Other objects, features, and advantages of the present invention will be apparent to one of ordinary skill in the art from the following detailed description and drawings.BRIEF DESCRIPTION OF DRAWINGS

[0054] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawings will be provided by the Office upon request and payment of the necessary' fee.

[0055] FIGS. 1A-1B show a diagram outlining / .wt / mm-medialed delivery of protein antagonists of type I Interferon (IFN-I) signaling into infected cells. Wildtype Plasmodium parasite infection and development within hepatocytes results in the transcription, production and secretion of IFN-I, causing the infected and neighboring hepatocytes and immune cells to impair the ability of CD8+ T cells to find and eliminate Plasmodium parasites in the liver (FIG. 1A).Schematic of a transgenic Plasmodium parasite encoding antagonists of IFN-I and expression within infected hepatocyte, thereby limiting IFN-I production and thereby enabling liver CD8+ T cells to effectively locate and eliminate Plasmodium parasites in the liver (FIG. IB).

[0056] FIGS. 2A-2C. show proof-of-concept: Transgenic Plasmodium delivers Cre recombinase into infected cells to alter the infected hepatocyte genome in vivo. Infection of ROSAmT / mG mice with Py p230p-Cre transgenic parasites or wildtype / normal Plasmodium yoelii parasites (Py XNL) (FIG. 2A). The RosamT / mGmouse expresses sequences for the red fluorescent protein (RFP) and suppresses sequences for the green fluorescent protein (GFP) under normal conditions, whereas removal of the RFP gene by Cre recombinase allows the cell to express GFP (FIG. 2B). Immunofluorescence imaging of liver sections harvested from ROS AmT / mGmice infected with transgenic (Py p230p-Cre) or wildtype / normal parasites (Py XNL) 42 hpi. Key: Plasmodium parasites (identified by white arrow in each panel), GFP (green), DNA stained with DAPI (blue) and RFP (red) (FIG. 2C).

[0057] FIG. 3 is a schematic diagram illustrating the integration of Interferon Regulatory Factor 3 (IRF3) antagonists into the Plasmodium yoelii genome using CRISPR-Cas9 technology7.

[0058] FIGS. 4A and 4B show a two-step schematic representation of CRISPR-Cas9-mediated knockin of HBV antigens and human IL-2. The insertion of the transgenes encoding HBV antigens and hIL- into the genome of P. falciparum SPZ-LARC2 by CRISPR-Cas9 is depicted in 2 steps. In the first step, HBpolAg and / or HBxAg are integrated into the Mei2 locus (FIG. 4A - “Construct 1”). In the second step, hIL-2, and HBsAg, and / or HBcAg antigens are integrated into the LINUP locus (FIG. 4B - “Construct 2"). In FIGS. 4A-4B. expression of the antigens is driven by the PfCSP promoter and their secretion to the cytosol of the liver cells is mediated by an export signal (EXP). Both Construct 1 and Construct 2 are directed to the target loci by homology-derived repair using 2 homologous arms flanking the integrated constructs. The vector harbors guide RNA (Sp) and Cas9 to mediate a double strand break at the desired locus.

[0059] FIG. 5 shows an image depicting the life cycle of Plasmodium.

[0060] FIGS. 6A and 6B show schematics for generation of PyLARC2 expressing mcIL-2eRa. FIG. 6A shows the first step to generate the PyLARC2cspmcIL-2eRa, wherein the AMei2 locus will be targeted to express the CSP signal peptide and PEXEL fused to the mcIL-2eRa cassette under the LISP1 promoter, followed by the LISP1 3’UTR. FIG.6B shows the step where the PyLARC2LISP2mclL-2eRa parasite targets the endogenous L1SP2 locus and a L1SP2-PyLARC2LISP2mcIL-2eRa fusion protein is thereby generated.

[0061] FIG. 7A shows a schematic representation of CRISPR-Cas9 strategy. Gene cassettes containing DNA encoding cIL-2eRa or HBcAg were constructed to be expressed under the liver stage UIS4 gene promoter and secreted to cytoplasm of hepatocytes using the PyCSP PEXEL export motif. The black arrows indicate the position of the primers used in the PCR assays confirming the integration. PCR was done with the indicated primers to confirm the integration of cIL-2eRa (FIG. 7B) or HBcAg (FIG. 7C) DNA into the mei2 locus. PCR was done on DNA from 2 populations (#1 and #2) from different transfections of WT Py. 3 PCR reactions done on each sample of DNA. They were a) WT mei2 gene (513 bp), b) 5’ of the insertion, amplifying from upstream to 5’ arm to the UIS4 promoter (1635 bp) and c) 3’ of the insertion, amplifying from inside the cIL-2eRa or HBcAg ORF to downstream of 3' arm (1050 and 1153 bp for cIL-2eRa and HBcAg, respectively). Both samples contained parasites with integrated cIL-2eRa or HBcAg ORFs.DETAILED DESCRIPTION DEFINITIONS

[0062] As used herein, the term “antigen” refers to a molecule capable of being bound by an antibody. An antigen is additionally capable of being recognized by the immune system and / or being capable of inducing a humoral immune response and / or cellular immune response leading to the activation of B- and / or T-lymphocytes. An antigen can have one or more epitopes (B-and / or T-cell epitopes). “Antigens” as used herein may also be mixtures of several individual antigens.

[0063] The term “antibody” is used herein in the broadest sense and specifically encompasses monoclonal antibodies (including full length monoclonal antibodies), polyclonal antibodies, multi-specific antibodies (e.g., bispecific antibodies), and antibody fragments such as variable domains and other portions of antibodies that exhibit a desired biological activity, e.g., PfHAP2p binding and or neutralizing.

[0064] As used herein, “attenuated” (alternatively, “arrested”) in the context of Plasmodium parasites or sporozoites refers to a live Plasmodium parasite that can infect its host,migrate through the bloodstream to and invade the liver, but compared to its wildtype homologue, is unable to develop beyond the liver-stage of infection, progress to the subsequent blood stage, or infect red blood cells.

[0065] As used herein, a “cytokine” is a biologically active molecule, such as interferon, interleukin, or growth factors, which is normally secreted by certain cells of the immune system and has an effect on other cells.

[0066] As used herein, an “effective amount” of a vaccine is a regimen comprising a dosage of one or more dose administrations, wherein the dosage is sufficient to generate the desired response, e.g., immunologic response.

[0067] As used herein, the term “epitope” refers to basic element or smallest unit of recognition by an individual antibody or T-cell receptor, and thus the particular domain, region or molecular structure to which said antibody or T-cell receptor binds. An antigen may consist of numerous epitopes while a hapten, typically, may possess few- epitopes. As used herein “correspond essentially to” refers to an epitope that will elicit an immunological response at least substantially equivalent to the response generated by the native epitope. An immunological response to a composition or vaccine is the development in the host of a cellular and / or antibody-mediated immune response to the polypeptide or vaccine of interest. Usually, such a response consists of the subject producing antibodies, B cell, helper T cells, suppressor T cells, and / or cytotoxic T cells directed specifically to an antigen or antigens included in the composition or vaccine of interest.

[0068] As used herein, a “functional” gene, is a gene that is capable of expressing a gene product that performs one or more of its wildtype function(s).

[0069] As used herein, the abbreviation “HBV” indicates one or more strains of Hepatitis B Virus.

[0070] As used herein, the abbreviation “CHB” indicates chronic Hepatitis B infection.

[0071] As used herein, “heterologous” with regard to a genetically engineered construct or vector or elements thereof, refers to a gene or nucleic acid sequence that is not derived from, or not of the same species as, the genome of the construct.

[0072] As used herein, an “immunomodulator” is a biologically active molecule which has an effect on certain cells of the immune system.

[0073] As used herein, an “immune response” refers to a humoral immune response or a cellular immune response or both, leading to the activation and / or proliferation of B- and / or T-lymphocytes and / or and antigen presenting cells. In some instances, however, the immune responses may be of low intensity7and become detectable only when using at least one substance in accordance with the invention. “Immunogenic” or “immunogen” refers to an agent used tostimulate the immune system of a living organism, so that one or more functions of the immune system are increased and directed towards the immunogenic agent. An “immunogenic polypeptide” is a polypeptide that elicits a cellular and / or humoral immune response, whether alone or linked to a carrier. Preferably, antigen presenting cells may be activated. Similarly, the terms “immunize” or “immunization”, and related terms, refer to conferring the ability to mount a substantial immune response (comprising antibodies and / or cellular immunity) against a target antigen or epitope subsequent to the initial presentation of an antigen. These terms do not require that complete immunity be created, but rather that an immune response be produced which is substantially greater than baseline.

[0074] The term "immunotherapeutic” refers to a composition for the treatment of diseases, disorders or conditions. More specifically, the term is used to refer to a method of treatment wherein a beneficial immune response is generated by vaccination or by transfer of immune molecules. An “immunologically effective amount” refers to an amount of a composition sufficient to induce an immune response in an individual when introduced into that individual. In the context of active immunization, the term is synonymous with “immunogenically effective amount.” The amount of a composition necessary to be immunologically effective varies according to many factors including the composition, the presence of other components in the composition, the antigen, the route of immunization, the individual, the prior immune or physiological state etc.

[0075] As used herein, “knock-in” with regard to a genetically engineered construct, refers to gene inserted into a recipient genome or genetic construct.

[0076] As used herein, “knock-out” with regard to a genetically engineered construct, refers to genes that are partially or completely removed from a recipient genome or genetic construct, such that the gene product of the knocked-out gene is no longer functional or absent.

[0077] As used herein, “LARC SPZ” is an abbreviation for “Late Arresting Replication Competent Sporozoite” in a Plasmodium-species mutant that is replication-competent through most of the liver-stage of development, but arrests (attenuates) late in development. This mutant can be created for example by genetic knockout of at least a portion or all of the PlasMei2 (Mei2) gene from the wildtype Plasmodium-species genome, resulting in the absence of the corresponding functional Mei2 gene product.

[0078] As used herein, “LARC2 SPZ” is an abbreviation for a Plasmodium-species mutant that is replication-competent through most of the liver-stage of development, but arrests (attenuates) late in development. This mutant was created by genetic knockout of at least a portion or all of the LINUP gene, resulting in a lack of a functional LINUP gene product, and at least aportion or all of the PlasMei2 (Mei2) gene from the wildtype Plasmodium-species genome, resulting in a lack of a functional Mei2 gene product.

[0079] As used herein, “nucleic acid” refers to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double-stranded form, composed of monomers (nucleotides) containing a sugar, phosphate and a base which is either a purine or pyrimidine. Unless specifically limited, the term encompasses nucleic acids containing known analogs of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and / or deoxyinosine residues (Batzer et al. (1991) Nucl. Acids Res.19:508; Ohtsuka et al. (1985) JBC 260:2605; Rossolini et al. (1994) Mol. Cell. Probes 8:91. A “nucleic acid fragment” is a fraction of a given nucleic acid molecule. Deoxyribonucleic acid (DNA) in the majority of organisms is the genetic material while ribonucleic acid (RNA) is involved in the transfer of information contained within DNA into proteins. The term “nucleotide sequence” refers to a polymer of DNA or RNA that can be single- or double-stranded, optionally containing synthetic, non-natural or altered nucleotide bases capable of incorporation into DNA or RNA polymers. The terms “nucleic acid,” “nucleic acid molecule,” “nucleic acid fragment,” “nucleic acid sequence or segment,” or “polynucleotide” may also be used interchangeably with gene, cDNA, DNA and RNA encoded by a gene.

[0080] As used herein, the terms “Plasmodium ' or “parasite” refer to any parasite that belongs to the genus Plasmodium. In some embodiments, the Plasmodium organism is of human host range, i.e. can infect human hosts, such as, for example, P. falciparum, P. vivax, P. ovale, P. malariae, and P. knowlesi. In certain aspects, the Plasmodium organism is P. falciparum. In certain aspects, the Plasmodium organism can infect other vertebrate hosts, such as nonhuman primates and rodents. Examples of such Plasmodium organisms include P. yoelii, P. berghei, P. chabaudi, P. vinckei, and P. cynomolgi.

[0081] With regard to wildtype Plasmodium species of human host range, ’’late liver stage” as used herein refers to the developmental period within the liver beginning around day 6 post hepatic infection, with a duration of from day 6-7, day 6-8, day 6-9, or day 6-10, and is characterized by cytomere formation, exo-erythrocytic merozoite formation, and breakdown of the parasitophorous vacuole membrane of the schizont.

[0082] As used herein, a “prophylactic” vaccine is used to reduce the likelihood or prevent infection of an individual by one or more specific infectious agent(s).

[0083] As used herein, “protein,” “peptide,” and “polypeptide” are used interchangeably herein. Polypeptide sequences specifically recited herein are written with the amino terminus on the left and the carboxy terminus on the right. As used herein, the terms “protein,” “peptide,” and “polypeptide” refer to a polymer in which the monomers are amino acid residues that are joined together through amide bonds. When the amino acids are alpha-amino acids, either the L-optical isomer or the D-optical isomer can be used, the L-isomers being preferred. The term polypeptide or protein as used herein encompasses any amino acid sequence and includes modified sequences such as glycoproteins. The term polypeptide is specifically intended to cover naturally occurring proteins, as well as those that are recombinantly or synthetically produced.

[0084] The phrase “recombinant protein” or “chimeric protein” or “engineered fusion protein” or “fusion protein,” are used interchangeably and includes proteins, in particular recombinant fusion proteins that are prepared, expressed, created or isolated by recombinant means, such as proteins expressed using a recombinant expression vector transfected into a host cell. The term “fusion protein” refers to a protein produced by recombinant technology which comprises segments, i.e., amino acid sequences, from heterologous sources, such as different proteins or different organisms. The segments are joined either directly or indirectly to each other via peptide bonds. By indirect joining it is meant that an intervening amino acid sequence, such as a peptide linker is juxtaposed between segments forming the fusion protein. A fusion protein is encoded by a nucleotide sequence, which is obtained by genetically joining nucleotide sequences derived from different regions of one gene and / or by joining nucleotide sequences derived from two or more separate genes.

[0085] Tn the context of the present invention, an “isolated” or “purified” polypeptide is a polypeptide that exists apart from its native environment and is therefore not a product of nature. A polypeptide may exist in a purified form or may exist in a non-native environment such as, for example, a transgenic host cell. For example, an “isolated” or “purified” protein, or biologically active portion thereof, is substantially free of other cellular material, or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized. A protein that is substantially free of cellular material includes preparations of protein or polypeptide having less than about 30%, 20%, 10%, 5%, (by dry weight) of contaminating protein. When the protein of the invention, or biologically active portion thereof, is recombinantly produced, preferably culture medium represents less than about 30%, 20%, 10%, or 5% (by dry weight) of chemical precursors or non-protein-of- interest chemicals. Fragments and variants of the disclosed proteins or partial-length proteins encodedthereby are also encompassed by the present invention. By "‘fragment’" or “portion” or “segment” or “domain” is meant a full length or less than full length of the amino acid sequence of, a polypeptide or protein. “Naturally occurring” is used to describe an object that can be found in nature as distinct from being artificially produced. For example, a protein or nucleotide sequence present in an organism (including a virus), which can be isolated from a source in nature, and which has not been intentionally modified by man in the laboratory, is naturally occurring.

[0086] A “variant” of a molecule is a sequence that is substantially similar to the sequence of the native molecule.

[0087] As used herein, “seroconversion” means a change from seropositive condition to seronegative condition.

[0088] As used herein, “therapeutic” refers to use to treat, reduce the symptoms of, or cure an individual with a disease (e.g., an infectious disease).

[0089] As used herein, a “vaccine” is a composition comprising an immunogenic component and a carrier such as a diluent, excipient, and the like.

[0090] The terms “administer,” “administering,” “administration,” and the like, as used herein, refer to methods that may be used to enable delivery of a therapeutic drug, agent, or composition (e.g., a vaccine), e.g., a Plasmodium-species sporozoite of human host range or vaccine composition disclosed herein, to a subject or the desired site of biological action. Administration techniques that can be employed with the agents and methods described herein are found in, e.g., Goodman and Gilman, The Pharmacological Basis of Therapeutics, current edition. Remington’s Pharmaceutical Sciences, current edition, Elsevier.

[0091] The terms “treat,” “treatment,” or “treating,” as used herein refers to, e.g., the reduction in severity of a disease or condition; the reduction in the duration of a disease course; the amelioration or elimination of one or more symptoms associated with a disease or condition; the provision of beneficial effects to a subject with a disease or condition, without necessarily curing the disease or condition.

[0092] The terms “subject.” “patient,” “individual,” and “host,” and variants thereof are used interchangeably herein and refer to any mammalian subject, including without limitation, humans, domestic animals (e.g., dogs, cats and the like), farm animals (e.g., cows, sheep, pigs, horses and the like), and laboratory animals (e.g., monkey, rats, mice, rabbits, guinea pigs and the like) for whom diagnosis, treatment, or therapy is desired, particularly humans. The methods described herein are applicable to both human therapy and veterinary applications. As used herein, the phrase “subject in need thereof’ includes subjects, such as mammalian subjects, that would benefit from administration of a therapeutic agent, e.g., a Plasmodium-species sporozoite of human host range or vaccine composition disclosed herein.

[0093] As used herein, the term "in vitro” refers to events that occur in an artificial environment, e.g., in a test tube or reaction vessel, in cell culture, in a Petri dish, etc., rather than within an organism (e.g., animal).

[0094] As used herein, the term “zn vivo” refers to events that occur within an organism (e.g., animal).

[0095] As used herein, "transgene” refers to a gene or functional fragment thereof, which is artificially introduced into the genome of another organism.

[0096] The term ‘‘promoter” as used herein refers to a DNA sequence recognized by the machinery of the cell, or introduced synthetic machinery, required to initiate the transcription of a gene or coding sequence. The term “promoter” is also meant to encompass those nucleic acid elements sufficient for promoter-dependent gene or coding sequence expression controllable for cell-type specific, tissue-specific or inducible by external signals or agents; such elements can be located in the 5' or 3' regions of the gene. In some aspects, the promoter is a constitutively active promoter, a cell-type specific promoter, or an inducible promoter.

[0097] “Percent (%) sequence identity” with respect to a reference polynucleotide or polypeptide sequence is defined as the percentage of nucleic acids or amino acids in a candidate sequence that are identical to the nucleic acids or amino acids in the reference polynucleotide or polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Alignment for purposes of determining percent nucleic acid or amino acid sequence identity can be achieved in various ways that are within the capabilities of one of skill in the art, for example, using publicly available computer software such as BLAST, BLAST-2, or MegAlign software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. For example, percent sequence identity values can be generated using the sequence comparison computer program BLAST. Hepatotropic Vectors

[0098] Targeting hepatotropic pathogens is challenging primarily because the liver maintains a unique immunologically tolerant microenvironment to avoid excessive inflammation from gut-derived products (Hepatic Immune Regulation and Its Involvement in Viral Hepatitis Infection, Knolle, Percy A. et al. Gastroenterology, Volume 146, Issue 5, 1193 - 1207). This tolerogenic environment is actively exploited by pathogens like Hepatitis B (HBV) and C (HCV) viruses to establish chronic infections and evade the host immune response. Persistent exposure to high levels of pathogen antigens in the liver leads to the functional impairment, exhaustion, or premature death of pathogen-specific T cells (both CD4+ and CD8+). This prevents effective pathogen clearance. Additionally, the liver environment promotes the development and expansionof immunosuppressive regulatory T cells (Tregs) and myeloid-derived suppressor cells (MDSCs), which actively suppress effector T cell function via anti-inflammatory cytokines like IL- 10 and TGF-p. Moreover, hepatotropic pathogens have evolved specific mechanisms to subvert the immune system. For instance, HCV and HBV can produce proteins that directly impair T cell function or disable innate immune signaling pathways within hepatocytes. In addition to professional antigen-presenting cells (APCs) like dendritic cells (DCs) and Kupffer cells (KCs), non-parenchymal cells like liver sinusoidal endothelial cells (LSECs) and hepatocytes can also present antigens. Antigen presentation by these non-professional APCs often occurs without the necessary co-stimulatory signals required for a full, effective immune response, leading to T cell anergy or tolerance rather than immunity. These factors mean that a delicate balance must be achieved when developing therapies (such as vaccines or immunotherapies) for hepatotropic pathogens, aiming to induce effective, pathogen-specific immunity.Plasmodium Sporozoites

[0099] Human malaria is an infectious disease caused by Plasmodium-species parasites and transmitted to humans from Plasmodium-species-infected Anopheles mosquitoes by the sporozoite developmental stage of the parasite. Plasmodium-species sporozoites are eukaryotic single cell organisms that infect the host and quickly migrate through the blood stream invading the liver. There, they continue to develop within hepatocytes, expressing parasite-specific antigens. Plasmodia are indirect parasites with sexual stages occurring in the mosquito, and asexual stages in the intermediate host (e.g., a human subject). The sporozoite is an asexual developmental stage of Plasmodia that resides in mosquito salivary glands immediately prior to passage to the human host during feeding (FIG.5). Sporozoites pass through the blood and invade the liver where, without causing disease symptoms, they replicate 25.000-75.000 times over 6-10 days from early to late liver stage and eventually mature into schizonts. At this point they exit the liver and return to the blood stream at the merozoite stage of development. Merozoites then infect red blood cells (erythrocytes), further amplify and destroy the erythrocytes, perpetuating the erythrocytic infection. It's at this erythrocytic stage that the parasite causes the signs, symptoms and pathology of malaria disease. Some merozoites continue to develop into male and female gametes that are then ingested by mosquitoes that feeds on an infected subject, and the cycle is repeated.

[0100] Plasmodium parasite genomes encode over 5,000 genes which are differentially transcribed throughout its life cycle, rendering Plasmodium a difficult target for traditional subunit vaccine approaches. Immunization with attenuated Plasmodium falciparum (Pf) sporozoites (SPZ), e.g. attenuated by radiation, chemotherapeutic agents, or genetically, and administered parenterally, preferentially when administered intravenously by direct venous inoculation (DVI),all traffic to hepatocytes, where they generate protective, liver-specific, cellular immune responses [17-20, 23-45], More than 30 clinical trials in the US, Europe, Africa, and Asia have generated extensive safety, immunology and efficacy data in > 3,000 5-month- to 61 -year-olds [17-20, 23-45], PfSPZ-based vaccines have induced 100% T cell mediated protection against challenge with highly variant Pf parasites for at least 3 months

[0020] , and protection against Pf infection in the field in Africa for at least 18 months [23, 30] (A randomized controlled trial showing safety and efficacy of a whole sporozoite vaccine against endemic malaria. Science translational medicine, 14(674), eabj3776. Sirima et al., in press and Sagara et al. unpublished).

[0101] Immunization with whole attenuated sporozoites that are unable to cause blood stage infection has been investigated as promising vaccine strategy. For example, genetic alteration of Plasmodium parasites by gene deletion has been used to generate transgenic parasites that arrest in hepatocytes during liver stage development. Arrest of parasite development results in the release of Plasmodium proteins to the host immune system, enabling these altered parasites to act as live attenuated vaccines. Further details regarding genetically attenuated parasites can be found in Minkah, N. K., Wilder, B. K., Sheikh, A. A. et al. Innate immunity limits protective adaptive immune responses against pre-erythrocytic malaria parasites. Nat Commun. 10, 3950 (2019), which is herein incorporated by reference in its entirety. Immunization of human volunteers with genetically attenuated Plasmodium parasites has been shown to lead to complete protection from parasite infection months later. (See Murphy, Sean C et al. “A genetically engineered Plasmodium falciparum parasite vaccine provides protection from controlled human malaria infection.” Science translational medicine vol. 14, 659 (2022), which is herein incorporated by reference in its entirety.)

[0102] However, this protective immune response is reduced in individuals who live in malaria-endemic regions of the world and young children, necessitating the development of improved genetically attenuated Plasmodium parasite vaccines. (See Jongo, Said A. et al. “Safety, Immunogenicity, and Protective Efficacy against Controlled Human Malaria Infection of Plasmodium falciparum Sporozoite Vaccine in Tanzanian Adults.” The American journal of tropical medicine and hygiene vol. 99, 2 (2018): 338-349; and Oneko, Martina et al. “Safety, immunogenicity and efficacy of PfSPZ Vaccine against malaria in infants in western Kenya: a double-blind, randomized, placebo-controlled phase 2 trial.” Nature medicine vol. 27,9 (2021): 1636-1645, both of which are herein incorporated by reference in their entirety.)

[0103] The asexual erythrocytic stages of Plasmodium spp. parasites are haploid and can be transfected. This allows for generation of transgenic parasites that can express foreign antigens (e.g. GFP, luciferase, LacZ, perfringolysin O) [47-67], or express Plasmodium spp. proteins from other Plasmodium species [68-77], Most of these transgenic parasites have been used as reporterparasites or as tools to assess immunogenicity. However, recombinant P. berghei SPZ expressing Pf circumsporozoite protein (CSP) have been developed as a vaccine and shown to induce protective immune responses in humans [74, 75, 77],

[0104] The immunogen of attenuated Sanaria® PfSPZ vaccines provides up to 100% protection against controlled human malaria infection (CHMI) and at least 18 months protection against Pf in the field despite inducing only modest CD8+ T cell responses in PBMCs. PfSPZ vaccines induce protective, tissue resident CD8+ T cells in the liver, because all PfSPZ traffic to hepatocytes, where they generally develop for 5-6 days and the attenuated SPZ immunogen of the vaccine arrests during liver stage [17-20],

[0105] Appropriately attenuated sporozoites cease development prior to reentry into the blood stream where they would infect red blood cells causing the signs symptoms and pathology of malaria. Attenuated Plasmodium-species sporozoites have proven useful as immunogens in highly efficacious attenuated whole parasite vaccines. Inventively, as disclosed herein, attenuated Plasmodium-species sporozoites can also be utilized as liver-specific vectors for the introduction of host immunity modifiers, heterologous antigens, particularly antigens related to hepatic infections and diseases, or both.Attenuated Plasmodium Sporozoites

[0106] Attenuated Plasmodium sporozoites have demonstrated potent immunogenicity in whole-parasite malaria vaccines. In murine HBV-transgenic models, intravenous administration of attenuated Plasmodium yoelii sporozoites induced a broad innate and adaptive immune response within the liver and led to a near-complete reduction in HBV replication, as measured by relaxed circular DNA (rcDNA) levels. These findings suggest that liver-localized immune activation may provide an effective therapeutic approach for HBV control.

[0107] Accordingly, in certain aspects, the presently disclosed vaccine platform combines genetic alteration / modification of Plasmodium parasites by gene deletion to arrest liver stage development with the delivery noa-Plasmodium protein modifiers of the host immune system into infected hepatocytes to enhance the immune response and improve vaccine efficacy.

[0108] The disclosed genetically modified Plasmodium parasites can also serve as cargo platforms for the deliver}' of novel proteins / antigens including hepatotropic viral proteins or tumor neoantigens into the liver. Examples of such hepatotropic viral proteins include, but are not limited to, Hepatitis B Virus (HBV) DNA polymerase, HBV major surface antigen, HBV e antigen, and HBV core antigen or HBVs antigen.

[0109] The present disclosure further provides vaccine compositions and methods for inducing an immune response in a vertebrate host against, a Plasmodium parasite. The host is vaccinated with a multiplicity of live attenuated transgenic Plasmodium parasites that encode anddeliver non-Plasmodium protein modifiers of the host immune system into infected hepatocytes to enhance the host immune response to malaria parasites, other hepatotropic infections, or hepatic maladies.

[0110] In certain aspects, the non-Plasmodium protein is an antagonist of interferon regulatory factor (IRF) or an interferon signaling protein. Nonlimiting examples non-l’lcisinodium protein modifiers include antagonists of IRF3 and / or INF-1 signaling proteins. To date, no whole parasite vaccine platform has delivered neo-antigens and / or proteins expressed in hepatotropic (liver-targeting) pathogens to mount antiviral immune responses against pathogens that infect the liver, such as the hepatitis viruses.[OHl] In aspects of the current disclosure, the Plasmodium -species sporozoites (e.g., an attenuated Plasmodium-s cies sporozoites disclosed herein) are used as liver-targeting vectors for the expression of heterologous antigens and / or immunomodulators useful for generating and / or enhancing liver-specific antigen expression and / or immune responses.

[0112] In some aspects, the attenuated Plasmodium-s ecies sporozoites comprises a nucleic acid sequence encoding a heterologous antigen and / or an immunomodulator inserted into the Plasmodium sporozoite genome. In some aspects, the species of Plasmodium sporozoites are P. falciparum. In some aspects, the Plasmodium sporozoite ceases developmental progression during late liver stage. In some aspects, the Plasmodium sporozoite lacks a functional LINUP gene (e.g., SEQ ID NO: 19). In some aspects, the full LINUP gene is knocked out from the Plasmodium sporozoite genome. In some aspects, a functional portion of the LINUP gene is deleted from the Plasmodium sporozoite genome. In some aspects, the Plasmodium sporozoite is a linup' knockout, e.g.. the Plasmodium sporozoites that comprise PfSPZ-LARC sporozoites. In some aspects, the sporozoite lacks a gene encoding a functional LINUP protein (e.g., SEQ ID NO: 37).

[0113] In some aspects, the Plasmodium sporozoite lacks a functional Mei2 gene (e.g., SEQ ID NO: 18). In some aspects, the full Mei2 gene is knocked out from the Plasmodium sporozoite genome. In some aspects, a functional portion of the Mei2 gene is deleted from the Plasmodium sporozoite genome. In some aspects, the sporozoite lacks a gene encoding a functional Mei2 protein (e.g., SEQ ID NO: 20).

[0114] In some aspects, the Plasmodium sporozoite lacks a functional LINUP gene (e.g., SEQ ID NO: 19) and / or a functional Mei2 gene (e.g., SEQ ID NO: 18). In some aspects, the LINUP gene and / or Mei 2 gene are deleted from the Plasmodium sporozoite genome. In some aspects, functional portions of the LINUP gene and / or Mei2 gene are deleted from the Plasmodium sporozoite genome. In some aspects, the Plasmodium sporozoites are a LINUP and Mei2knockout (i.e., meiZ / linup), e.g., the Plasmodium sporozoites comprise PfSPZ-LARC2 (mei2~ / linup") sporozoites.

[0115] PPSPZ-LARC2 (mei2' / linup'') sporozoites express up to 2,500 -3,500 proteins not expressed in early arresting radiation attenuated PfSPZ, arrest at the late liver stage, 5-6 days after administration, and do not cause Pf blood stage infection. Thus, as disclosed herein. PfSPZ -LARC2 vectors are non-pathogenic, and particularly, do not cause malaria.

[0116] PfSPZ can be genetically engineered to be replication competent during most of liver stage and arrest their development late in liver stage (e.g., Late Arresting Replication Competent - “LARC’'). PfSPZ mutants with this characteristic include PfSPZ-Afe / 2 (lacking a functional Mei2 gene (mei2~)

[0093] ; PfSPZ-LARC (lacking a functional LINUP gene (linup')), and PfSPZ-LARC2 (lacking both a functional Mei2 and a functional LINUP gene (mei2~ / linup')) [86-87], In some aspects, the sporozoite arrest at days 6-7, days 6-8, days 6-9, or days 6-10 during liver stage development.

[0117] The immunogen of Sanaria® PPSPZ-LARC2 Vaccine comprises Pf sporozoites with deletions of both the LINUP and Mei2 genes (linup' / mei2')

[0046] , In certain aspects of the disclosure the PfSPZ-LARC2 Vaccine is used as the vector to express Hepatitis B virus (HBV) protective antigens or antigenic fragments thereof, or functional human Interleukin-2 (hIL-2) or a derivative, or both.

[0118] In some aspects, one or more heterologous transgenes encoding one or more antigens of a human hepatic pathogen (e.g., a hepatitis virus) are inserted into the Plasmodium sporozoite genome (e.g., PfSPZ-LARC2 genome knockin).

[0119] In some aspects, the knockin insertion is at the site or region where the LINUP and / or Mei2 gene is deleted in PISPZ-LARC2. In some aspects, the human hepatic pathogen comprises hepatitis B virus (HBV). In some aspects, the one or more antigens of the human hepatic pathogen comprise one more of the HBV antigens, e.g., HBcAg, HBsAg, HBpolAg, HBxAg or antigenic fragments thereof.

[0120] In some aspects of the present invention transgenic Plasmodium parasites are generated that encode noa-Plasmodium protein modifiers of the host immune system that enhance the immune response of the host and / or enhance pathogen antigenicity (e.g. to malaria parasites) within infected hepatocytes, as well as the immune responses to other hepatotropic infections, or hepatic maladies. In contrast to existing whole parasite vaccines, which induce an immune response that is dependent on endogenous or naturally encoded parasite factors, the presently disclosed parasite vaccine platform combines naturally encoded anti-parasite immunity with the inclusion of know n modifiers of host immunity, such as Interferon Regulatory' Factors (IRFs). Non-limiting examples of IRFs include IRF3, IRF1, IRF7, and other regulators of Type 1Interferon (IFN-1) sensors and kinases shown or predicted to induce IFN-1 signaling, such as melanoma differentiation-associated gene 5 (MDA5), DNA-dependent protein kinase (DNA PK), mitochondrial antiviral-signaling protein (MAVS), and TANK-binding kinase 1 (TBK1).

[0121] In certain aspects, the transgenic Plasmodium parasites encode and secrete antagonists of IFN-1 signaling into infected host hepatocytes. These transgenic Plasmodium parasites can be generated, for example, by inserting Interferon Regulatory Factor 3 (1RF3) antagonists into the non-essential / dispensable P230p locus within the Plasmodium genome. Nonlimiting examples of IRF3 antagonists include protein sequences from the following viral antagonists of IRF3: Flavivirus Nonstructural Protein 1 (NS1), Human Immunodeficiency Virus Viral Protein R (HIV VPR). West Nile Virus (WNV) NS1. Severe Acute Respiratory Syndrome Coronavirus 2 Nonstructural Protein 3 (SARS Cov-2 NSP3), SARS Cov-2 NSP13, SARS Cov-2 NSP15, SARS Cov-2 Open Reading Fram 6 (ORF6), SARS Cov-2 ORF3b, SARS Cov-2 NSP1, Ebolavirus VP35, and Ebola virus Nucleoprotein (NP). Integration of IRF3 antagonists into the Plasmodium genome can be achieved using CRISPR-Cas9 recombination technology (see Example 3 and FIG.3). A novel DNA sequence is generated containing the 5’ untranslated region (UTR) of the dispensable P230p gene at the N terminus, then the promoter sequence of the Plasmodium liver stage protein UIS4 (up-regulated in infective sporozoites gene 4), the sequence of the Plasmodium circumsporozoite protein (CSP) PEXEL {Plasmodium export element), the selected IRF3 antagonist, the U1S4 3’UTR, and then lastly the 3’UTR downstream of the endogenous P230p gene (SEQ ID NOs: 40 and 41). This sequence is cloned into a plasmid that is then transfected into Plasmodium blood stage schizonts. Transfected schizonts are then intravenously injected in commercially available Swiss Webster (SW) mice. Drug treatment (Pyrimethamine in the drinking water) ensures that only transgenic parasites that have acquired the plasmid and its encoded pyrimethamine resistance cassette are retained.

[0122] In some aspects, the IFN-1 and / or IRF3 antagonists are operably linked to the promoter region of the Plasmodium liver stage gene UIS4 and to the PEXEL sequence to facilitate transport into the infected hepatocyte. In addition, a nuclear localization sequence (NLS) can be appended to the antagonists to target nuclear components of the IFN-I signaling cascade. (See Mueller, Ann-Kristin et al. “Plasmodium liver stage developmental arrest by depletion of a protein at the parasite-host interface.’' Proceedings of the National Academy of Sciences of the United States of America vol. 102.8 (2005): 3022-7; and Gabriela, Mikha et al. "‘Sequence elements within the PEXEL motif and its downstream region modulate PTEX dependent protein export in Plasmodium falciparum.” Traffic (Copenhagen, Denmark) vol. 25,1 (2024), both of which are herein incorporated by reference in their entirety).Immune Modulation by Means of Attenuated Plasmodium Sporozoite Administration

[0123] Direct intra-hepatic immune modulation provides a novel approach for therapeutic immunization. The unique tropism of malaria parasite sporozoites for hepatocytes provides a singular opportunity for liver-specific immune modulation, even absent the addition of antigens or cytokines specific to the pathogen. For example, a single intravenous administration of the mouse host range Plasmodium yoelii (Py) SPZ to HBV-trans genic mice, provided near complete reduction in HBV replication, with a concomitant rigorous innate immune reaction in the liver

[0021] , Antiviral cytokine production resulted in influx of natural killer cells, macrophages, and T cells. HBV gene expression and replication were suppressed, likely due to enhanced production of interferon (IFN)-gamma, IFN-alpha and IFN-beta [99- 101], The exquisite targeting of the liver by attenuated Plasmodium SPZ. essentially all of which reach the liver sinusoids and invade hepatocytes, is critical to their success in effecting cure, suggesting that intense attenuated Plasmodium SPZ infection of the liver in human subjects chronically infected with HBV might similarly influence the course and pathogenesis of HBV infection in humans. Effector mechanisms for clearance of HBV in humans (IFNgammaproduced by hepatitis B surface antigenspecific cytotoxic T lymphocytes (CTLs) leading to the release of nitric oxide

[0102] ) are similar to effector mechanisms for clearance of infected hepatocytes in human malaria (IFNgammaproduced by Plasmodium-specific CTLs also leading to the release of nitric oxide [17-18]). Other mechanisms, including cytolysis, have also been demonstrated.

[0124] Certain aspects of the disclosure are directed to methods of immune modulation of CHB-specific antigens and HBV replication in CHB-infected subjects, comprising administration of an attenuated Plasmodium-species SPZ of human host range, where attenuation is achieved by radiation attenuation (PfSPZ) or genetic manipulation (PfSPZ Mei2, PfSPZ LARC or PfSPZ LARC 2).

[0125] Certain aspects of the disclosure are directed to Plasmodium sporozoites of human host range which are genetically attenuated to arrest the Plasmodium sporozoite’s developmental progression in late liver stage and prior to red blood cell infection, said sporozoites comprising a heterologous transgene encoding one or more human hepatic pathogen antigens or antigenic fragments thereof, or a human cytokine, or both. In some aspects, the sporozoite species is P. falciparum. In some aspects, the sporozoites ceases developmental progression during late liver stage. In some aspects, the sporozoites lack a functional LINUP gene, a functional Mei2 gene, or both.

[0126] In some aspects. Plasmodium-species attenuated SPZ, e.g., genetically attenuated Plasmodium-species PfSPZ -LARC sporozoites, e.g., PfSPZ-LARC2 sporozoites, are utilized as the platform for targeting liver-specific expression of heterologous antigens of chosen pathogens. In some aspects, Plasmodium-species attenuated SPZ, e.g., genetically attenuatedPlasmodium-species PfSPZ-LARC sporozoites, e.g., PfSPZ-LARC2 sporozoites, are utilized as the platform for targeting liver-specific expression of auxiliary cytokines, e.g., IL-2 or engineered analogues thereof. In some aspects, PfSPZ-LARC2 comprising a heterologous HBV-specific antigen, or comprising 1 or more auxiliary' cytokines, or both, can directly deliver a large payload (genome of PfSPZ is 23 mb on 14 chromosomes) comprising multiple HBV antigens, or the full HBV genome, as well as multiple immune modulators. The sporozoite delivery approach disclosed herein is distinct from all current vaccine strategies.Advantages of Liver-Specific Immune Modulation For a HBV therapeutic Vaccine

[0127] In some aspects, the Plasmodium-species sporozoites (e.g., an attenuated Plasmodium-species sporozoites disclosed herein) are used as liver-targeting or hepatotropic vectors for the expression of an HBV antigen. In some aspects, the Plasmodium-species sporozoites (e.g., an attenuated Plasmodium -species sporozoites disclosed herein) comprises a nucleic acid sequence encoding a HBV antigen (e.g., inserted into the Plasmodium sporozoite genome). In some aspects, the Plasmodium-species sporozoite liver-targeting vector provides liver-specific antigen expression and / or an immune response.

[0128] The sinusoidal vasculature in the liver slows the circulation of blood cells, which allows circulating T cells to more easily recognize HBV antigens on underlying liver cells. However, the lack of efficient antigen presentation by the hepatocyte, allowing T cells to recognize HBV antigens expressed on liver cells, and a sub-optimal cytokine milieu, can inhibit activation and differentiation [2], In addition, the duration of HBV infection, often spanning decades, weakens the antiviral potential of HBV-specific T cells in systemic circulation. Current approaches based on peripheral immunization that is not hepatocyte-vectored are limited by suboptimal trafficking of T cells to the liver, by the tolerogenic immunoregulatory milieu that exists in the liver, and by the constraints placed on the systemic administration of cytokines to overcome dysregulated T cell responses in the liver imposed by dose-limiting systemic toxicity that does not permit high intrahepatic levels.

[0129] In contrast, liver-vectored immune modulation using attenuated Plasmodiumspecies SPZ of human host range disclosed herein offers a novel approach for therapeutic immunization: (1) precise targeting of the liver; (2) robust innate immune stimulation without systemic reactogenicity; (3) the potential to augment antigen-specific immunity by engineering the attenuated Plasmodium-species SPZ to express hepatitis viral antigens; (4) the potential to augment antigen-specific immunity by engineering the PfSPZ to express immunostimulatory molecules as adjuvants.Immunomodulators

[0130] In some aspects, the Plasmodium-species sporozoites (e.g., an attenuated Plasmodium-species sporozoites disclosed herein) are used as liver-targeting vectors for the expression of an immunomodulatory (e.g., an IRF, an IRF antagonist, an interferon signaling protein, Interleukin 2 (IL -2), or an engineered fusion protein thereof). In some aspects, the Plasmodium-species sporozoites (e.g., an attenuated Plasmodium-species sporozoites disclosed herein) comprises a nucleic acid sequence encoding an immunomodulator (e.g., inserted into the Plasmodium sporozoite genome). In some aspects, the Plasmodium-species sporozoite livertargeting vector provides a liver-targeted immune response.

[0131] In certain aspects of the disclosure. Interleukin 2 (IL-2), or an engineered fusion protein thereof, is utilized as an immunomodulator to overcome immune exhaustion / dysregulation and enhance antigen specific T cell responses by stimulating natural killer (NK) cells and effector CD4+and CD8+T cells. However, IL-2 can also stimulate immunosuppressive regulatory' T cells (Tregs)

[0103] , Thus, an engineered IL-2 fusion protein is used, designated cIL-2eRa and comprised of a circularly (c) permuted IL-2 with the extracellular domain (e) of IL-2Ra designed to selectively activate effector lymphocytes bearing the intermediate-affinity IL-2R receptor, and not to activate Tregs

[0104] , Genetically attenuated P. falciparum LARC2 human malaria parasites expressing human (h) cIL-2eRa, hepatitis B core antigen (HBcAg) or both hcIL-2eRa and HBcAg, are utilized. In other aspects of the disclosure, other immunomodulators are substituted for hcIL-2eRa, or additionally provided. These include IL- 12 for potent T-cell stimulation, and IL-15, 18, and 21, broad pro-inflammatory cytokines, for CD8, NK cell and B cell proliferation.

[0132] In some aspects, cIL-2eRa (e.g., SEQ ID NO: 38) is used as an immunomodulator. In some aspects, the immunomodulator encodes a protein comprising an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% to SEQ ID NO: 38.

[0133] Certain aspects of the disclosure are directed to Plasmodium sporozoites of human host range which are genetically attenuated to arrest the Plasmodium sporozoite’s developmental progression in late liver stage and prior to red blood cell infection, said sporozoites comprising a heterologous transgene encoding a human cytokine or an engineered fusion protein thereof, one or more human hepatic pathogen antigens or antigenic fragments thereof, or both. In some aspects, the sporozoite species is P. falciparum. In some aspects, the sporozoites ceases developmental progression during late liver stage. In some aspects, the sporozoites lacks a functional LINUP gene, a functional Mei2 gene, or both.

[0134] In some aspects, the Plasmodium sporozoite genome (e.g., the genome of PfSPZ- LARC2) comprises a nucleic acid sequence encoding a human cytokine (e.g., hIL-2). In someaspects, the Plasmodium sporozoite comprises PfSPZ-LARC2 (mei2⁻ / linup⁻) and a transgene encoding hIL2 or a functional fragment thereof. In some aspects, the Plasmodium sporozoite comprises PfSPZ-LARC2 (mei2 / linup') and a transgene encoding one more of the HBV antigens selected from HBcAg, HBsAg, HBpolAg, HBxAg or antigenic fragments thereof.

[0135] Certain aspects of the disclosure are directed to methods utilizing SPZ vaccine-vectored delivery of a human cytokine to a human subject, e.g., as an adjuvant, for enhancing an antigenic immune response. In some aspects, the immune response is directed to preexisting HBV antigens from a CHB infection. In other aspects, the immune response is directed to concomitantly delivered HBV antigens. In still other aspects, the immune response is directed to the Plasmodium-specific antigens of the SPZ vaccine itself.

[0136] Certain aspects of the disclosure are directed to a Plasmodium-species sporozoite of human host range which is attenuated to arrest the Plasmodium-species sporozoite developmental progression at late liver stage and prior to red blood cell infection (e.g., PfSPZ-LARC2). wherein said Plasmodium-species sporozoite comprises a polynucleotide comprising (i) a heterologous transgene encoding a human immunomodulator, a functional fragment thereof, or an engineered analogue thereof (e.g., hIL-2 or cIL-2eRa), which is operably linked to a promoter and optionally an export signal; (ii) a heterologous transgene encoding one or more antigens of hepatitis B virus (HBV) or antigenic fragments thereof, which is operably linked to a promoter and optionally an export signal. In some aspects, the polynucleotide further comprises a 3’ UTR and / or a 5’ UTR.Hepatitis B Virus

[0137] HBV is a hepatotropic, partially double-stranded DNA virus that replicates through reverse transcription of a pre-genomic RNA intermediate. Following entry into hepatocytes, the HBV genome is converted into covalently closed circular DNA (cccDNA), which serves as a transcriptional template for production of viral proteins and progeny viral DNA. Persistence of cccDNA, integration of HBV DNA into the host genome, and impaired innate and adaptive immune responses contribute to the difficulty in eradicating HBV infection, particularly in chronically infected individuals.

[0138] Current therapeutic options for chronic HBV infection include interferons (IFNs) and nucleos(t)ide analogues (NAs), which effectively suppress viral replication and reduce disease progression, including cirrhosis, liver failure, and hepatocellular carcinoma (HCC). However, these treatments rarely eliminate cccDNA or integrated HBV DNA and therefore do not achieve complete viral eradication. As a result, patients remain at risk of developing HCC despite longterm antiviral therapy.

[0139] A sterilizing cure, characterized by complete elimination of cccDNA and integrated HBV DNA, remains elusive. In contrast, a functional cure — defined by loss of Hepatitis B surface antigen (HBsAg), seroconversion, and undetectable serum HBV DNA — has emerged as a more attainable therapeutic goal. Presently, such functional cures are observed in fewer than 5% of patients following IFN or NA therapy. Efforts are ongoing to achieve higher rates of functional cure by combining antiviral agents targeting novel viral pathways with immunomodulatory therapies aimed at restoring both innate and adaptive immune functions.

[0140] Extensive human and animal studies indicate that the outcome of HBV infection is strongly influenced by the kinetics, breadth, and functionality of HBV-specific adaptive immune responses. HBV-specific CD8+T cells serve as primary effectors of viral clearance through cytolytic and cytokine-mediated mechanisms, while HBV-specific CD4+T cells provide essential help for CD8+T cell and B cell responses. Nevertheless, HBV infection induces substantial T cell exhaustion and dysfunction, resulting in ineffective viral control.

[0141] Recent research suggests that circulating HBsAg levels are not directly correlated with T cell magnitude or functionality. Moreover, studies in HBV-infected mouse models have shown that reducing or eliminating HBsAg does not necessarily restore HBV-specific T cell responses. Instead, hepatocellular priming of naive HBV-specific CD8+T cells result in incomplete differentiation and effector function, a process that can be partially reversed by interleukin-2 (IL-2) treatment. Similarly, analyses of peripheral T cells from chronically infected patients demonstrate antigen-specific differences in proliferative capacity and cytotoxic potential, with uniformly reduced antiviral cytokine production.

[0142] Various immunomodulatory approaches, including oral Toll-like receptor 7 (TLR7) and Toll-like receptor (TLR8) agonists, have shown promise in preclinical models of HBV infection but have failed to translate into clinical efficacy, often due to dose-limiting toxicity. Accordingly, there remains an unmet need for therapeutic strategies capable of inducing durable antiviral immune responses in the liver with acceptable safety profiles.Antigens of Hepatic Pathogens

[0143] Certain aspects of the disclosure are directed to a sporozoite comprising one or more heterologous transgenes encoding antigens of human hepatic pathogens or antigenic fragments thereof. In some aspects, the human hepatic pathogen is a hepatitis virus (e.g., HAV, HBV, or HCV). In some aspects, the human hepatic pathogen antigen comprises one or more HBV antigens HBxAg, a HBpolAg, a HBsAg, a HBcAg, any antigenic fragment thereof, or any combination thereof.

[0144] In some aspects, the antigen of the PfSPZ-LARC2 HBV knock-in is the entire hepatitis B virus (HBV) genome.

[0145] In some aspects, the HBV antigen is derived from the HBV pol protein (e.g., SEQ ID NO: 1), the HBV large S protein (e.g., SEQ ID NO: 2), the HBV X protein (e.g., SEQ ID NO: 3), or the HBV core protein (e.g., SEQ ID NO: 4).

[0146] In some aspects, the HBV antigen coding sequence is codon optimized. In some aspects, the transgene encoding the HBV antigen is a HBV pol protein coding sequence (e.g., SEQ ID NO: 21), a HBV large S protein coding sequence (e.g., SEQ ID NO: 22), a HBV X protein coding sequence (e.g., SEQ ID NO: 23), or a HBV core protein coding sequence (e.g., SEQ ID NO: 24). In some aspects, the transgene comprises a nucleic acid sequence having at least 85%, 90%, 95%, 96%. 97%, 98%, 99%, or 100% to SEQ ID NO: 21, 22, 23, 24, or any combination thereof. In some aspects, the transgene comprises a nucleic acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% to SEQ ID NO: 21. In some aspects, the transgene comprises a nucleic acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% to SEQ ID NO: 22. In some aspects, the transgene comprises a nucleic acid sequence having at least 85%. 90%. 95%, 96%, 97%, 98%, 99%, or 100% to SEQ ID NO: 23. In some aspects, the transgene comprises a nucleic acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% to SEQ ID NO: 24.

[0147] In some aspects, the antigen of the human hepatic pathogen is a protein or a portion of a protein derived from HBV (e.g., any one of the amino acid sequences of SEQ ID Nos: 1-4).

[0148] In some aspects, the human hepatic pathogen antigen comprises one or more of a protein or a portion of a protein derived from HBpol (e.g., the amino acid sequence of SEQ ID NO: 1). In some aspects, the HBpolAg comprises an amino acid sequence having at least 85%, 90%. 95%. 96%, 97%, 98%, 99%, or 100% to SEQ ID NO: 1. In some aspects, the transgene encoding HBpolAg comprises a nucleic acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% to SEQ ID NO: 21.

[0149] In some aspects, the human hepatic pathogen antigen comprises one or more of a protein or a portion of a protein derived from HBs (e.g., the amino acid sequences of SEQ ID NO: 2). In some aspects, the HBsAg comprises an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% to SEQ ID NO: 2. In some aspects, the transgene encoding HBsAg comprises a nucleic acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% to SEQ ID NO: 22.

[0150] In some aspects, the human hepatic pathogen antigen comprises one or more of a protein or a portion of a protein derived from HBx (e.g., the amino acid sequences of SEQ ID NO: 3). In some aspects, the HBxAg comprises an amino acid sequence having at least 85%, 90%, 95%. 96%, 97%, 98%, 99%, or 100% to SEQ ID NO: 3. In some aspects, the transgeneencoding HBxAg comprises a nucleic acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% to SEQ ID NO: 23.

[0151] In some aspects, the human hepatic pathogen antigen comprises one or more of a protein or a portion of a protein derived from HBc (e.g., the amino acid sequences of SEQ ID NO: 4). In some aspects, the HBcAg comprises an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% to SEQ ID NO: 4. In some aspects, the transgene encoding HBcAg comprises a nucleic acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% to SEQ ID NO: 24.

[0152] While some reports have focused on dissecting T cell responses against the HBV surface antigen (HBsAg) or core antigen (HBcAg), the proteins X (HBxAg) and polymerase (HBpolAg) represent alternative targets, as both are vital for viral persistence; HBxAg is expressed only in infected hepatocytes and is involved in progression to HCC. HBpolAg is more immunogenic than HBsAg in HBV transgenic mice, and high frequencies of Pol-specific T cells are associated with viral control after discontinuation of viral replication inhibitors in patients.

[0153] While the HBV surface antigen HBsAg and the HBV core antigen HBcAg induce protective antibody responses, they are not considered a strong producer of protective T cell immune responses

[0078] , which are critical to recognize and result in the elimination of infected cells. HBsAg vaccines are not effective therapeutic vaccines against chronic HBV infection. Recent evidence indicates that the Pol and X proteins and core antigen are inducers of robust cellular immune responses, and that these cellular immune responses are critical for a successful therapeutic vaccine against chronic HBV [11, 78, 79], In some aspects, the full HBV genome is transfected into and expressed from PfSPZ-LARC2.

[0154] In certain aspects of the disclosure, in order for the antigens to generate cellular immune responses in the liver, they are expressed during liver stage development, and upon death of the PfSPZ-LARC2 within hepatocytes, taken up by antigen presenting cells (e.g., in the liverdraining lymph nodes) for induction of CD8 T cell responses, or in case of hIL2, released from the infected hepatocytes for optimal enhancement of local T cell responses and potentially crosspriming.

[0155] In some aspects, a heterologous promoter is used to express the antigen. In some aspects, a promoter is selected from the group consisting of PfCSP, PfEXPl, PfLSAl, and PfHSP70 promoters. Based on expression of other foreign antigens (e.g. GFP) by Pf developing in hepatocytes, the PfCSP promoter is preferred as it is active throughout liver stage development. Thus, the pre-erythrocytic stage constitutive PfCSP promoter, signal sequence and export motif is used to drive expression of the foreign antigens.

[0156] In some aspects, the PfSPZ-LARC2 further comprises a. Plasmodium falciparum (Pf) PfCSP, PfEXPl, PfLSAl, or PfHSP70 promoter, wherein said promoter is configured to drive expression of one or more heterogeneous transgenes (e.g., HBV antigens). In some aspects, the promoter is PfCSP.

[0157] In some aspects, the promoter comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%. 96%, 97%, 98%, 99% or 100% sequence identity to any one of SEQ ID NOs 25, 29, 30, or 31.

[0158] In some aspects, the promoter comprises a sequence having at least 95%. 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 25. In some aspects, the promoter comprises a sequence having at least 95%. 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 29. In some aspects, the promoter comprises a sequence having at least 95%. 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 30. In some aspects, the promoter comprises a sequence having at least 95%. 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 31.

[0159] In some aspects, the antigen of the human hepatic pathogen comprises hepatitis B virus (HBV) HBxAg or functional fragment thereof, wherein the HBxAg or functional fragment thereof is expressible during liver stage sporozoite infection.

[0160] In some aspects, the antigen of the human hepatic pathogen comprises HBV HBpolAg or functional fragment thereof, wherein the HBpolAg or functional fragment thereof is expressible during liver stage sporozoite infection.

[0161] In some aspects, the antigen of the human hepatic pathogen comprises HBV HBsAg or functional fragment thereof, wherein the HBsAg or functional fragment thereof is expressible during liver stage sporozoite infection.

[0162] In some aspects, the antigen of the human hepatic pathogen comprises HBV HBcAg or functional fragment thereof, wherein the HBcAg or functional fragment thereof is expressible during liver stage sporozoite infection.

[0163] In some aspects, one or more of the antigens of the human hepatic pathogen are selected from the group consisting of HBV HBxAg or functional fragment thereof, HBpolAg or functional fragment thereof, HBsAg or functional fragment thereof, and HBcAg or functional fragment thereof, and optionally also hIL2 cytokine.Methodology For Creation of Knockin Strains

[0164] Certain aspects of the disclosure are directed to genetically engineered attenuated strains of Plasmodium-species, preferably Plasmodium falciparum, and in certain aspects, expressing one or more heterologous antigens. These strains can comprise heterologous antigens of pathogens targeting the liver, preferably heterologous antigens of Hepatitis B Virus,particularly, the antigens including: 1) HBpolAg; 2) HBxAg; 3) HBcAg; and 4) HBsAg. and optionally, also including hIL2 or an engineered analogue (FIGS. 4A-4B). In some aspects, expression of the transgene is preferably driven by the PfCSP promoter. The PfCSP promoter has been shown to be active throughout liver stage development. In some aspects, a promoter is chosen from the group comprising PfCSP. PfEXPl, PfLSAl, or PfHSP70 promoters.

[0165] In some aspects, export motif (EXP) is used for secretion of the antigens into the cytosol of the liver cells. Expression cassettes can be designed in which inserts target the two LARC2 knockout sites (pFC-yFCU plasmids) Mei2

[0086] and linup

[0087] , to which the insert antigens will be directed. These constructs are designed for plasmid-free, marker-free transfections as demonstrated with PILARC2. In some aspects, to ensure sporozoite-stage surface expression of these antigens, a 967 base pair fragment of the PfCSP promoter is amplified

[0088] plus the first 77 amino acids containing the signal peptide and two PEXEL export motif sequences

[0089] , fused to the 5 ' end of the antigens. In some aspects, HBV antigens can use ribosome skipping sequences to express multiple polypeptides from the same promoter. In some aspects, the viral 2A peptide (V2A) can be used for this purpose to express multiple HBV antigens (or hIL2) within the Mei2 and LINUP loci.

[0166] In alternative aspects, other non-essential loci, e.g., Pfs47 or cg6, can be used, which can be targeted for genetic modification without inhibiting sporozoite production in mosquitoes [90, 91], In certain aspects, the disclosure provides novel nucleic acid sequences (SEQ ID NOs: 39-41) incorporated into the P230p gene of Plasmodium yoelii blood stage schizonts. In certain aspects, the novel nucleic acid sequence is at least 80% (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%. 92%, 93%, 94%, 95%. 96%, 97%, 98%, 99% or 100%) identical to any one of SEQ ID NOs: 39-41. For example, integration of IRF3 antagonists into the Plasmodium yoelii genome can be achieved using CRISPR-Cas9 recombination technology (see Example 3 and FIG. 3). A novel DNA sequence is generated containing the 5’ untranslated region (UTR) of the dispensable P230p gene at the N terminus, then the promoter sequence of the Plasmodium liver stage protein UIS4 (up-regulated in infective sporozoites gene 4), the sequence of the Plasmodium circumsporozoite protein (CSP) PEXEL (Plasmodium export element), the selected IRF3 antagonist, the UIS4 3’ UTR, and then lastly the 3’ UTR downstream of the endogenous P230p gene (SEQ ID NO: 40 and SEQ ID NO: 41). This sequence is cloned into a plasmid that is then transfected into Plasmodium yoelii blood stage schizonts. Transfected schizonts are then intravenously injected in commercially available Swiss Webster (SW) mice. Drug treatment (Pyrimethamine in the drinking water) ensures that only transgenic parasites that have acquired the Plasmid and its encoded pyrimethamine resistance cassette are retained.

[0167] In certain aspects, the IFN-1 and / or IRF3 antagonists are operably linked to the promoter region of s Plasmodium liver stage gene UIS4 and to the PEXEL sequence to facilitate transport into the infected hepatocyte. In addition, a nuclear localization sequence (NLS) can be appended to the antagonists to target nuclear components of the IFN-I signaling cascade. (See Mueller, Ann-Kristin et al. “Plasmodium liver stage developmental arrest by depletion of a protein at the parasite-host interface. " Proceedings of the National Academy of Sciences of the United States of America vol. 102,8 (2005): 3022-3027; and Gabriela, Mikha et al. “Sequence elements within the PEXEL motif and its dow nstream region modulate PTEX-dependent protein export in Plasmodium falciparum." Traffic (Copenhagen, Denmark) vol. 25,1 (2024), both of which are herein incorporated by reference in their entirety).

[0168] In some aspects, to further regulate and improve expression, a 3’ UTR, such as that of PfCSP (SEQ ID NO: 26) or HSP70 (SEQ ID NO: 30), can be added. In some aspects, for preparation of the knockin plasmid, a 20-bps single guide RNA is inserted for the endonuclease Cas9 to induce double strand breaks in the target sequences.Vaccine Compositions and Methods of Use

[0169] Certain aspects of the disclosure are directed to a composition (e.g., a vaccine composition) comprising the Plasmodium sporozoites disclosed herein (e.g., PfSPZ LARC2-HBV, PfSPZ hIL2 and PfSPZ LARC2-HBV-hIL2) and a carrier, e.g.. Plasmodium-sy A s sporozoites of human host range, which are attenuated to arrest the Plasmodium sporozoite’s developmental progression in late liver stage and prior to red blood cell infection, said sporozoites comprising one or more heterologous transgenes encoding one or more antigens of a human hepatic pathogen (e.g., a hepatitis virus), an immunomodulatory, optionally, a human cytokine (e.g.. hIL-2 or an engineered homologue such as hcIL-2eRa) or both.

[0170] In some aspects, the human hepatic pathogen comprises hepatitis A (HAV), hepatitis B (HBV), hepatitis C (HCV), or any combination thereof. In some aspects, these vaccine compositions comprise PfSPZ-LARC or PfSPZ -LARC2, which comprise the coding sequence for one more of the HBV antigens HBcAg, HBsAg. HBpolAg. HBxAg or antigenic fragments thereof.

[0171] In certain aspects, these vaccine compositions comprise PFSPZ-LARC or PfSPZ -LARC2 and a gene encoding hIL-2 or an engineered homologue such as hcIL-2eRa, and which additionally comprise one more of the HBV antigens HBcAg, HBsAg, HBpolAg, HBxAg or antigenic fragments thereof.

[0172] In some aspects, these vaccine compositions provide for expression in a subject of a HBV antigen derived from the HBV pol protein (e.g., SEQ ID NO: 1), the HBV large Sprotein (e.g., SEQ ID NO: 2), the HBV X protein (e.g., SEQ ID NO: 3), or the HBV core protein (e.g., SEQ ID NO: 4).

[0173] In some aspects, the HBV antigen coding sequence is codon optimized. In some aspects, the transgene encoding the HBV antigen is a HBV pol protein coding sequence (e.g., SEQ ID NO: 21), a HBV large S protein coding sequence (e.g.. SEQ ID NO: 22), a HBV X protein coding sequence (e.g., SEQ ID NO: 23), or a HBV core protein coding sequence (e.g., SEQ ID NO: 24). In some aspects, the transgene comprises a nucleic acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% to SEQ ID NO: 21, 22, 23, 24, or any combination thereof. In some aspects, the transgene comprises a nucleic acid sequence having at least 85%, 90%. 91%. 92%. 93%. 94%. 95%, 96%, 97%, 98%, 99%, or 100% to SEQ ID NO: 21. In some aspects, the transgene comprises a nucleic acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% to SEQ ID NO: 22. In some aspects, the transgene comprises a nucleic acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%. 95%, 96%, 97%, 98%. 99%, or 100% to SEQ ID NO: 23. In some aspects, the transgene comprises a nucleic acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% to SEQ ID NO: 24.

[0174] In some aspects, the antigen of the human hepatic pathogen is a protein or a portion of a protein derived from HBV (e.g., any one of the amino acid sequences of SEQ ID Nos: 1-4).

[0175] In some aspects, the human hepatic pathogen antigen comprises one or more of a protein or a portion of a protein derived from HBpol (e.g., the amino acid sequence of SEQ ID NO: 1). In some aspects, the HBpolAg comprises an amino acid sequence having at least 85%, 90%. 91%. 92%. 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% to SEQ ID NO: 1. In some aspects, the transgene encoding HBpolAg comprises a nucleic acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% to SEQ ID NO: 21.

[0176] In some aspects, the human hepatic pathogen antigen comprises one or more of a protein or a portion of a protein derived from HBs (e.g., the amino acid sequences of SEQ ID NO: 2). In some aspects, the HBsAg comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% to SEQ ID NO: 2. In some aspects, the transgene encoding HBsAg comprises a nucleic acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%. or 100% to SEQ ID NO: 22.

[0177] In some aspects, the human hepatic pathogen antigen comprises one or more of a protein or a portion of a protein derived from HBx (e.g., the amino acid sequences of SEQ ID NO: 3). In some aspects, the HBxAg comprises an amino acid sequence having at least 85%, 90%, 91%, 92%. 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% to SEQ ID NO: 3. In someaspects, the transgene encoding HBxAg comprises a nucleic acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% to SEQ ID NO: 23.

[0178] In some aspects, the human hepatic pathogen antigen comprises one or more of a protein or a portion of a protein derived from HBc (e.g., the amino acid sequences of SEQ ID NO: 4). In some aspects, the HBcAg comprises an amino acid sequence having at least 85%, 90%.91%, 92%, 93%. 94%. 95%, 96%, 97%, 98%, 99%, or 100% to SEQ ID NO: 4. In some aspects, the transgene encoding HBcAg comprises a nucleic acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% to SEQ ID NO: 24.

[0179] In some aspects, the carrier is selected from the group consisting of water, PBS, saline, human serum albumin, or any combination thereof.

[0180] In some aspects, vaccine compositions comprise an attenuated Plasmodium SPZ, including the knocked-in heterologous antigens and host immunity modifier SPZ disclosed herein, and one or more adjuvants. An adjuvant is a substance that enhances the immune response when administered together with an immunogen, antigen, or in this case with the sporozoite vector.

[0181] In some aspects, an adjuvant is co-administered (e.g., separately or in the same composition) with the vaccine. In certain aspects, the adjuvant is 7DW8-5 [97-98],

[0182] Certain aspects of the disclosure are directed to a method of expressing a transgenic human hepatic pathogen antigen or a human cytokine intra-hepatically in a subject, the method comprising administering to the subject an effective amount of a Plasmodium sporozoite (e.g., PfSPZ LARC2-HBV and PfSPZ LARC2-HBV-hIL2 or a composition disclosed herein.

[0183] Certain aspects of the disclosure are directed to a method of generating an immune reaction to a transgenic human hepatic pathogen or a human cytokine intra hepatically in a subject, the method comprising administering to the subject an effective amount of a Plasmodium sporozoite (e.g., PfSPZ LARC2-HBV and PfSPZ LARC2-HBV-hIL2) or a composition disclosed herein.

[0184] Certain aspects of the disclosure are directed to a method of preventing or reducing the likelihood of an infection of a human pathogen in a subject comprising administering to the subject an effective amount of a Plasmodium sporozoite (e.g., PfSPZ LARC2-HBV and PfSPZ LARC2-HBV-hIL2) or a composition disclosed herein.

[0185] Certain aspects of the disclosure are directed to a method of treating a hepatic infection in a subject in need thereof comprising administration to the subject an effective amount of a Plasmodium sporozoite (e.g., PfSPZ LARC2-HBV and PfSPZ LARC2-HBV-hIL2) or a composition disclosed herein.

[0186] In some aspects, the subject is a human subject.

[0187] In some aspects, the administration is by intravenous (IV) direct venous injection (DVI). In some aspects, administration is intramuscular, subcutaneous, transdermal or by implantation.

[0188] In some aspects, the subject suffers from a Hepatitis B viral infection.

[0189] In some aspects, the Hepatitis B viral infection is a chronic infection.

[0190] In some aspects, the methods disclosed herein prevent or reduce the likelihood of liver cancer (HCC) in a subject having or at risk of having a chronic hepatitis infection.

[0191] In some aspects the methods disclosed herein modulate the repertoire of HBV-specific T cell responses, e.g. magnitude and / or diversity (CD8 regulatory, gamma / delta, and the like).

[0192] In some aspects the methods disclosed herein modulate B cell responses, either directly or through T cells.

[0193] In some aspects, Plasmodium-species attenuated SPZ, e.g., genetically attenuated PfSPZ-LARC or PISPZ-LARC2, are utilized as the platform for targeting liver-specific expression of heterologous antigens of chosen pathogens, or antigenic fragments thereof, as well as auxiliary cytokines in the methods described herein. In certain aspects, these transgenic Plasmodium-species sporozoites expressing heterologous antigens against hepatocytic pathogens, and particularly purified Plasmodium-species genetically attenuated sporozoites, are prepared aseptically [87, 92-93], Methodology for aseptic preparation is known in the art as exemplified by

[0038] ,

[0194] Genetically attenuated transgenic Plasmodium-species sporozoites can be administered in a dosage regimen of one to three doses, and usually are administered parenterally, including subcutaneous, intradermal and intravenous administration, preferably by intravenous direct venous inoculation (DVI) methodology. A suitable dose of genetically attenuated Plasmodium sporozoites, such as P. falciparum genetically attenuated sporozoites (e.g., PfSPZ LARC2-HBV and PfSPZ LARC2-HBV-hIL2), per inoculation may be between about 10,000 to about 10 million sporozoites, preferably between 100,000 and 1,000,000 sporozoites. In some aspects, the vaccine is administered with an adjuvant, for example 7DW8-5 as described in [39; 97; 98], and such administration in conjunction with adjuvant would likely reduce and number of sporozoites required per dose.

[0195] Such vaccines are useful prophylactically for prevention of, or reduction of severity in the likelihood of infection, by the pathogen to which the transgene product is directed. In an embodiment, PfSPZ LARC2-HBV and PfSPZ LARC2-HBV-hIL2 are useful for the prevention, reduction of severity in the likelihood of infection, of HBV infection, its manifestations, symptoms or its pathology, and in the progression of CHBV infection to HCC.

[0196] In some aspects, compositions and vaccines comprising aseptically prepared genetically attenuated purified sporozoites provide partial, enhanced, or full protection in human and other mammalian subjects not previously exposed to a HBV-causing pathogen, or exposed, but not fully protected. These compositions and vaccines are similarly useful to reduce the chance of becoming ill when one is infected, reduce the severity of the illness, reduce the concentration of virus in the infected person, and reduce mortality rates from HCC in populations exposed to HBV.

[0197] In certain aspects, these vaccines are used therapeutically to treat chronically HBV-infected individuals to eliminate or reduce the presence of HBV-specific antigens, particularly HBsAg. and concomitant seroconversion. In certain aspects, the seroconversion results in a function cure of HBV.

[0198] The prevention and / or treatment of HBV, either acute or chronic infection, may be readily ascertained by the skilled practitioner by evaluation of clinical or pathological manifestations associated with HBV infection. Thus, according to the methods of the present invention, the subject shows improved or absent clinical signs, symptoms or pathological manifestations of HBV follow ing administration of a vaccine comprising purified live attenuated Plasmodium transgenic sporozoites.

[0199] In some aspects, the administering step results in infection of a hepatocyte of the subject, and development of the genetically attenuated transgenic Plasmodium parasite through liver stage, providing the expression of transgenic antigens as well an array of Plasmodium-specific antigens, and the generated immune response ameliorates or protects against infection from a subsequent w ildtype parasite challenge, or in the case of chronically infected individuals, a reduction in expression of pathogen-specific antigens. Accordingly, in some aspects comprising PfSPZ-LARC2-HBV-hIL-2, the disclosed methods confer protective immunity sufficient to reduce or prevent the symptoms of HBV in at least 60% of subjects, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% of subjects, following exposure to wildtype HBV, or in the case of chronically infected individuals, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% reduction in the expression of pathogen-specific antigens.

[0200] Effective and optimal dosage ranges for vaccines and immunogens can be determined using methods known in the art. Guidance as to appropriate dosages to achieve an anti-malarial effect is provided from the exemplified assays disclosed herein. More specifically, results from the immunization pattern described herein and in cited references can be extrapolated by persons having skill in the requisite art to provide a test vaccination schedule. Volunteer subjects are inoculated with varying dosages at scheduled intervals and test blood samples are evaluated for levels of protection against malaria upon subsequent challenge with infectiveparasites. Such results can be used to refine an optimized immunization dose and dosage regimen (schedule) for effective immunization of mammalian, specifically human, subjects.

[0201] An immune response in a subject can be measured by standard tests including, but not limited to the assessment of humoral and cellular immune responses, including, but not limited to: measurement of antigen specific or parasite stage specific antibody responses; direct measurement of peripheral blood lymphocytes by means known to the art; natural killer cell cytotoxicity assays

[0040] cell proliferation assays

[0041] immunoassays of immune cells and subsets [42, 43] and skin tests for cell mediated immunity

[0044] , Various methods and analyses for measuring the strength of the immune system have been described, for example,

[0045] ,

[0202] The vaccines provided comprise aseptic and non-aseptic compositions (preferably aseptic) of purified live attenuated transgenic Plasmodium sporozoite substantially free of attendant material, and compositions with a pharmaceutically acceptable diluent, excipient, or carrier. Methods of aseptic preparation of Plasmodium sporozoites are known in the art

[0095] as are methods of purification of Plasmodium sporozoites

[0096] , both of which are incorporated by reference in their entirety. These vaccines are effective in preventing or mitigating malaria upon subsequent challenge with infectious parasites. Methods of formulating pharmaceutical compositions and vaccines are well known to those of ordinary skill in the art [see, e.g., 46],

[0203] Comprehended by the invention are vaccine compositions, aseptically prepared or otherwise, comprising purified, live attenuated or non-attenuated Plasmodium sporozoites along with appropriate diluent and buffer. Diluents, commonly Phosphate Buffered Saline (PBS), or Normal Saline (NS), are of various buffer content pH and ionic strength. Such compositions may also include an excipient such as serum albumin, particularly human serum albumin. Serum albumin may be purified from naturally occurring sources such as human blood or be produced by recombinant DNA or synthesis technologies. Such compositions may also include additives such as antioxidants e.g., ascorbic acid, sodium metabisulfite, and / or preservatives or cryopreservatives. Incorporation of the material into particulate preparations of polymeric compounds such as polylactic acid, polyglycolic acid, etc. or into liposomes may also be used. (See, e.g.,

[0046] pages 1435-1712 which are herein incorporated by reference).

[0204] In order to determine the effective amount of the vaccines, the ordinary skilled practitioner, considering the therapeutic context, age, and general health of the recipient, will be able to ascertain proper dosing. The selected dosage depends upon the desired therapeutic effect, on the route of administration, and on the duration of the treatment desired. Experiments to determine levels for dosages can be ascertained by one of ordinary skill in the art by appropriate human clinical trials in which various dosage regimens are evaluated for their capacity to elicit protection against malaria.

[0205] Disclosed vaccines and disclosed methods of using these vaccines may be useful as one component in a vaccine regimen, each component in turn comprising a discrete vaccine to be administered separately to a subject. Regimens may include sequential immunization with attenuated Plasmodium species sporozoites and other types of Plasmodium vaccines, so-called, prime-boost strategies. This may include attenuated sporozoites as a prime, and Plasmodium-related recombinant proteins or proteins in adjuvant as a boost or vice versa. This may also include Plasmodium-rQ\aie< DNA vaccines or a recombinant virus, such as adenovirus, that express Plasmodium-relatd proteins, as a prime and purified, attenuated sporozoites vaccine as a boost, or vice versa. It may also include sequential or mixed immunization with attenuated Plasmodium species sporozoites and some form of erythrocytic stage parasites, including, killed and live attenuated. A vaccine complex comprising separate components may be referred to as a vaccine regimen, a prime / boost regimen, component vaccine, a component vaccine kit or a component vaccine package, comprising separate vaccine components. For example, a vaccine complex may comprise as a component, a vaccine comprising purified, aseptic, live attenuated sporozoites. The complex may additionally comprise one or more recombinant or synthetic subunit vaccine components, including but not limited to recombinant protein, synthetic polypeptide, DNA encoding these elements per se or functionally incorporated in recombinant virus, recombinant bacteria, or recombinant parasite. A vaccine component may also include aseptic attenuated axenic sporozoites that are allowed to develop to the early liver stage extracellularly.

[0206] Vaccines according to the disclosure can be administered, e.g., intradermally, subcutaneously, intramuscularly, intraperitoneally, and intravenously.

[0207] Dosage is empirically selected to achieve the desired immune response in the host. As used herein, "'immune response” means an acquired and enhanced degree of protective immunity, preferably complete or sterile protection, against subsequent exposure to wildtype Plasmodium sporozoites.

[0208] In certain aspects vaccine compositions as descri bed herein also comprise, m one or more adjuvants. An adjuvant is a substance that enhances the immune response when administered together with an immunogen, or antigen. A number of cytokines or lymphokines have been shown to have immune modulating activity, and thus are useful as adjuvants, including, but not limited to, the interleukins I -a, 1-p. 2, 4, 5, 6, 7, 8 and 10, 12 (see, e.g., U. S. Pat. No.5,723,127), 13. 14, 15, 16, 17 and 18 (and its mutant forms); the interferons-a, p and y; granulocyte-macrophage colony stimulating factor (GM-CSF) (see. e.g., U. S. Pat. No. 5.078,996 and ATCC Accession Number 39900); macrophage colony stimulating factor (M-CSF): granulocyte colony stimulating factor (G-CSF); and the tumor necrosis factors a and 0. Still other adjuvants that are useful with the immunogenic compositions described herein includechemokines, including without limitation, MCP-1, MIP-la, MIP-lp, and RANIES; adhesion molecules, such as a selectin, e.g., L-selectin, P-selectin and E -sei ectin; mucin-like molecules, e.g., CD34, GIyCAM-1 and MadCAM-1; a member of the integrin family such as LFA-1, VLA- 1, Mac-1 and pl 50, 95; a member of the immunoglobulin superfamily such as PEC AM, ICAMs, e.g,, 1CAM-1, ICAM-2 and ICAM-3, CD2 and LFA-3; co-stimulatory molecules such as CD40 and CD40L; growth factors including vascular growth factor, nene growth factor, fibroblast growth factor, epidermal growth factor, B7.2, PDGF, BL-1, and vascular endothelial growth factor; receptor molecules including Fas, TNF receptor, Fit, Apo-1, p55, WSL-1, DR3, TRAMP, Apo-3, AIR. LARD. NGRF, DR4. DR5, KILLER, TRAIL-R2, TRICK2, and DR6; and Caspase (ICE).

[0209] Still other adjuvants include glycolipid adjuvants including those disclosed in U. S. Patent Nos. 9,278,125 and 9,642,909. Assigned to the applicant and incorporated herein by reference. Preferably, the glycolipid adjuvant is 7DW8-5.

[0210] In certain aspects, the composition (e.g., vaccine composition) is a liquid composition

[0211] The present disci osure further provides a modified human hepatocyte comprising a Plasmodium derived gene construct encoding a protein or antigen antagonist of an IRF or an interferon signaling protein.EXAMPLES EXAMPLE 1Design of Plasmodium parasite-mediated delivery of protein antagonists of type I Interferon signaling into infected cells

[0212] Wildtype Plasmodium parasite infection and development within liver cells (hepatocytes) results in the activation and translocation of the Interferon Regulatory Factor 3 (IRF3) transcription factor into the nucleus. Activated IRF3 promotes the transcription of type I Interferon (IFN-I), leading to the production and eventual secretion of IFN-I proteins into the microenvironment surrounding the infected hepatocyte. IFN-I binds to the IFN-I receptor (IFNAR) on the infected cell and neighboring cells (hepatocytes and immune cells), causing them to impair the ability of CD8+ T cells to find and eliminate Plasmodium parasites in the liver (FIG.1A) (See Minkah, N. K., Wilder, B. K., Sheikh, A. A. et al.).

[0213] Transgenic Plasmodium parasites are designed to encode and secrete antagonists of IFN-I signaling into the infected hepatocyte. IRF3 antagonists (green) are inserted into the non-essential / dispensable p230p locus within the Plasmodium genome. Transcription of this constructis driven by the Plasmodium liver stage promoter (UIS4 promoter), while the PEXEL sequence facilitates the export of IRF3 antagonists into the infected hepatocyte. Antagonism of IFN-I by the antagonist limits IFN-I production by the infected hepatocyte, and, in turn, enables liver CD8+ T cells to effectively locate and eliminate Plasmodium parasites in the liver. This approach can significantly improve current malaria vaccines and serve as platform to deliver other immune modifiers into the host cell (FIG. IB).EXAMPLE 2Proof of concept for development of transgenic Plasmodium parasite vaccine platform

[0214] Transgenic Plasmodium parasites were shown to deliver Cre recombinase into infected cells to alter the infected hepatocyte genome in vivo. ROSAmT / mG mice were infected with 300,000 Py P230p-Cre (SEQ ID NO: 39) transgenic parasites or wildtype / normal Plasmodium yoelii parasites (Py XNL). 42 hours post infection (hpi), livers were harvested for microscopy (FIG. 2A)

[0215] Commercially available RosamT / mG mice are engineered to express sequences for the red fluorescent protein (RFP) and the green fluorescent protein (GFP). Under normal conditions, RFP is expressed while GFP is turned off, and thus each cell looks red under a fluorescence microscope. Importantly, the RFP gene is flanked by sequences (loxP sites - black arrowheads) that can be recognized by Cre recombinase. Binding of Cre recombinase to these loxP sites results in the removal of the RFP gene from the cell’s genome allowing that Cre-expressing cell to then express GFP. Proof of concept is demonstrated when the transgenic parasite delivers Cre recombinase into the nucleus of the infected cell, resulting in the loss of RFP expression and subsequent expression of GFP by that infected cell (FIG.2B).

[0216] Immunofluorescence imaging was conducted on liver sections harvested from ROSAmT / mG mice infected with transgenic (Py P230p-Cre) (SEQ ID NO: 39) or wildtype / normal parasites (Py XNL) 42 hpi. Transgenic Py parasites delivered nuclear-targeted Cre recombinase into hepatocy tes, driving GFP expression solely within the infected cell. This is conclusive evidence that the transgenic Py parasites designed and generated by the present inventors can deliver distinct proteins into the infected cell (FIG. 2C).EXAMPLE 3Integration of Interferon Regulatory Factor 3 (IRF3) antagonists into the Plasmodium yoelii genome using CRISPR-Cas9 technology

[0217] Integration of IRF3 antagonists into the P. yoelii genome can be achieved using CRISPR-Cas9 technology. The IRF3 antagonist preceded by CSP-PEXEL under UIS4 promoter is integrated into the dispensable P230p locus by homologous recombination. PCR amplified 5’ and 3’ UTRs as homologous regions of the target loci are cloned into CRISPR Cas9 plasmidsflanking the human DHFR cassete. UIS4-promotor-CSP-PEXEL-IRF3 antagonist sequence is ligated between 5’ and 3’ homology arms to create a Py_CSP-PEXEL-IRF3 (SEQ ID NO: 41) antagonist containing plasmid (FIG. 3). This Plasmid is transfected into blood stage schizonts recovered by density' gradient centrifugation from overnight in-vitro culture of P. yoelii 17XNL blood stage parasites. After transfection, parasites are intravenously injected into SW mice. Pyrimethamine is used for the positive selection and downstream cloning of the recombinant parasites. Transgenic parasites are genotyped with integration specific oligonucleotide primers to confirm integration of the UIS4-promoter-CSP-PEXEL-IRF3 antagonist cassete into the P. yoelii 17XNL genome. Integration from the 5' end is detected by PCR amplification using the primers that anneal in the genomic sequence 5' to the integration site (5’ integration test). Integration from the 3' end is detected by PCR using the primers that anneal in the sequence 3' to the integration site (3 ’ Integration test).EXAMPLE 4Safety and Efficacy of SanaricP' PfSPZ Vaccine with regard to treatment of subjects with CHB

[0218] Sanaria® PfSPZ Vaccine consists of a GMP-manufactured suspension of metabolically active, non-replicating (live, radiation atenuated) PfSPZ of the NF54 strain of Pf, purified from infected, aseptic Anopheles stephensi mosquitoes and formulated in phosphate buffered saline (PBS) with human serum albumin (HSA) (‘“Diluent”) and cryoprotectant. In this clinical trial 8 adult volunteers with CHB infection receive 3 doses of 4.5 x 105of PfSPZ Vaccine, 8 receive 3 doses of 9.0 x 105, and 8 receive 3 doses of 1.8 x 106. Safety is evaluated by examination of adverse clinical events relative to controls receiving normal saline. Efficacy is evaluated by comparing diminished levels of HBsAg (SEQ ID NO: 2) in volunteers receiving vaccine, and the expected 30% of participants receiving the vaccine may seroconvert (develop antibodies to HBsAg).EXAMPLE 5Murine model of Persistent HBV Infection - Measure Efficacy of PySPZ-LARC2 expressing the murine IL-2 ortholog (mcIL-2eRa) (PySPZ-LARC2-mcIL-2eRa) in Transgenic Mice

[0219] Dr. Lishan Su and colleagues at the University of Maryland (UMD) have developed a model of persistent HBV infection using a liver-tropic adeno-associated virus (serotype 8) as a vector for 1.3 copies of genoty pe D full-length HBV 1.3 (AAV -HBV 1.3) which is capable of viral replication in the liver and secretion of HBV [105.106], Plasmodium yoelii SPZ-LARC2 expressing the murine IL-2 ortholog (mcIL-2eRa) (denoted PySPZ-LARC2-mcIL-2eRa) is assessed for its capacity to reduce HBV burden in these mice. This provides the foundation for subsequent studies.EXAMPLE 6Generation of a PJLARC2 SPZ strains expressing human engineered IL-2 fusion protein designated cIL-2eRa comprised of a circularly (c) permuted IL-2 with the extracellular domain (e) ofIL-2Ra

[0220] A genetically engineered strain of Pf is developed using CRISPR-Cas9 to mediate knock in of an engineered IL-2 fusion protein, human cIL-2eRa (hIL-2) (SEQ ID NO: 38). hlL-2, is integrated into the LIN UP locus (SEQ ID NO: 19) (FIG. 4B - construct 2). Expression of the antigens is driven by the PfCSP promoter and their secretion to the cytosol of the liver cells is mediated by export signal (EXP). The viral ribosome skipping sequence V2A is inserted to improve expression of multiple antigens from the same promoter. The 3’ UTR, HSP70 (SEQ ID NO: 30), is inserted to improve expression. Constructs are directed to the target loci by homology-derived repair using 2 homologous arms flanking the integrated constructs. The vector harbors guide RNA (Sp) and Cas9 to mediate double stranded break at the desired locus.EXAMPLE 7Generation ofPfLARC2 SPZ strains expressing HBV antigen (HBc), hIL-2 cytokine (PJLARC2- HBV- hcIL-2eRa). and both HBcAg and hcIL-2eRa

[0221] A genetically engineered strain of Pf is developed using CRISPR-Cas9 to mediate knock in of HBV antigens and human IL-2 (SEQ ID NO: 38). The insertion of the HBV antigens and hIL-2 to the genome of / 7-LARC2 by CRISPR-Cas9 is performed in 2 steps. In the first step. HBpolAg (SEQ ID NO: 21) and / or HBxAg (SEQ ID NO: 23) are integrated into the Mei2 locus (FIG. 6A - construct 1). In the second step, hIL-2(SEQ ID NO: 38), and HBsAg ((SEQ ID NO: 22), and / or HBcAg (SEQ ID NO: 24) antigens are integrated into the LINUP locus (FIG. 6B - construct 2). Expression of the antigens is driven by the PfCSP promoter and their secretion to the cytosol of the liver cells is mediated by export signal (EXP). The viral ribosome skipping sequence V2A is inserted to improve expression of multiple antigens from the same promoter. The 3’ UTR, HSP70 (SEQ ID NO: 30), is inserted to improve expression. Both constructs are directed to the target loci by homology-derived repair using 2 homologous arms flanking the integrated constructs. The vector harbors guide RNA (Sp) and Cas9 to mediate double-strand break at the desired locus.EXAMPLE 8PfLARC2-HBV-IL2 strain produces fertile stage V Pf gametocytes that can infect mosquitoes and produce Pf sporozoites (SPZ)

[0222] The PfLARC2-HBV-IL2 strains are assessed for robust and successful growth through the parasite life cycle and infectivity in mosquitoes. Methodology: To assess whether the PILARC2-HBV-IL2 strains can produce viable sporozoites, gametocytogenesis is induced in these lines using established protocols [17, 80], including optimization for male: female (M: F)gametocyte ratios during gametocytogenesis, oocyst formation in midguts, prevalence and survivability in infected aseptic mosquitoes. Asexual growth is monitored with 2 concentrations of hypoxanthine (33 pM or 330 pM) in RPMI with 10% human serum. Oocyst scores of >20 oocysts / midgut with > 70% prevalence is indicative of adequate infection. On day 14 after infection, mosquitoes are dissected for sporozoite assessments (>50,000 sporozoites per mosquito). PfSPZ-LARC2-HBV-lL2 are purified and cryopreserved using established methods

[0080] ,

[0223] Data analysis: Statistical analyses are performed according to the data generated. Basic descriptive statistics of general data trending are analyzed using GraphPad Prism (currently version 9.4.1) and Excel, using parametric and non-parametric comparisons as appropriate. The skewed distribution of infections in mosquitoes

[0081] require analysis by nonparametric comparisons of oocysts and PfSPZ intensities and prevalence rates in a minimum of 30 mosquitoes per group per time point

[0082] , 7) optimal M: F gametocyte ratios of 1:2; 2) Yields of > 20 oocysts / mosquito; 3) Prevalence of oocysts of >70% in aseptic mosquitoes; 4) Yields of > 50,000 sporozoites / mosquito. Vialed and cryopreserved PfSPZ-LARC2-HBV-IL2 are stored in liquid nitrogen vapor phase.EXAMPLE 9Creation ofPyLARCl-cIL-2eRa and PyLARC 1 -HBcAg

[0224] LARC1 is similar to LARC2 except only the mei2 gene is deleted. Using Plasmodium yoelii (Py) LARC1 parasites, a DNA cassette was constructed to express modified human IL2 (cIL-2eRa) (SEQ ID NO: 38) and hepatitis B core antigen (HBcAg) (SEQ ID NO: 24) by Py in the liver (FIG. 7A). cIL-2eRa was created by synthesizing a gene in which the 4 amino acids that are responsible for the binding of IL2 to the alpha receptor (I12Ra) were replaced with Alanine. In previous studies, this mutation was show n to reduce expansion of regulatory T-cells (Tregs) while activating normal level of effector T-cells in mice and in human cells [107-109], This cIL-2eRa mutant gene was fused to PyCSP PEXEL export motif

[0110] to be secreted from the parasite to the cytoplasm of hepatocytes with protein expression driven by the Py liver stage UIS4 promoter

[0111] . The cassette was placed between 2 homologous arms, targeting the replacement of the mei2 gene as was done previously

[0112] , A similar construct was constructed with HBcAg (SEQ ID NO: 24) replacing the cIL-2eRa. Both constructs (cIL-2eRa and HBcAg) were ligated into a Plasmodium CRISPR vector containing guide RNA and codon-optimized CAS9, and transfected into wildtype Py, as reported

[0112] , Drug resistant parasites emerged, and DNA was extracted from the populations to confirm insertions by PCR. PCR done on populations of parasites integrated with the cIL-2eRa (FIG. 7B) or HBcAg (FIG. 7C) ORFs, and on DNA from WT Py, indicated transfections were successful, and constructs were integrated at targetedmei2 locus, creating PyLARCl-cIL-2eRa and PyLARC 1-HBcAg parasites. The products were cloned using limiting dilution as done routinely (not shown).

[0225] Although the foregoing specification and examples fully disclose and enable the present invention, they are not intended to limit the scope of the invention, which is defined by the claims appended hereto.

[0226] All publications, patents and patent applications are incorporated herein by reference. While in the foregoing specification this invention has been described in relation to certain embodiments thereof, and many details have been set forth for purposes of illustration, it will be apparent to those skilled in the art that the invention is susceptible to additional embodiments and that certain of the details described herein may be varied considerably without departing from the basic principles of the invention.

[0227] Although the foregoing specification and examples fully disclose and enable the present invention, they are not intended to limit the scope of the invention, which is defined by the claims appended hereto.

[0228] Embodiments of this invention are described herein, including the best mode known to the inventors for carry ing out the invention. Variations of those embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law; Moreover, any combination of the abovedescribed elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.TABLE 1. SEQUENCESSEQ Description SequenceID NO1 AHV90191 MPLSYQHFRKLLLLDDEAGPLEEELPRLADEGLNRRVAEDLNLGNLN.1 - VS1PWTHKVGNFTGLYSSTVPVFNPEWQTPSFPH1HLQEDI1NRCQQYV polymerase GPLTVNEKRRLKLIMPARFYPNLTKYLPLDKGIKPYYPEHAVNHYFKT [Hepatitis RHYLHTLWKAGILYKRETTRSASFCGSPYSWEQELQHGRLVFQTSTR B virus] HGDESFCSQSSGILSRSPVGPCVRSQLKQSRLGLQPQQGSLARGKSGR SGSIRARVHPTTRRSFGVEPSGSGHIDNSASSTSSCLHQSAVRKTAYSH LSTSKRQSSSGHAVELHNIPPSSARSQSEGPIFSCWWLQFRNSKPCSDY CLTHIVNLLEDWGPCTEHGEHNIRIPRTPARVTGGVFLVDKNPHNTTE SRLVVDFSQFSRGSTHVSWPKFAVPNLQSLTNLLSSNLSWLSLDVSAA FYHIPLHPAAMPHLLVGSSGLPRYVARLSSTSRNINYQHGTMQDLHDS CSRNLYVSLLLLYKTFGRKLHLYSHPIILGFRKIPMGVGLSPFLLAQFT SAICSVVRRAFPHCLAFSYMDDVVLGAKSVQHLESLFTSITNFLLSLGI HLNPNKTKRWGYSLNFMGYVIGSWGTLPQEHIVLKIKQCFRKLPVNR PIDWKVCQRIVGLLGFAAPFTQCGYPALMPLYACIQSKQAFTFSPTYK AFLCKQYLNLYPVARQRSGLCQVFADATPTGWGLAIGHRRMRGTFV APLPIHTAELLAACFARSRSGAKLIGTDNSVVLSRKYTSFPWLLGCAA NWILRGTSFVYVPSALNPADDPSRGRLGLYRPLLHLPFRPTTGRTSLY AVSPSVPSHLPDRVHFASPLHVAWRPP2 AHV90192 MGGWSSKPRQGMGTNLSVPNPLGFFPDHQLDPAFGANSNNPDWDFN.1 - large S PNKDHWPEANQVGAGAFGPGFTPPHGGLLGWSPQAQGILTTVPAAPP protein PASTNRQSGRQPTPISPPLRDSHPQAMQWNSTTFHQALLDPRVRGLYF [Hepatitis PAGGSSSGTVNPVPTTASPISSIFSRTGDPAPNMENTTSGFLGPLLVLQA B virus] GFFLLTRILTIPQSLDSWWTSLNFLGGAPTCPGQNSQSPTSNHSPTSCPP ICPGYRWMCLRRFIIFLFILLLCLIFLLVLLDYQGMLPVCPLLPGTSTTS TGPCKTCTIPAQGTSMFPSCCCTKPSDGNCTCIPIPSSWAFARFLWEWA SVRFSWLSLLVPFVQWFVGLSPTVWLSVIWMMWYWGPSLYNILSPFL PLLPIFFCLWVYI3 AHV90193 MAARVCCQLDPARDVLCLRPVGAESRGRPVSGPFGTLPSPSSSAVPAD.1 - X HGAHLSLRGLPVCAFSSAGPCALRFTSARRMETTVNAHQVLPKVLHKprotein RTLGLSAMSTTDLEAYFKDCVFKDWEELGEEIRLKVFVLGGCRHKLV [Hepatitis CSPAPCNFFTSAB virus]AHV90194 MDIDPYKEFGASVELLSFLPSDFFPSIRDLLDTASALYREALESPEHCSP.1 - core HHTALRQAILCWGELMNLATWVGSNLEDPASRELVVSYVNVNMGL protein K1RQLLWFH1SCLTFGRETVLEYLVSFGVW1RTPPAYRPPNAPILSTLPE [Hepatitis TTVVRRRGRSPRRRTPSPRRRRSQSPRRRRSQSRESQCB virus]KJ173426. CTCCACAACATTCCACCAAGCTCTGCTAGATCCCAGAGTGAGGGG 1 - CCTATATTTTCCTGCTGGTGGCTCCAGTTCCGGAACAGTAAACCCT Hepatitis B GTTCCGACTACTGCCTCACCCATATCGTCAATCTTCTCGAGGACTG virus GGGACCCTGCACCGAACATGGAGAACACAACATCAGGATTCCTAG isolate GACCCCTGCTCGTGTTACAGGCGGGGTTTTTCTTGTTGACAAGAAT C2_CLP(3) CCTCACAATACCACAGAGTCTAGACTCGTGGTGGACTTCTCTCAAT, complete TTTCTAGGGGGAGCACCCACGTGTCCTGGCCAAAATTCGCAGTCCC genome CAACCTCCAATCACTCACCAACCTCTTGTCCTCCAATTTGTCCTGGC TATCGCTGGATGTGTCTGCGGCGTTTTATCATATTCCTCTTCATCCT GCTGCTATGCCTCATCTTCTTGTTGGTTCTTCTGGACTACCAAGGTA EGTTGCCCGTTTGTCCTCTACTTCCAGGAACATCAACTACCAGCAC GGGACCATGCAAGACCTGCACGATTCCTGCTCAAGGAACCTCTAT GTTTCCCTCTTGTTGCTGTACAAAACCTTCGGACGGAAACTGCACT TGTATTCCCATCCCATCATCCTGGGCTTTCGCAAGATTCCTATGGG AGTGGGCCTCAGTCCGTTTCTCCTGGCTCAGTTTACTAGTGCCATTT GTTCAGTGGTTCGTAGGGCTTTCCCCCACTGTTTGGCTTTCAGTTAT ATGGATGATGTGGTATTGGGGGCCAAGTCTGTACAACATCTTGAGT CCCTTTTTACCTCTATTACCAATTTTCTTTTGTCTTTGGGTATACATT TGAACCCTAATAAAACCAAACGTTGGGGCTACTCCCTTAACTTCAT GGGATATGTAATTGGAAGTTGGGGTACTTTACCGCAGGAACATATT GTACTAAAAATCAAGCAATGTTTTCGAAAACTGCCTGTAAATAGA CCTATTGATTGGAAAGTATGTCAAAGAATTGTGGGTCTTTTGGGCT TTGCTGCCCCTTTTACACAATGTGGCTATCCTGCCTTAATGCCTTTA EATGCATGTATACAATCTAAGCAGGCTTTCACTTTCTCGCCAACTT ACAAGGCCTTTCTGTGTAAACAATATCTGAACCTTTACCCCGTTGCCCGGCAACGGTCAGGTCTCTGCCAAGTGTTTGCTGACGCAACCCCCGTTTTGCTCGCAGCCGGTCTGGAGCGAAACTTATCGGAACCGACAGGGTGTGCTGCCAACTGGATCCTGCGCGGGACGTCCTTTGTCTACGGGGACTCTACCGTCCCCTTCTTCATCTGCCGTTCCGGCCGACCACG GGGCGCACCTCTCTTTACGCGGTCTCCCCGTCTGTGCCTTCTCATCT GCCGGACCGTGTGCACTTCGCTTCACCTCTGCACGTCGCATGGAGA CCACCGTGAACGCCCACCAGGTCTTGCCCAAGGTCTTACATAAGA GGACTCTTGGACTCTCAGCAATGTCAACGACCGACCTTGAGGCATA CTTCAAAGACTGTGTGTTTAAAGACTGGGAGGAGTTGGGGGAGGA GATTAGGTTAAAGGTCTTTGTACTAGGAGGCTGTAGGCATAAATTG GTCTGTTCACCAGCACCATGCAACTTTTTCACCTCTGCCTAATCATCGGCTTTGGGGCATGGACATTGACCCGTATAAAGAATTTGGAGCTTCCAATGTTAGTATCCCTTGGACTCATAAGGTGGGAAACTTTACTGGG CTTTATTCTTCTACTGTACCTGTCTTTAATCCTGAGTGGCAAACTCC CTCCTTTCCTCACATTCATTTACAGGAGGACATTATTAATAGATGT CAACAATATGTGGGCCCTCTTACAGTTAATGAAAAAAGGAGATTAGCCCTTGGACAAAGGCATTAAACCGTATTATCCTGAACATGCAGTTCTGGCATTCTATATAAGAGAGAAACTACACGCAGCGCCTCATTTTG TGGGTCACCATATTCTTGGGAACAAGAGCTACAGCATGGGAGGTT GGTCTTC C AAAC CTCGAC AAGGC ATGGGGAC GAATCTTTCTGTTC C CAATCCTCTGGGATTCTTTCCCGATCACCAGTTGGACCCTGCGTTC GGAGCCAACTCAAACAATCCAGATTGGGACTTCAACCCCAACAAG GATCACTGGCCAGAGGCAAATCAGGTAGGAGCGGGAGCATTCGGG CCAGGGTTCACCCCACCACACGGCGGTCTTTTGGGGTGGAGCCCTC AGGCTCAGGGCATATTGACAACAGTGCCAGCAGCACCTCCTCCTG CCTCCACCAATCGGCAGTCAGGAAGACAGCCTACTCCCATCTCTCC ACCTCTAAGAGACAGTCATCCTCAGGCCATGCAGTGGAA PF3D7 12 TATTGTTGTTGTTGTTTGCCTTGG49700_Guide 3FPF3D7 12 AAACCCAAGGCAAACAACAACAAC49700_Guide 3RPF3D7 12 TATTATATGTGCTACTCCATATAC49700_Guide 5FPF3D7 12 AAACGTATATGGAGTAGCACATAT49700_Guide 5RPF3D7 12 ATACTAGTTTTTATTTTTTATATGAACCATTTAAGAC49700_5UTRF PF3D7 12 TACTTTTCCCCAACCCGGGTATAGGCGCGCCTCTTTCAACAAAAAA 49700_5U TAATATATGTGTACTRR PF3D7 12 TTGTTGAAAGAGGCGCGCCTATACCCGGGTTGGGGAAAAGTAAAA 49700_3U TTCGCGATAAAAATCTRFPF3D7 12 FAGCGGCCGCAATATATATGTGATTTTATTAAGTACGG 49700_3UTRR PF3D7 12 FAAATGAGGGATAAAACCTCAAAAAG49700_TestF PF3D7 12 AATATGTTTCAAACGGTTAAATGTAAG49700_TestR PF3D7 12 CTTAACAACCATCTTCTTGACAATAGGG49700_ORF F PF3D7 12 GTTATGTGATTAACTTTCGCTTGATAC49700_ORF RPlasmodiu ATGCATAAGA TAGATGAAAA GATAAATATG AATAATGTAA m AAGGCACTAG AATTATTCATGTAAAAAATA CATATATATC falciparum FCCATACATA CTACACAATA TAAACAATAAPlasMei2 FGAAGGCCCATTAAAAAATA TAGACAAAAT ATTAAAAGAA gene GAAAAAGATA ATATAAATTT AAAGATCAATAATGTTAATA ATAAGGAAAC TAATAATGAT AAGCACACAT TAAAATATTC AACATTATGTAATGATCTAA ATATAATGCA TACACAAAAT AAAGAAGAAG AAATTGAATT AAATACAATTTCAACGGTTA FACACATGAA TTCTGATGAA GATAGTGATA AAGATAATGA TACCATAATAAATGAAAACA ACGATTTGTT AATTGATAAT ATAAAGAAAT ATCATGCCGT TAAAGATAATCATAATAATA FTATTTATAA TGATAAATAT AATACAATTA ATAATAATTC AGTCATTAATGATGTATGTA ATTCTATTCA TTTTAATAAT AATTCATATA TAACTAACTT TAATTTAAATCATAATTTTT CTGTGTGTAA TCGTACTTTA AATAACACCT GCACAAGTCA GGTAAATTTGAGAAATAATA TGAACAGCAA TAAAAAAACTAATGATAACA ATAAAAATCT GAACAATGAAATTAATAAGAAAATCAATAA TGATAATATT ATAAACGAAT TTGATAATAT T A AT A AT A AGA A A AAT A ATT ATATTGATTG TTCCGTTTAC AAATGTGAGG ATGAAATACC CCTTGGTACCATATTAAATA TCCAAGATTT GGATATACAT AATAGAAATA ATATGAACAA TTGTAATAATAATATTAATA ATAAGAGTAA GATTCTTACA ACAGTTATGC TTAGAAATAT TCCAAACAAATATACACAAA ATATGTTGAT GGATGTTATG AATGAACATT TTAAAGGTTT ATATGATTTTTTTTATTTAC CAATTGATTT TAGAAATAAA TGTAATGTTG GATATGCTTT TATTAATTTCATACATCCAT ATTATGCTGA ATTGTTTATC AAATTTTTTA ATAATTATAA ACTCAATGCATTTAAAAGTA ACAAAGTCTG TTCTGTTACT TGGGGAAGAG TACAAGGATT AAAGGCAAATATTGAGCATT ATAGAAATTC AGCGATTATG ACCATACCAA TACCACAATA TAAACCTATACTTTTTCAAA ATGGTATAAC TGTTTCGTGG CCTGAATCAG ATGGGCCTTT ACCATCCATCAAATTAAGAT CTCAAAAATT TTAAPlasmodiu ATGAAAAATGAAGATTCTACATATAAAGAAGATAAGTATTCAAAA m TATTCAAAAGAGAATATCCAAAAAATATGTGCTACTCCATATACCG falciparum GATCTAATATAAGTAACACAGTAGAATCTGTTTATTTTGTTAATTA LINUP TAGAGCTGTCGTAGTAAAAACAAAACATTATTATGTTACTCAAAACgene TCTTATATGCTTTTTGGTGATGTCTTTAAAATATATAATTTAAAAGG AGAGATAAAAAAAATACATGCATTTTTTCCAGGAGATGAAAATAA TTTATCGGATACATTAATAATGTTAAAAACAACAATAGTTGCTGTT ACTACATCTAAAGGAGAAGCTTTGCGTTTGGCTGATGAAATTAATA TGACATGTAAAATATGTCATAAGAAAAGAAAAGCAACCAAATTTT CTATAGAAGGATTTCTAAGAAAAGTTAGATGTGACACCTGTAGGG TTAAACCACGAAGTGTAATTTGTAAAAATATAAATAACAATAAAA AAACAAACAATTCAAAAAATAACACAAATAGTAATAATAGTAATA ATAATAATAATAATAATATAAATAATAATAATAATAATAATAATA ATAGTAATAATAGTAATAATAAAAATGATTCATTAAAACCAGATG AATGTAATATACAGAGTAATCAAAATAAACATCTTACAAAAAATA AAATTATTAAATATGAAAATATAGATGACGAAAATATAAAAAATG AAAAATTTAAAAATATGAATATCAAAAAAGTTAATACAAAAAATTGTAATATCACAAATAGTAAGGCAAAAAAAACTGATAATAATATACCATCTTAATAATAATCAACTTAACAACCATCTTCTTGACAATAGGG CCTTAAATATAATAATAGTAATAATAATTACTCAGCTAGCCAAAAT CAAATTTTTTTTTCAGAGGAAGAAAATAGAACTCATTTTCTAGAGACTTAACCAAAATAATATAGACGGAGAATATGGGAATAATAATTTG CAATAAAAAATGATGTTAATATAAATAATATAAGTAATAATAATACTACAAATACTCACATGACTAACCTTTCTTATATTAATAGAAATGA CCAAAATAAAAATAATAATATGAATAATGATACGGATAGCAATATATCATATTAATGGTAACAATAATATTAATAGTAACAATAATATTAACAAAAAATATTCAAAATATTCAAAATATACAAAATGTACAAAATAAATCATATATAAAGGATAATAAAGAATATATGTATGAAAATAATA AGAAGGATGAACACGAAGATAAAAACCAATATCATGACCACCAA GGCAAACAACAACAACAACAACAACAACAACAACAACAACAACA ACAACAACAACAACAACAACATCATAATAATAATAATATAAGTCA FACAAAAAATAATTATTACAATAATAAACAGCAGTTCTTATTTTCT ACTTTATCAAAACAATATAATACTAATGTCAATATTTCATTTGCTA ATATAAACAATTCTTTATCAAATATTTGTAAGAATGTCTCTAAAAA FTATAATCTCAAAGATACAGTTTTAAATATGGAAAAGTAAPlasmodiu MHKIDEKINMNNVKGTRIIHVKNTYISPYILHNINNNEGPLKNIDKILK m EEKDNINLKINNVNNKETNNDKHTLKYSTLCNDLNIMHTQNKEEEIEL falciparum NTISTVIHMNSDEDSDKDNDTIINENNDLLIDNIKKYHAVKDNHNNTIY PlasMei2 NDKYNTINNNSVINDVCNSIHFNNNSYITNFNLNHNFSVCNRTLNNTC amino acid FSQVNLRNNMNSNKKTTSIDNNKNLNNEINKKINNDNIINEFDNINNKK NWIDCSVYKCEDEIPLGTILNIQDLDIHNRNNMNNCNNNINNKSKILT TVMLRNIPNKYTQNMLMDVMNEHFKGLYDFFYLPIDFRNKCNVGYA FINFIHPYYAELFIKFFNNYKLNAFKSNKVCSVTWGRVQGLKANIEHY RNSAIMTIPIPQYKPILFQNGITVSWPESDGPLPSIKLRSQKF AHV90191 ATGCCATTAAGTTATCAACATTTTAGAAAATTATTATTATTAGATG.1 ATGAAGCAGGACCATTAGAAGAAGAATTACCAAGATTAGCAGATG polymerase AAGGATTAAATAGAAGAGTAGCAGAAGATTTAAATTTAGGAAATT [Hepatitis TAAATGTAAGTATACCATGGACACATAAAGTAGGAAATTTTACAG B virus] GATTATATAGTAGTACAGTACCAGTATTTAATCCAGAATGGCAAAC codon ACCAAGTTTTCCACATATACATTTACAAGAAGATATAATAAATAGA optimized TGTCAACAATATGTAGGACCATTAACAGTAAATGAAAAAAGAAGA nucleotide TTAAAATTAATAATGCCAGCAAGATTTTATCCAAATTTAACAAAAT sequence ATTTACCATTAGATAAAGGAATAAAACCATATTATCCAGAACATG CAGTAAATCATTATTTTAAAACAAGACATTATTTACATACATTATG GAAAGCAGGAATATTATATAAAAGAGAAACAACAAGAAGTGCAAGTTTTTGTGGAAGTCCATATAGTTGGGAACAAGAATTACAACATGGAAGATTAGTATTTCAAACAAGTACAAGACATGGAGATGAAAGTTT TTGTAGTCAAAGTAGTGGAATATTAAGTAGAAGTCCAGTAGGACC ATGTGTAAGAAGTCAATTAAAACAAAGTAGATTAGGATTACAACC ACAACAAGGAAGTTTAGCAAGAGGAAAAAGTGGAAGAAGTGGAAGTATAAGAGCAAGAGTACATCCAACAACAAGAAGAAGTTTTGGAGCAAGTAGTTGTTTACATCAAAGTGCAGTAAGAAAAACAGCATATA GTCATTTAAGTACAAGTAAAAGACAAAGTAGTAGTGGACATGCAG GATTGGGGACCATGTACAGAACATGGAGAACATAATATAAGAATA CCAAGAACACCAGCAAGAGTAACAGGAGGAGTATTTTTAGTAGAT GCAGTACCAAATTTACAAAGTTTAACAAATTTATTAAGTAGTAATT AAATTATCAACATGGAACAATGCAAGATTTACATGATAGTTGTAGT GAAAATTACATTTATATAGTCATCCAATAATATTAGGATTTAGAAAAGAGGAACAAGTTTTGTATATGTACCAAGTGCATTAAATCCAGCA GATGATCCAAGTAGAGGAAGATTAGGATTATATAGACCATTATTA CATTTACCATTTAGACCAACAACAGGAAGAACAAGTTTATATGCA GTAAGTCCAAGTGTACCAAGTCATTTACCAGATAGAGTACATTTTG CAAGTCCATTACATGTAGCATGGAGACCACCATAA HBsAG - ATGGGAGGATGGAGTAGTAAACCAAGACAAGGAATGGGAACAAA AHV90192 TTTAAGTGTACCAAATCCATTAGGATTTTTTCCAGATCATCAATTA.1 large S GATCCAGCATTTGGAGCAAATAGTAATAATCCAGATTGGGATTTTA protein ATCCAAATAAAGATCATTGGCCAGAAGCAAATCAAGTAGGAGCAG [Hepatitis GAGCATTTGGACCAGGATTTACACCACCACATGGAGGATTATTAG B virus] GATGGAGTCCACAAGCACAAGGAATATTAACAACAGTACCAGCAG codon CACCACCACCAGCAAGTACAAATAGACAAAGTGGAAGACAACCA optimized ACACCAATAAGTCCACCATTAAGAGATAGTCATCCACAAGCAATG nucleotide CAATGGAATAGTACAACATTTCATCAAGCATTATTAGATCCAAGA sequence GTAAGAGGATTATATTTTCCAGCAGGAGGAAGTAGTAGTGGAACA GTAAATCCAGTACCAACAACAGCAAGTCCAATAAGTAGTATATTT AGTAGAACAGGAGATCCAGCACCAAATATGGAAAATACAACAAGT GGATTTTTAGGACCATTATTAGTATTACAAGCAGGATTTTTTTTATT AACAAGAATATTAACAATACCACAAAGTTTAGATAGTTGGTGGAC AAGTTTAAATTTTTTAGGAGGAGCACCAACATGTCCAGGACAAAA TAGTCAAAGTCCAACAAGTAATCATAGTCCAACAAGTTGTCCACC AATATGTCCAGGATATAGATGGATGTGTTTAAGAAGATTTATAATA TTTTTATTTATATTATTATTATGTTTAATATTTTTATTAGTATTATTA GATTATCAAGGAATGTTACCAGTATGTCCATTATTACCAGGAACAA GTACAACAAGTACAGGACCATGTAAAACATGTACAATACCAGCAC AAGGAACAAGTATGTTTCCAAGTTGTTGTTGTACAAAACCAAGTG ATGGAAATTGTACATGTATACCAATACCAAGTAGTTGGGCATTTGC AAGATTTTTATGGGAATGGGCAAGTGTAAGATTTAGTTGGTTAAGT TTATTAGTACCATTTGTACAATGGTTTGTAGGATTAAGTCCAACAG TATGGTTAAGTGTAATATGGATGATGTGGTATTGGGGACCAAGTTT ATATAATATATTAAGTCCATTTTTACCATTATTACCAATATTTTTTT GTTTATGGGTATATATATAA AHV90193 ATGGCAGCAAGAGTATGTTGTCAATTAGATCCAGCAAGAGATGTA.1 X protein TTATGTTTAAGACCAGTAGGAGCAGAAAGTAGAGGAAGACCAGTA[Hepatitis AGTGGACCATTTGGAACATTACCAAGTCCAAGTAGTAGTGCAGTA B virus] CCAGCAGATCATGGAGCACATTTAAGTTTAAGAGGATTACCAGTA codon TGTGCATTTAGTAGTGCAGGACCATGTGCATTAAGATTTACAAGTG optimized CAAGAAGAATGGAAACAACAGTAAATGCACATCAAGTATTACCAA nucleotide AAGTATTACATAAAAGAACATTAGGATTAAGTGCAATGAGTACAA sequence CAGATTTAGAAGCATATTTTAAAGATTGTGTATTTAAAGATTGGGA AGAATTAGGAGAAGAAATAAGATTAAAAGTATTTGTATTAGGAGG ATGTAGACATAAATTAGTATGTAGTCCAGCACCATGTAATTTTTTT ACAAGTGCATAA AHV90194 ATGGATATAGATCCATATAAAGAATTTGGAGCAAGTGTAGAATTA.1 - core TTAAGTTTTTTACCAAGTGATTTTTTTCCAAGTATAAGAGATTTATT protein AGATACAGCAAGTGCATTATATAGAGAAGCATTAGAAAGTCCAGA [Hepatitis ACATTGTAGTCCACATCATACAGCATTAAGACAAGCAATATTATGT B virus] TGGGGAGAATTAATGAATTTAGCAACATGGGTAGGAAGTAATTTA codon GAAGATCCAGCAAGTAGAGAATTAGTAGTAAGTTATGTAAATGTA optimized AATATGGGATTAAAAATAAGACAATTATTATGGTTTCATATAAGTT nucleotide GTTTAACATTTGGAAGAGAAACAGTATTAGAATATTTAGTAAGTTT sequence TGGAGTATGGATAAGAACACCACCAGCATATAGACCACCAAATGC ACCAATATTAAGTACATTACCAGAAACAACAGTAGTAAGAAGAAG AGGAAGAAGTCCAAGAAGAAGAACACCAAGTCCAAGAAGAAGAA GAAGTCAAAGTCCAAGAAGAAGAAGAAGTCAAAGTAGAGAAAGT CAATGTTAAPfCSP AATTGTATGTAAATTAAAATTTTAATTGTTAAAATAATAAAAAGTG 5’UTR AATTTATAAAAAAAAGCACGAATGAAACTATTTAATTTTCTTTTGG (also the GCATTTTATTATTATTATAATTTTTTAAATAATATGTTGCATTTATC PfCSP ATGCCTGGTGTGTATTTTATAACTATTATGTAAGAACATACCTAAA Promoter) AGATGAAATTAATAAAAATGCTATGATCAGGAAGCATGAGCACAT ATATATCATATGTTTAATCAAACATTTTATGGGATTATTGTAAATA TAACATGCACATTTTGTATAAGTTCCTTAATTTTTTTTTTTTTTTTTT TGGGGGGGGAGGGGTAAAGGGGGGCTTAATTATAAAACAGAAATT ATTCTTATCTTACATGCACATATAAAAAAATGGATTGGTGGTAAAC CAAAAAAATAAATTCACTATATGTTCTTAAGGAAGCATATAATGTT TTCCTTTTTTTTTACATGCAGATATAAAAAGGTAGAAGAACTTACGAGAAGCTCTATATTTTACACATGCGATTTGGATATATATTTTTTTTTTTGTAACTTTCTATCATACTTGTCATAAATTCTGAATTATCAAATAA CTCAAATATATTTCATAATATCATAATGCTACAATAATAAAAATAT TTTTAATATATATTAAAAAAAAAAATTATATATATATATTTATATA TATATAATATACCTATATACATACACTATTTTTCATTATAATTTTTTrTTTTTTTGTGTTTTTTTATATATTTTGGAAATATGTAACGTATAAAAAACAAGACAAATATAATATATACTATTAATAAATAAGATATAGT TCTTGTTTTAATATTAATTAAAAGAAAATTTTTGTGAATATATAAA AAAAAAAAAAAAAAAAAAGAAAATTATAAATAAATATATATATTC GTGTAAAAATAAGTAGAAACCACGTATATTATAAATTACAATTCPfCSP ATAAAGAACACATCTTAGTTTGAGTTGTACAATATTTATAAAAATA 3’UTR TATACTACTTTTTTTCTTAATTTTCATTTTTCTTTATATTTTCCTATTT AATTTATTTTTTTGTGAATATTTAATTACGTTTGCGATTAATTGTAG AAATATATATGTATATACTATATTTATAGAATGTGTTATTCTCAAA AACAACAACAAAAAAAAAAAAAAAAAAAAAAAAAAAGAAAAAA GGATTAAAAGTAAAATAGTTATAAATATTTTCAAAAATATTTATAA CACAAAAAATACTTCGAAGTTCATTTAACATTTTTGTTTATTTATTT ATTTATATATTTCATTTTTACGTATTTATATTATAAAATGGTGTATC FTAAAAATAGTGAACTATATATATAAAATATTAATTTAAAAAAATT ATAACTTTCTTTTTATTTTCTAAAATAACTTAAAAATTATATGTTTA AGAAAGGGGTAAATTATAATATTTGTATAAATATATAAACATAGA FATATTAAATAAAATAACAAATGTACTATATTTGTGCATAAGACGT ATACGCTTCATATAATACATATATATATATATATATATATATATAT ATATATTAATTCTATTTTATATATAGAGAAACATTTATGTTACACA ACTGAAACCTTTAAATGATCCTAAATTAAAAAAGGAACAATATAC AATTAGTCATTTAACATAATTTACGTATTATACATTATATGGCTTAT AAATAATTGAATTAATTATTATTAAAAATATAATACAATAATATAT ATTATAAGGAAAAAAAGCTAAAATATTAAATCCCAATTTATTTTAT ATATTTCATAATGTATATAATAATATATATAATGTTATGAAAATAT ATAATATATTATCTATCCTATACTAAAAGCTAAAATATTTPfCSP MMRKLAILSVSSFLFVEALFQEYQCYGSSSNTRVLNELNYDNAGTNL Signal YNELEMNYYGKQENWYSLKKNSRSLGENDDsequenceand PEXELmotifsamino acidsequencePfCSP ATGATGAGAAAATTAGCTATTTTATCTGTTTCTTCCTTTTTATTTGT Signal TGAGGCCTTATTCCAGGAATACCAGTGCTATGGAAGTTCGTCAAAC sequence ACAAGGGTTCTAAATGAATTAAATTATGATAATGCAGGCACTAATT and PEXEL TATATAATGAATTAGAAATGAATTATTATGGGAAACAGGAAAATT motifs GGTATAGTCTTAAAAAAAATAGTAGATCACTTGGAGAAAATGATG nucleotide ATsequenceHSP70 CGCATAAATATCTGGTGAAATACAAACAAGAATAAATTCAACAAT promoter, ATTTATATATTTTTTATTTATTATATATATATAATATTTTTTTATGAT NF54 ACTACTATTTATATATATATATATATATTTATATGTATTATATATCT CGTTTATAACAAATTGAAAAATATAAAATATATTTATATTCAAATA AAAGTAAAAATAAAGTAATTATATTATATATATTATTAATACTTAT TATATATATATTATATAACATAATATTTTAATGGTTTATTTATAATA ATATATACTATATATGTTATATATAAATAAATACCATAGTTCTTAT ATATATATATTTTTTATATATTGTAATATTATAAAAAAATTCGTAA AATATAATTTTTTTTATTTTATTATATATTTAACATTAGTAATTATTT ATATTATTTATATTTATATACATAATATAATATATGTAAAAAAAAT AAAATAAAATAAAATAAAATAATATCTTATACAAATATAATAATA AATTATTATATATAAATAAAATATATATAATATATTTACAAAATAA ATAAAAAATAGTAAATATTTATATAATATTAATAAATAAATATATT CTATATATTTTTTATAAAGATAAATAAAATAATAATAATATATTAT AAAATAATATATTTATAAATATATATTTTTTATATATTTCCAAAAA AAAAAAAAAAGATAAAATATTTTCGTATCTTAAGAATAATTTTTCT TATATTAATTATATTATATTATATTATAATTATTTAAAAATATATTA TTTGTAAAAGTTTTATAATTATATAATAAAATAGTATAAATATAAT ATATTATTATATATATATAATATATAAATATATTTATATTTATTATT ATTATTATTATATATATATATATATATACAAAAGAATTAAGAAAAAPfHSP70 ACTAATAAAAAAAAAAAACATTAAACAGGACAAATATAAAAATAT 3’UTR ATATATTATAAAAATATATATATATATATATATATTTTTTTTTTTTTTTTTACATTTTTGTAAATTAATATATATTGAATACATATATGCCAAACAAAAAAGAAAAATATATCTACATATATTTATATATATATATATAT ATATATATATATTCATGTGTAATAATATATACAATATTTATACATA TAAAAATATTTTTTATATTTTCGGTTATATAAATAAACAAGCTATTT ATGGATCACACAAATATAATTATATATTTGTATATGTATTTTTATAT ATATATATATATATATGTATGTGTAATTATTTATTTTTTTTTTCCTCA TAAAATTATAATAAACTTTTGTACTGCTTAGAAATTAAAAAATTAA TAAATTAATAAATCATTAAAGGTTTTTATTTAATTTGAAAAATAAGrTTTTTTTTAAAGAATATACATCCATACAAAATGTTTTTAAAAATATATGTATTATTAGGCATGAAATATATATATTATAAATATATATATAT ATATATTTTTTACAAGTTGAAACACCTTAAAATTGTTCTTTTTTTTTTTTTTTTTTTTTGTCATGGGATTATTCAGGAGTATTATTATTTATAAATATTTTAAAAAAATATCCCATATGAATGAATATATAAAAAAAAA ATTAAATAGTTTATGTTGATATTTAATAAGTTTAAATAGTATTTGA ATTGTATGCATTTTTAGCAATATAAAACATATATATATATGTATAT ATATATATTATATTTAAAATATAAT NF54 GTTAATATGAATATATATAAAATATTTATATACATAAAAAAATAAA EXP1 TATATTATATAATAATATTAATATATGAAATGTAATTTTTTTACAA promoter GATATAGTATATTTTATTATGGTTCATTTTATATAAAATAAAAATT ATATAGTTAAAGGTTAAAAAATAGTTTTAAATATATATTTATATAT AATTATATTTATAGTAATAATAATAAAAAATATATATATATATATA TATATTGCTGATTTTAAAAAATAACAAAATATTTTATATAATATAT AAATATTATATAAAACAAAAATAAGAACATTTTTTTCATTCCTATT TAATATAAAAATTTATTATATTTATATATTTAAGTTTTTTCATAATA TTTAAGTTTTATATCTATCTAAATTCAAAATTTCATAGTTTTTTTTTTTTTTTTTCTTTTTCCTTCTTTTATAATATAATATATTATATATGTTATTTATTATTTTTTATACCTTAATATTTTTTTAAATATATATATATTTTT TTATAAAATCATTTATATTTATATATATAAATAAATATATCACACTT TATAATTAAAAAAAAAAATAAAAAAAAAATATATATATATTTTTTT TAAATATTTATCCAGCAAAATAATTTTTATATATAGATTTCAAAAT ATTTAATTATCTAAATAAATTTAATTAAAAATTTTTATAACATATTT TATTTAAGATTTTATAATAATTAAGTTTTAATTTCTTTTGATCCAAAGTTTTTAATAATTAAAGTAAAAATTCAAAACACAAAAAAAAAGTATTATATATATATATATATTTATATGTTTATATTTTTATGTGTTTATTTAAATGTTCATGTAATAAAATAAATATGTAAATAAAATTCTTTATATTTTTTGAGAAATGTTATATATATATATATATATATATATAGAATAT ATTTATAAAGCATGTATAACAATTTTAATCAATTTTCTATAGAAACCTTTTTTTTCTTCTTCTCTTATAGTTTGTAGATTTTTAATTTATTTAATATATTCAAA NF54 AAGACCTTTTATGTTTTAACAAGTTATAAAAAGTATATAGGATGTG LSA1 AAAAAAATAATAATGCATTACATGACATTATTAATAATTCGATATG promoter TCCAATTTATTTTCTTTCTATAATTAGTGATGATATATATATATATA TATATATACAACATTTAATTTAACGTTACATAAATATTCTAAATAT TTTCGCCTTATACCGAATAAATCGAATGTTCTTACCTTTTTATAATG AATGTCACATGTTATATTGTGTCTCAGCATGATTAAAATTATATTT AAAAATATGAATTAGGTTGTTGGATTTTTATAATTAATATTATATA TATATGTGAATATATTTTTTGTGTATGTATTATTTATTGAAAGAAGA GTCATTAGATTTGTAAATTAAGCAATTACATAAAAATGTAAAATAT AATATTTCCTATATTTTATTTTATTTATTGAAGTACATTATACAAAA ATAAATATATATATTTAAAAATAGTGAGGAAAAAATCTAATTGTGT GTTTCTACTTATGATATTCTTTTAATGAAATGGAAAGAATAAAAAT AAAATGATATACTTATATAATTTAAAAAAAATATATATAATTCGTT TTTGTTAATTACTTTATTGTTGTTCTTCCTTTGTTATTATTATACTTT TTTTAGTATAATAATTTTATATAGATGTTTCAGTTTTTTTACATAGG GAAATTAAATTGTATTAGAATAAATGATTTTGTTGCATAAATAAAA GTAGTAAATGAAAAAAATAGTTTTTTTTTTTGTTTACATAAATAGA CATTTTAATAATATTAAACACTAACATATGTTTAAATGATCTATAA ATATTTGTGTAGTTAAAATAAAAAAAACGTAGATTTTTTAAAATCA TAAAATTATTTTTTATTTGTTTTATATATTTTTATTAAAAAAGTATA CATCTTCCTTCTTTACTTCTTAAAPf3D7_12_ TCTTTCAACAAAAAATAATATATGTGTACAAAAAATAAAATATGT v3 | GTAAAAAAAATAAAATATGTGTAAAATATATATATATATATATAA Plasmodia TGATATGAACAAGAATATTTTTATAAGGGTACTTATATAAAGGTTG m TATAATAATCCATAAACTAATTAAAATTATTATCATCGAACAATTA falciparum TTATTTCTTTTTATTTTATAATATTTATATTATATTATTTTTTACTTTT 3D7 | TTTGTACTTTTCTTACTTGATCATATATAAACATATGAATAATAATA 2026190 to GGGAATAAAATGGTAAAGACTCATATATATATATGTATATATATATATATATATATATATATTTAAAAAAATATATAAAAGAAAAAAAAAATAAAAAGAAAAATAAAAAGCATTTCTGCCGTTTCAAATATAGAAC2027125 - TCGTATTTTATTGCGTTTATATTTTTAGGTATTCTCTCTCTCTCTCTA 5’ arm TATATATATATATATATATATATATGTGTGAGTACGGTGAAGAATC AATTACACAATAATATTTTTCAATAGTTAAAAATATATTTCAATTT CTTTTAATCATATATATAATAAAAGAAACATAAAAATTTTTTATTT TGAATAAACAAATATATAAATATTAATTATATTTACAATAATAAAC AGTGTCAAACAAAAAAAATAAAATATTTAAAAAATATATTTTTTTA TATAAATATATATTCGTAACAATCTACATGTAAAAAAAAAAATTA AAATTATAATACTATAATAAAAATAAATATATGTATAGATTTATAT ATTATATTAATATTATATTAATGCGGATTTTTTTATGTAACAGAAA AATTTGACGTATATTCATAAACATTTAGATATGTGTATAATATGTA CATATATATATATATATATATATATATATATGTCTTAAATGGTTCAT ATAAAAAATAAAAAPf3D7_12_ AATATATATGTGATTTTATTAAGTACGGAAAATATTTATTATATAT v3 | ATATAAAAAAAAAAAAAAAAAAAACCATTTTATAGTTATTTTAAA Plasmodiu ATTTAAATTATTTATATATTTATCCATATATTTATTTTTTAAATTGCTCTTTTTTTTTTATAAATATATTGTTAATCAAATGAATAATTAAAAGAmfalciparum ACGGACATAGTACATTTGTTTGTTCTACCCTTCAAGGGAAATAATT 3D7 | TAAAACGAACTACATGATGATAATATATATATATATATATTATATA 2022536 to TATATATATTTTTTTTTTGGAAGAATGCAAAAAAAAAAAAAAAAA 2023464 AAAAAATTCTATTATAATATATTTTTTGTATTTTATTATGTGTGTCA - 3’ arm AAAAAAAAAAAAAAAAAACATATAAAGAAATAAAATAAAAAAAA AATATATTTTATATTTAACATGAATATACTTAATAATATGAGTCTTTrTTTTTATTTATTTTAAACTATTGCAATATATATGTCTTTTCAAGACTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTCTTTTTATAAATGATTATTATATTGTATATATGTTCTTTGAGTCTGTATATAATTCACTGAAGCCA TTTTTGATAATTAAAAAGAAAAAAAAATTATTATATAGTATAAACT AAAAAATGATACATTGAAAAAAAAGGTAGACAACACATTTTATTT TGAAAGATTTAAAAAGGAACATGTGTATGGGGATGTGTATATGTA TATGTATATATATATATATATATATATATATATATATGTTTAAATCC TTTTAAGAATAATGTGACAACCTATTTTGTAAAAAAAAAAAAAAA AAATAATAATAAACACACTATAGTAGTTTTCTTTAAAATGATTGAT TTTAATTTTTAATTTTTAATTTTTGATTTTTATCGCGAATTTTACTTT ICCPF3D7 06 CATTTATAATAATACATACAATTTTGAATTATAGGATTTTTTTCTTT 23400 GATGTATTATATGTATTAATATTATTATTTTTATAAAATGAATATTT Mei2-like TAATTATTATATTTTAATTTCATTTAAAGTATTTTAAAATTATTATT RNA- ATAATAAAAATATTAGATGCATCTTGTGCTTACATATAACATTTGC binding ATGTGTGGAAATATTCTCTTTCCATAAAAAGATACAACTTCTCTTG protein - 5’ TTTTTGTTTTATTTATTTTGATTATTGAGAAATAATATAAGAATATT Arm TAACCAATGAATTAAATTAAAAATGGTATTCTTTTTAAAAATGAAA AAAAAAAAAAAAAAACGTCAATGTAAGAACAAAAAAGTGACAAA TTCATTTGGTATTTTTTATATAATATATTTTTTTTAATTTATTTATTT ATTCCTTTATATATATATATATATATATATATATATATATACATACA TACATATATATATAATTTATATATAATTTATATATATTATATTAAAT GATATGTTTATAAGAAAAAATAAAATTATGTGAATATTTTAATTGT TATAATTGATTATAAATTATTTGTGTTTTTATTTTGTATAATTGTAT GCTCCAACATTTAATGTGTATGCTAAATATCGATGGAATATTATTA AATATATAATTCTTAAAAATTGAGATTTTACATATTTTTTTTTATTC CCGTTTTTAAGCTGTTTCATAAAAAAAAAAAAAATTCATAAAGATA TGAAATCCATATTTTTTTAAATATTTGGAAAAAGGAATATAACATG TAATTATATAATATATATATATATATATATATATATATAATTTGGA CTAATCATATTGATATAAAAAAATATTTTTATAATAAAAAATAATA AATAAATAAATAATAGAGGAGAGACATAACCAAAAAGTGTTGTAA ATAATACATTAATATATATATATATATAGATATATATATATTTTTTTrTTTTTTTTTTTCTCCTTTTATCAAAPF3D7 06 AATTCAAAGATGTATATAGACACAAAAAAAAAAAAAAAATAATA 23400 AAAAAATAAAAATATATATATATATTTATATATTTATGTATCATTG Mei2-like TTTAAAAAAATTAACAAATTAACCTTCTTATGTTTTTACAATTTACA RNA- TACTGATTATATATATATACGTGTATAATTTACTATTCATCTTATCT binding TATATATTATCTTCTTTTATTTATTTATTTATTTATTTGTTTTTTTTTT protein - 3’ TTTTTCTTTTTCTTTTTCTTTTTCTTTTTCCGCATTAAATATAATGTA Arm TCAAAATGATTATTTTAATTATATGAAAATAAAATATATCTGTTAA AAATAATAATGTGTAAAAAAAATAAAACTTATTTATATACATTTAT ATGTGATAATTATATATTATTATGTATATATATTAATTTATTTTTAT TTTATTGATTAAAATATAATATTAATGTTTTTATTTGTATATGAATA TATATTGTCAATGCTTTATATATATTTAAAATATATGAAGGAATTTTATTTTTTATATTCTTGTCTATATATATATATATATATATATATATATATGTATATATATTTTTTATTTCTTAACAAATTAAATCTAGTTTCTTT ATTATTATATAACTTTATCAACATTTAAAATAAATAAAATAATAAT TTGTGAATATATAATTATCAAATGTTTAATTGCTTTTTTATATCCCT FATAAAAACATATTTCGAAAAATATACATATATATATATATATATA FATATATATATATATATATATATATGTTTGAACCGTTAAGTAAATA ATATGGATAGTAATATTCTAATACTTTTATGAATTCCCTCGTAATTT TAATAATTGCCACAGCTTAATTTGAAATAAATGTTTCCTTGTATTA ATAATAATATTTATATTAAATATACATGGGCTTCTATATTATAATA FAAATATTTTTTAATTTACATATTTCTTATTGTACATCTCATAAAAA TATGAATATATTAATTTAAAATPlasmodiu MKNEDSTYKEDKYSKYSKENIQKICATPYTGSNISNTVESVYFVNYRA m VVVKTKHYYVTQNSYMLFGDVFKIYNLKGEIKKIHAFFPGDENNLSD falciparum TLIMLKTTIVAVTTSKGEALRLADEINMTCKICHKKRKATKFSIEGFLR LINUP KVRCDTCRVKPRSVICKNINNNKKTNNSKNNTNSNNSNNNNNNNINNamino acid NNNNNNNSNNSNNKNDSLKPDECNIQSNQNKHLTKNKIIKYENIDDE (Pf_124970 NIKNEKFKNMNIKKVNTKNCNITNSKAKKTDNNIQNDMNNNHPNNN 0) FVNNNILDNNHINNNILDNNHINNNILDNNHINNNHLNNNHLNNNHL NNNHLNNNHLNNNHFNNNHLNNNQLNNHLLDNRDKIQSLHYKHAN SNSVLKYNNSNNNYSASQNQIFFSEEENRTHFLENDPNKRDEIISMIIEL IKYISYMQKALIKASEGNFLIENPDINVENVSKLVHSAQQLCNKLLNQ NNIDGEYGNNNLCAPTSLLLHNNEKGNTIKNDVNINNISNNNNNNNY NFYNNNNYNNNNINYNNFSNNNQLKIKYTKMYQAKVNHITRTSNLNN NNNNNNNNTNNTDMFYNMNNPNDMSTTNTHMTNLSYINRNDQNKN NNMNNDTDSNIYINNKSNDNNIINNINNMNIMNNINNINNIKNINNINN IKNINNINNINSINSINNMNHMNHINGNNNINSNNNINSNNNINSNNNIN SNNNINSNNNINSNNNILNNTNERNKYNIENEYSKNIQNIQNIQNVQNN QSYVIPYYYNNRHNINSSTSLDTSTNDQKNKSYIKDNKEYMYENNKK DEHEDKNQYHDHQGKQQQQQQQQQQQQQQQQQQQHHNNNNISHT KNNYYNNKQQFLFSTLSKQYNTNVNISFANINNSLSNICKNVSKNYNL KDTVLNMEKAmino SKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITF Acid SQS11STLTGGSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLT sequence of FKFYMPKKATELKHLQCLEEELKPLEEVLNLAQGSGGGSELCDDDPPEIPHATFKAMAYKEGTMLNCECKRGFRRIKSGSLYMLCTGNSSHSSWengineered DNQCQCTSSATRNTTKQVTPQPEEQKERKTTEMQSPMQPVDQASLPG HCREPPPWENEATERIYHFVVGQMVYYQCVQGYRALHRGPAESVCK MTHGKTRWTQPQLICTGhIL-2analoguecIL-2eRaP230p_CR GAAGATTTTATCATTATTCGAGTCAGAGTACATCAAACTTATAAAA E AAATATTAGGAATAAGTAGTGATAAAAATGATAAACCATATTTTG AAGAAATTATTAGTGAAGAAAAAGATATATATGAATTTGAAGGATP230p ATTTAGAAAAAGTAATTGGAATCAAATTAGAAGGATATGAATTAG5’UTR- ATCCACCAAATTGTTTTAAAAGTGTATATGAAGATGATAAAAGAA UIS4TATATTTAGAAGTAGAATATCATTATTCAAAATGTATTAACTTAGAPromotor- CAGAAAAAATTATAAATTAAGATTTTATTTTTTATCACAATATTTT CSP GCTGATTATGAATTAAAGTTTTCATGTAATATAATTAATATAAAAA PEXEL- CTCAAAAAAAAAAAACTGTTACATTTGGTGATGGAATGGCAACAT CRE-UIS4CTGATCACATTTTAAAAATAGTTGATGATGATAGTAAAACGATTAA3’UTR- ATATTTTAACGATATACCTTATCAGATGTGTAATTTTGATTATAATTP230p TAAGCAAATTAAGTGAAATACAAATATGTGAAAAAACAATAAATG3’UTR AATTTAGTTTATTCATGTATAATTGTGAGCAAATAACAGATAATAG AATCGTATATGGTAAAGAACCTATTAATACCATAAAATATTTAAGT AATGTATTTCCTATAAATAAATTTACAGATTTATTTTTTAATACAAA AGATATAGATATACCAGAAATAAATGAACAATTTAAAGGTTTTAAATTTTTTATGACCTCATTTATAAATCATGGATCATATCCACTAACCTTTTTCTTTTAATGTATTAATTAGTGTAATAATTTTGTAAAAACTTTA TTCATTTATTTATTTTCCGTACTACATTGGGTTCATAATACCATAAT TATTATATAAACCACATTAAATAATTTGTAATTTATTCAAGGGTAT TAAAAAAAACATATATAAAATGCATATATCCACATATGGTTCATTA TAGGATGAATAAAAATGGGAACAACATATGAATTATTATATCATG AAAATAATGAAAAAACATAAATTATATGGATATATACATATATAA ATATATTAAAAAATAATAATAAATAAATAAAATGTATTATAAACC TTATAAAATAAGTGATGTTGTGGATAATCCACGAAATATGCCATAA ATAGACACTGAACAAATTAGTGGTTCTTAATATTTTTTTGGATACATGCGGATATTACCATTGACAGATGATTTATTTTTTGTTATTTTTAAAGAAAATCAGTTATTTAAATTTTAACTGAAGAAATTAAATAAGTATA CATTTTAGTTGTAGCGTCACTTTTATTAGTTGATTCTCTACTTCCAG GATATGGACAAAATAAAAGTGTCCAAGCCCAAAGAAACTTAAACG AGCTATGTTACAATGAAGAAAATGATAATAAATTGTATCACGTCCT CTGGAAAATGCTTCTTTCAGTATGTCGTTCTTGGGCAGCTTGGTGT CAATACAGCAGCATTTAGGACAATTGAATATGCTTCACCGTAGAA GTGGATTGC C AAGGCC AAGTGATAGTAATGC AGTTTC ATT AGTGAT GAGGAGAATACGTAAAGAGAATGTTGATGCAGGAGAGCGTGCAA CTCTTATGGAGAACTCAGACAGGTGCCAGGACATTCGTAATTTGGC GCACGTATAAGAGTAAAGGACATTTCTAGGACAGATGGTGGAAGGGAGAAAAGAAGGAAAAACATTATAACAAAATCACATTTTAACCATCAAACCTGGACTGATTATAGAAGAGCAATTATTTTATTACATGTTC GAACAGATTTAAAAGATAGACCAGTTTCATTTTGTGATTTTAGAAA CCGAAAATACAAAAAAAATGCCAGAAAAATGTTTTACTCAAGTAT GTAGATAAAGAAACAGAACAAATTGTAAAAACTGTTTTAGTAAAAP230p- GAAGATTTTATCATTATTCGAGTCAGAGTACATCAAACTTATAAAA HIV-VPR AAATATTAGGAATAAGTAGTGATAAAAATGATAAACCATATTTTG AAGAAATTATTAGTGAAGAAAAAGATATATATGAATTTGAAGGATP230p ATTTAGAAAAAGTAATTGGAATCAAATTAGAAGGATATGAATTAG5’UTR- ATCCACCAAATTGTTTTAAAAGTGTATATGAAGATGATAAAAGAA UIS4TATATTTAGAAGTAGAATATCATTATTCAAAATGTATTAACTTAGAPromotor- CAGAAAAAATTATAAATTAAGATTTTATTTTTTATCACAATATTTT CSP GCTGATTATGAATTAAAGTTTTCATGTAATATAATTAATATAAAAA PEXEL- CTCAAAAAAAAAAAACTGTTACATTTGGTGATGGAATGGCAACAT-VPR- CTGATCACATTTTAAAAATAGTTGATGATGATAGTAAAACGATTAA UIS4ATATTTTAACGATATACCTTATCAGATGTGTAATTTTGATTATAATT3’UTR- TAAGCAAATTAAGTGAAATACAAATATGTGAAAAAACAATAAATGP230p AATTTAGTTTATTCATGTATAATTGTGAGCAAATAACAGATAATAG3’UTR AATCGTATATGGTAAAGAACCTATTAATACCATAAAATATTTAAGT AATGTATTTCCTATAAATAAATTTACAGATTTATTTTTTAATACAAATTTTCTTTTAATGTATTAATTAGTGTAATAATTTTGTAAAAACTTTA TTCATTTATTTATTTTCCGTACTACATTGGGTTCATAATACCATAAT TATTATATAAACCACATTAAATAATTTGTAATTTATTCAAGGGTAT TAAAAAAAACATATATAAAATGCATATATCCACATATGGTTCATTA TAGGATGAATAAAAATGGGAACAACATATGAATTATTATATCATG AAAATAATGAAAAAACATAAATTATATGGATATATACATATATAA ATATATTAAAAAATAATAATAAATAAATAAAATGTATTATAAACC TTATAAAATAAGTGATGTTGTGGATAATCCACGAAATATGCCATAA ATAGACACTGAACAAATTAGTGGTTCTTAATATTTTTTTGGATACA TGCGGATATTACCATTGACAGATGATTTATTTTTTGTTATTTTTAAA TTATACATATTCATAGTTTATATAGTCCTCAAAAAATAGGATGTTT TATTCTTTTATAGCTATATTTTATGGTTGATCCTTTCCTTTTATGGTG TTTCATAAAAATTTTATTGAGCTATATATAATCCAATAAAAAAAGG TGATTGAATTTTGAAATATATTAAACTTTTTTTATAATAAAATAAA EATAATTATTTTTAAATAATATATATTATATATATATATTATATATT ATATATTTTTTTTTATCTTTACACAGAATTTTTTTTATAGAGTCCAATATATATAATTAGTTATATATATACACCACCATAAATAATTATAAGGAAAATCAGTTATTTAAATTTTAACTGAAGAAATTAAATAAGTATA CATTTTAGTTGTAGCGTCACTTTTATTAGTTGATTCTCTACTTCCAG GATATGGACAAAATAAAAGTGTCCAAGCCCAAAGAAACTTAAACGCTTGGACAGCACATATATGAGACATATGGAGATACTTGGGCAGGA GTTGAGGCCATAATAAGAATTTTGCAACAGCTTCTTTTTATTCACTT CCAGAATTGGGTGAGTACATAGCATTATGAGGGTAATTCAGAAAGCGAAAAGAAATAGAAGAAGAAAAAAAATTATATATAATATATTTGAGCAATTATTTTATTACATGTTCGAACAGATTTAAAAGATAGACCA GTTTCATTTTGTGATTTTAGAAAAGGTGAATTATATAATTATTTAAATATCAATTTGAATGTCATTGTGTAGATAAAGAAACAGAACAAAT TGTAAAAACTGTTTTAGTAAAATATGTAAATGAAGATGAAATATAT GATTATAATGATTTACCATTGGTTAATCATAAATCTATTGTTGCAC ATCCAAATAAAACACATCTATGTGACTTTATGACATCTGATAATAT GTTATCACCTAAAAAAGAAGATTCAGTAAATTATGTTTGTAATGTA TTTCCAAAACCATTAGAATATGTAGCATTACATTGTCCAACCAATA TAGTAGATGTAGAAAATGAAGATGATATTTCAAGACTATCAAAGG AAATGCATAAAGAAGAAATAAAAGCGGAATTAAGAATCAAACTTCP230p- GAAGATTTTATCATTATTCGAGTCAGAGTACATCAAACTTATAAAA WNV-NS1 AAATATTAGGAATAAGTAGTGATAAAAATGATAAACCATATTTTG AAGAAATTATTAGTGAAGAAAAAGATATATATGAATTTGAAGGATP230pATTTAGAAAAAGTAATTGGAATCAAATTAGAAGGATATGAATTAG5’UTR- ATCCACCAAATTGTTTTAAAAGTGTATATGAAGATGATAAAAGAA UIS4TATATTTAGAAGTAGAATATCATTATTCAAAATGTATTAACTTAGAPromotor- CAGAAAAAATTATAAATTAAGATTTTATTTTTTATCACAATATTTT CSP GCTGATTATGAATTAAAGTTTTCATGTAATATAATTAATATAAAAA PEXEL- CTCAAAAAAAAAAAACTGTTACATTTGGTGATGGAATGGCAACAT WNV- CTGATCACATTTTAAAAATAGTTGATGATGATAGTAAAACGATTAA NS1-UIS4ATATTTTAACGATATACCTTATCAGATGTGTAATTTTGATTATAATT3’UTR- TAAGCAAATTAAGTGAAATACAAATATGTGAAAAAACAATAAATGP230p AATTTAGTTTATTCATGTATAATTGTGAGCAAATAACAGATAATAG3’UTR AATCGTATATGGTAAAGAACCTATTAATACCATAAAATATTTAAGT AATGTATTTCCTATAAATAAATTTACAGATTTATTTTTTAATACAAA AGATATAGATATACCAGAAATAAATGAACAATTTAAAGGTTTTAAATTTTTTATGACCTCATTTATAAATCATGGATCATATCCACTAACCTTTTTCTTTTAATGTATTAATTAGTGTAATAATTTTGTAAAAACTTTA TTCATTTATTTATTTTCCGTACTACATTGGGTTCATAATACCATAAT TATTATATAAACCACATTAAATAATTTGTAATTTATTCAAGGGTAT TAAAAAAAACATATATAAAATGCATATATCCACATATGGTTCATTA TAGGATGAATAAAAATGGGAACAACATATGAATTATTATATCATG AAAATAATGAAAAAACATAAATTATATGGATATATACATATATAA ATATATTAAAAAATAATAATAAATAAATAAAATGTATTATAAACCTTATAAAATAAGTGATGTTGTGGATAATCCACGAAATATGCCATAAGAAAATCAGTTATTTAAATTTTAACTGAAGAAATTAAATAAGTATACATTTTAGTTGTAGCGTCACTTTTATTAGTTGATTCTCTACTTCCAG GATATGGACAAAATAAAAGTGTCCAAGCCCAAAGAAACTTAAACGGCAAGAGCTTAGATGTGGTTCAGGTGTATTCATACACAATGACGTA GAAGCATGGATGGACAGGTATAAGTTCTACCCTGAAACTCCACAA GGACTTGCTAAAATAATTCAAAAAGCACATGCAGAGGGAGTGTGT GGTCTTCGTTCAGTTTCTCGTCTTGAGCATCAAATGTGGGAGGCTAACATTCGTAATTGACGGTCCTGAAACCGAAGAATGTCCTACCGCTACGAATGCGATTCAAAAATTATTGGAACAGCAGTAAAAAACAACAT GGCCGTGCATTCAGATTTAAGTTACTGGATTGAGTCAGGATTAAAT GATACTTGGAAGCTAGAGAGGGCCGTGTTAGGAGAAGTTAAGTCAGAATCAGACCTTATAATTCCAATTACCCTTGCTGGACCAAGAAGTA GGGATGAGGGAAGAGTGGAAATTGACTTCGATTACTGTCCTGGAA CAACCGTGACTATTTCAGATTCTTGTGAGCACAGAGGACCTGCAGCCAGAAAGAGAACAAATTGTTTTATGATAAATATATTTATAAAATAGTGTATAAAAAACTTTTACAATATATTTTTTTATTTTGAACGTATATAATCTGGCTTCGAAAAGAAATAGAAGAAGAAAAAAAATTATATAT GAAAATTTTTTTTATGTATTTTTGAGAGTAATTTTTAGTGTTTGTTTCTGATAATATGTTATCACCTAAAAAAGAAGATTCAGTAAATTATGTCCAACCAATATAGTAGATGTAGAAAATGAAGATGATATTTCAAGACTATCAAAGGAAATGCATAAAGAAGAAATAAAAGCGGAATTAAG AATCAAACTTCPy_mei2_U GGTATGTATATTC ATGTGC CTTTTTTGAAGAGC ATTTTTAATATTGA IS4 CTTTAGTTTTATTATTGTTGTAACTATTATACATAACAAATAAAAA promoter? AACATTTAATTTAAGAATATATAATTCATAAAAAAAATTTTGTTCA yCSPPEXE TATATTCATATTTGGTTGTTTTTTTTTTTTTTTTCCAATAGTATGGTA L hcIL- TTTTTCATTATACAACTTAACTACATTTTCTATTCTGTTATATTACA 2eRa ETACTCTCATTAATAGTGTAACATTAACAACGTAAAATTCTGAATA CATGGTTTTTCTTCTAAAAAGGTAAATATAGAATATGTATGCTTTA TATATATTTTAATGTCAAAAAAAAATAAAAATAATGCTATATTTTT AATATATTTTTTAATCATTTTATACGTTTTATTTTAATAATTCTGTG ATATAATTTGGCTACATTTAATTACCTTGCCGGTCAGAAAAAAAAA CATAAAAATATGCATGTACATAAAAAATATAATCATTACACACAA AAAATATATATATACATACATATAAAGAGAAAATAAGACTTAAAA TAATTCTGACGTATGAATATAGAAATAGAAAAGTTGTAAATATAA ATAAGGACGGACAAAACCATGGTTCTTTGAGCAAATACTGAACAA EAGGGAATGCTTCTATGTAACTGTGGATATATATGTACGTAATAAA ATAATTTTTTGTCCATAAAAATATCTTTAACAGCTACTATACAAAA GCAATGAATTGGTTAATACATTTTTGTCGTAAATAAAATAAATGAA TTAATGGTACAAAATTACAATAAAAAGGGAGCAAGTTTATTGTATT TTGTTTAATTTATGTCCTTTCCATTTTATTAATACAATTATGATATT CACTAATTCGTATTAAACATATCTTTTGCATGTACCAAAGGTTTGG GGAATAAGCATATGAATCCCATTAAATTTAATGATATTCTGCGATT TTTCTTGTATTTACTATTAAATATAATGGATTCATTTTTTGATGCAT GCAATTTTTTCTTTTAATGTATTAATTAGTGTAATAATTTTGTAAAA ACTTTATTCATTTATTTATTTTCCCTATTATATTGGGTTCATAATAC CATAATTATTATATAAACCACATTAAATAATTTGTAATTTATTCAA GGGTATTAAAAAAAACATATATAAAATGCATATATCCACATATGG ETCATTATAGGATGAATAAAAATGGGAACAACATATGAATTATTAT ATCATGAAAATAATGAAAAAACATAAATTATATGGATATATACAT ATATAAATATATTAAAAAATAATAATAAATAAATAAAATGTATTA TAAACCTTATAAAATAAGTGATGTTGTGGATAATCCACGAAATATGCCATAAATAGACACTGAACAAATTAGTGGTTCTTAATATTTTTTTGGATACATGCGGATATTACCATTGACAGATGATTTATTTTTTGTTATTCTTAAAAATTTTTTATTATTACATATTTATACATAAAAAAAATAAACATATATACCTTTCAGCACATAATTACATCTGAATAAAATGATGAA GAAGTGTACCATTTTAGTTGTAGCGTCACTTTTATTAGTTGATTCTCGGCCTAGGATGTATAGAATGCAATTATTAAGTTGTATAGCATTAAGGTGAAACAACATTTATGTGTGAATATGCAGATGAAACAGCAACAAGCTATTGTTTATAAAATTATAGAATTTTACAACTATGCTAATTTATTCCTGATCATATATATATACACTCATTTATAATATTTTAGTTTTAAACGTTTATTCCATATATCTAAAATTATTACTATTGCCAATGCGAAAACAGGCTATATGTTTCCCTTTTATTACATGTTTGAATTTGTGTATTATT ATTGTTATTCACATAATAATAAATGTGAAATGATGACCCGAGTTGT GAAGGTGATATCCTATATTATGTATGTTCATATGTATGCGTGTTCA TATGTATGCGTGTTCATATGTATGCGTGTTCATATGTATGCGTGTTC ATATGTATGCGTGTTCATATGTATGCGTGTTCATATGTATGTGTGTT CATGTATATGCTTGTTCATATGTATGTGTGTGTGTGAATGACGAGC GGTATTTATATTACAGGTACATAAATTTGCACACATATTGATAATT ATATTTTCCCTGTACATTTTATTTCCCTCTTTTTAATATATTACTTTA TATAACATGTATGTGAGTGCTTATAAATAAAATTGTGTAAAATTCG TTGGTAACTTATTATTACAACTGTTGTTATTTTGCTTATTTATTACT ATTTATTTTTTTAGCCTTAAACTGGTGCCTCTTTGATATGTTCTCTT GTATATGTCCACGAGTTATTTTGGPf linup P tttattttatatgaaccattaagacatatatatatatatatatatatatatatatgtacatattatacacatatctaaat fCSP_prom gtttatgaatatacgtcaaattttctgttacataaaaaaatccgcataatataatataatataatatataaatctatac oter PFCS atatattattttatatagtatataattaattttttttacatgtagatgtacgaatatatattatataaaaaaatat PPEXEL_ attttttaaatattttattttttttgtttgacactgtttattattgtaaatataattaatattatatatttgtttattcaaaataaa HBcAg aaattttatgttctttattatatatatgattaaaagaaatgaaatatatttaactattgaaaaatattattgtgtaat gatcttcaccgtactcacacatatatatatatatatatatatatatagagagagagagagaatacctaaaaatataa acgcaataaaatacgagtctatattgaaacggcagaaatgcttttattttcttttattttttttttcttttatatatttt taaatatatatatatatatatatatatatacatatatatatatgagtctttaccattttattccctattattattcatatgtttat atatgatcaagtaagaaaagtacaaaaaaagtaaaaaataatataatataaatatataaaataaaaagaaataat aattgttcgatgataataaUUaattagtltatggatlatlatacaacclttatataagtacccttataaaaatattcltgt tcatatcatatatatatatatatatattacacatattattttttacacatattttatttttgtacacatatatatttt gtgaaagaggcgcgCTATATGTTTGAGCTTATTTCAATTGTTGTGTTTTAT TTTAATTATTTTTAAGTATACATTATAAGTAGGTGTAATATTAACAT AAGTACTTGCTTTTTCCTTTTAATATTATTTTATAAAGCTTTTATAA TATATTTTTGTTTTATTTCATATTTATAGTTAGTAAAATATAATTTCCTTTTTAAAGCACGTGATAAAGT^TTGTATGTAAATTAAAATTTTAATTGTTAAAATAATAAAAAGTGAATTTATAAAAAAAAGCACGAA TGAAACTATTTAATTTTCTTTTGGGCATTTTATTATTATTATAATTTT TTAAATAATATGTTGCATTTATCATGCCTGGTGTGTATTTTATAACT ATTATGTAAGAACATACCTAAAAGATGAAATTAATAAAAATGCTA TGATCAGGAAGCATGAGCACATATATATCATATGTTTAATCAAACATTTTATGGGATTATTGTAAATATAACATGCACATTTTGTATAAGTTCCTTAATTTTTTTTTTTTTTTTTTTGGGGGGGGAGGGGTAAAGGGGG GCTTAATTATAAAACAGAAATTATTCTTATCTTACATGCACATATACGATTTGGATATATATTTTTTTTTTTGTAACTTTCTATCATACTTGTCGCTATGGAAGTTCGTCAAACACAAGGGTTCTAAATGAATTAAATTgatggatatagatccatataaa; aatttggagcaagtgtagaatattaagttttttaccaagtgatttttttccaagtataagagatttatagatacagca agtgcattatatagagaagcatagaaagtccagaacatgtagtccacatcatacagcataagacaagcaata;ggattaaaaataagacaattattatggtttcatataagttgtttaacatttgg aagagaaacagtattagaatattagtaagttttggagtatggataagaacaccaccagcatatagaccaccaaaaccaagtccaagaagaagaagaagtcaaagtccaagaagaagaagaagtcaaagtagagaaagtcaatgtT AACTCGAGcccgggCCTGCAGGtggggaaaagtaaaatcgcgataaaaatcaaaaataaa aattaaaaattaaaatcaatcattttaaagaaaactactatagtgtgtttattatattttttttttttttttacaaaataggccatacacatgtcctttaaatctttcaaaataaaatgtgtgtctaccttttttttcaatgtatcattttagtttatact atataataatttttttcttttaatatcaaaaatggcttcagtgaatatatacagactcaaagaacatatatacaata:gaaaagacatatatattgcalicit tatatgtlttttttt lit lit lit tgacacacataataaaatacaaaaaatatattataalagaat lit tttttttt lit lit lit tgcattctccaaaaaaaaaatatatatatatataatatatatatatatatatatcatcatgtagtcgttaaatattt cccttgaagggtagaacaaacaaatgtactatgtccgttcttttaatattcatttgattaacaatatatttataaaaaa aaaagagcaatttaaaaaataaatatatggataaatatataaataattaaattttaaaataactataaaatggttttttt ttttttttttttatatatatataataaatattccgtactaataaaatcacatatatatPf linup P tttattttttatatgaaccatttaagacatatatatatatatatatatatatatatatgtacatattatacacatatctaaat fCSP_prom gtttatgaatatacgtcaaatttttctgtacataaaaaaatccgcataatataatataatataatatataaatctatac oter PFCS atatattatttttattatagtattataatttaattttttttttacatgtagattgtacgaatatatatttatataaaaaaatat PPEXEL_ atttttaaatatttatttttgtttgacactgtttatatgtaaatataataatatttatatattgtttatcaaaataaa HBsAg aaatttatgtttctttatatatatatgataaaagaaatgaaatatatttaactatgaaaaatattatgtgtaatt gattctcaccgtactcacacatatatatatatatatatatatatatagagagagagagagaatacctaaaaatataa acgcaataaaatacgagttctatatttgaaacggcagaaatgctttttatttttcttttattttttttttcttttatatatttttt taaatatatatatatatatatatatatatacatatatatatatgagtctttaccatttatccctattattatcatatgtttat atatgatcaagtaagaaaagtacaaaaaaagtaaaaaataatataatataaatatataaaataaaaagaaataat aatgttcgatgataataatttaatagttatggattatatacaaccttatataagtacccttataaaaatatctgt tcatatcatatatatatatatatatattttacacatatttattttttttacacatattttatttttgtacacatatattatttttt gttgaaagaggcgcgCTATATGTTTGAGCTTATTTCAATTGTTGTGTTTTAT TTTAATTATTTTTAAGTATACATTATAAGTAGGTGTAATATTAACAT AAGTACTTGCTTTTTCCTTTTAATATTATTTTATAAAGCTTTTATAA TATATTTTTGTTTTATTTCATATTTATAGTTAGTAAAATATAATTTCCTTTTTAAAGCACGTGATAAAGT^TTGTATGTAAATTAAAATTTTAATTGTTAAAATAATAAAAAGTGAATTTATAAAAAAAAGCACGAA TGAAACTATTTAATTTTCTTTTGGGCATTTTATTATTATTATAATTTT TTAAATAATATGTTGCATTTATCATGCCTGGTGTGTATTTTATAACT ATTATGTAAGAACATACCTAAAAGATGAAATTAATAAAAATGCTA EGATCAGGAAGCATGAGCACATATATATCATATGTTTAATCAAACA TTTTATGGGATTATTGTAAATATAACATGCACATTTTGTATAAGTTC CTTAATTTTTTTTTTTTTTTTTTTGGGGGGGGAGGGGTAAAGGGGG GCTTAATTATAAAACAGAAATTATTCTTATCTTACATGCACATATA AAAAAATGGATTGGTGGTAAACCAAAAAAATAAATTCACTATATG ETCTTAAGGAAGCATATAATGTTTTCCTTTTTTTTTACATGCAGATA TAAAAAGGTAGAAGAACTTACGAGAAGCTCTATATTTTACACATG CGATTTGGATATATATTTTTTTTTTTGTAACTTTCTATCATACTTGTCATAAATTCTGAATTATCAAATAACTCAAATATATTTCATAATATCAGCTATGGAAGTTCGTCAAACACAAGGGTTCTAAATGAATTAAATTATTAGGATTTTTTCCAGATCATCAATTAGATCCAGCATTTGGAGCA GGCCAGAAGCAAATCAAGTAGGAGCAGGAGCATTTGGACCAGGATCAAATAGACAAAGTGGAAGACAACCAACACCAATAAGTCCACCATCCACAAAGTTTAGATAGTTGGTGGACAAGTTTAAATTTTTTAGGAG GAGCACCAACATGTCCAGGACAAAATAGTCAAAGTCCAACAAGTA GATGTGTTTAAGAAGATTTATAATATTTTTATTTATATTATTATTAT GTTTAATATTTTTATTAGTATTATTAGATTATCAAGGAATGTTACCA GTATGTCCATTATTACCAGGAACAAGTACAACAAGTACAGGACCACAATACCAAGTAGTTGGGCATTTGCAAGATTTTTATGGGAATGGGC AAGTGTAAGATTTAGTTGGTTAAGTTTATTAGTACCATTTGTACAA TGGTTTGTAGGATTAAGTCCAACAGTATGGTTAAGTGTAATATGGA TGATGTGGTATTGGGGACCAAGTTTATATAATATATTAAGTCCATT TTTACCATTATTACCAATATTTTTTTGTTTATGGGTATATATATAAC TCGAGcccgggC CT GC AGGttggggaaaag t aaaatt cgcgataaaaatcaaaaattaaaaat taa aaattaaaatcaatcattttaaagaaaactactatagtgtgtttattattattttttttttttttttttacaaaataggttgtca cattattcttaaaaggatttaaacatatatatatatatatatatatatatatatatacatatacatatacacatccccatac acatgtcctttaaatctttcaaaataaaatgtgtgtctacctttttttcaatgtatcatttttagttatactatataat aatttttttctttaatatcaaaaatggctcagtgaatatatacagactcaaagaacatatatacaatataataat cattataaaaagaaaaaaaaaaaaaaaaaaaaaaaaaaaaaagtctgaaaagacatatatattgcaatagttt aaaataaataaaaaaaaagactcatattataagtatatcatgttaaatataaaatatattttttttttattttatttctttat atgttttttttttttttgacacacataataaaatacaaaaaatatattataatagaattttttttttttttttttttgcatt cttccaaaaaaaaaatatatatatatataatatatatatatatatatatcatcatgtagtcgttttaaattattccctg aagggtagaacaaacaaatgtactatgtccgttcttttaattattcatttgattaacaatatatttataaaaaaaaaag agcaatttaaaaaataaatatatggataaatatataaataatttaaattttaaaataactataaaatggttttttttttttttt ttttatatatatataataaatattttccgtacttaataaaatcacatatatattPf linup P tttattttttatatgaaccattaagacatatatatatatatatatatatatatatatgtacatatatacacatatctaaat fCSP_prom gttatgaatatacgtcaaatttttctgttacataaaaaaatccgcataatataatataatataatatataaatctatac oter PFCS atatatttatttttattatagtattataattttaatttttttttttacatgtagattgttacgaatatatatttatataaaaaaatat PPEXEL h attttttaaatattttatttttttgtttgacactgtttattattgtaaatataattaatattatatatttgtttattcaaaataaa CIL-2eRa aaattlttatgtltcttltattatatatatgaltaaaagaaattgaaatatattlttaactattgaaaaalaltattgtgtaatt gatcttcaccgtactcacacatatatatatatatatatatatatatagagagagagagagaatacctaaaaatataa acgcaataaaatacgagtctatatttgaaacggcagaaatgctttttatttctttatttttttttcttttatatatttttt taaatatatatatatatatatatatatatacatatatatatatgagtctttaccattttattccctatattattcatatgtttat atatgatcaagtaagaaaagtacaaaaaaagtaaaaaataatataatataaatatataaaataaaaagaaataat aatgttcgatgataataatttaatagtttatggatatatacaaccttatataagtacccttataaaaatatctgt tcatatcatatatatatatatatatattacacatattattttttacacatattattttgtacacatatatattt gttgaaagaggcgcgCTATATGTTTGAGCTTATTTCAATTGTTGTGTTTTAT TTTAATTATTTTTAAGTATACATTATAAGTAGGTGTAATATTAACAT AAGTACTTGCTTTTTCCTTTTAATATTATTTTATAAAGCTTTTATAA TATATTTTTGTTTTATTTCATATTTATAGTTAGTAAAATATAATTTC CTTTTTAAAGCACGTGATAAAGTAATTGTATGTAAATTAAAATTTTAATTGTTAAAATAATAAAAAGTGAATTTATAAAAAAAAGCACGAACTTAATTTTTTTTTTTTTTTTTTTGGGGGGGGAGGGGTAAAGGGGG GCTTAATTATAAAACAGAAATTATTCTTATCTTACATGCACATATACGATTTGGATATATATTTTTTTTTTTGTAACTTTCTATCATACTTGTCACTATTTTTCATTATAATTTTTTTTTTTTTTGTGTTTTTTTATATATTTGCTATGGAAGTTCGTCAAACACAAGGGTTCTAAATGAATTAAATTCCAGCAAGTAGTAGTACAAAAAAAACACAATTACAATTAGAACATGTATTAGAATTAAAAGGAAGTGAAACAACATTTATGTGTGAATATGCAGATGAAACAGCAACAATAGTAGAATTTTTAAATAGATGGATAACATTTGCACAAAGTATAATAAGTACATTAACATAACTCGAGcccgg gCCTGCAGGttggggaaaagtaaaattcgcgataaaaatcaaaaattaaaaattaaaaattaaaatcaat catttaaagaaaactactatagtgtgtttattatattttttttttttttttacaaaataggttgtcacattattctaaaag gatttaaacatatatatatatatatatatatatatatatatacatatacatatacacatccccatacacatgtcctttta aatctttcaaaataaaatgtgtgtctacctttttttcaatgtatcattttttagttatactatataataattttttttcttttt aattatcaaaaatggctcagtgaattatatacagactcaaagaacatatatacaatataataatcatttataaaaag aaaaaaaaaaaaaaaaaaaaaaaaaaaaaagtcttgaaaagacatatatattgcaatagtttaaaataaataaaa aaaaagactcatattattaagtatattcatgttaaatataaaatatatttttttttatttattctttatatgtttttttttttttt tttgacacacataataaaatacaaaaaatatatataatagaattttttttttttttttttgcatctccaaaaaaaa aatatatatatatataatatatatatatatatattatcatcatgtagttcgtttaaatattccctgaagggtagaaca aacaaatgtactatgtccgttcttttaattattcatttgattaacaatatatttataaaaaaaaaagagcaatttaaaaa ataaatatatggataaatatataaataatttaaatttaaaataactataaaatggttttttttttttttttttttatatatatata ataaatatttccgtacttaataaaatcacatatatattPf_Mei2_P aatatagatgcatctgtgctacatataacatttgcatgtgtggaaatatctcttccataaaaagatacaactct fCSP_prom cttgttttgttttattatttgatattgagaaataatataagaatattaaccaatgaataaattaaaaatggtatctt oter PFCS tttaaaaatgaaaaaaaaaaaaaaaaaacgtcaatgtaagaacaaaaaagtgacaaattcatttggtattttttata PPEXEL_ taatatatttttttaattatttatttattcctttatatatatatatatatatatatatatatatatacatacatacatatatatat HBpolAg aatttatatataattatatatatatataaatgatatgttataagaaaaaataaaatatgtgaatatttaatgtata atgattataaatatttgtgttttattgtataatgtatgctccaacattaatgtgtatgctaaatatcgatggaatat tattaaatatataattcttaaaaattgagattttacatatttttttttattcccgtttttaagctgtttcataaaaaaaaaaaa aattcataaagatatgaaatccatatttttaaatatttggaaaaaggaatataacatgtaatatataatatatatata latatatatatatatalaatttggaclaatcalaltgalataaaaaaataUtUataalaaaaaataataaataaataaat aatagaggagagacataaccaaaaagtgtgtaaataatacataatatatatatatatatagatatatatatattttt tttttttttttctcctttatcaaaatgcataagaGCGCGC AT AT AT AT AT AT AT AT AT AT A TATATATATATATATATGTTTGAGCTTATTTCAATTGTTGTGTTTTA TTTTAATTATTTTTAAGTATACATTATAAGTAGGTGTAATATTAACA TAAGTACTTGCTTTTTCCTTTTAATATTATTTTATAAAGCTTTTATA ATATATTTTTGTTTTATTTCATATTTATAGTTAGTAAAATATAATTT CCTTTTTAAAGCACGTGATAAAGTAATTGTATGTAAATTAAAATTT TAATTGTTAAAATAATAAAAAGTGAATTTATAAAAAAAAGCACGA ATGAAACTATTTAATTTTCTTTTGGGCATTTTATTATTATTATAATT TTTTAAATAATATGTTGCATTTATCATGCCTGGTGTGTATTTTATAA CTATTATGTAAGAACATACCTAAAAGATGAAATTAATAAAAATGCTATGATCAGGAAGCATGAGCACATATATATCATATGTTTAATCAAACATTTTATGGGATTATTGTAAATATAACATGCACATTTTGTATAAGGGGCTTAATTATAAAACAGAAATTATTCTTATCTTACATGCACATACATAATGCTACAATAATAAAAATATTTTTAATATATATTAAAAAAAAAAATTATAAATAAATATATATATTCGTGTAAAAATAAGTAGAAA CCACGTATATTATAAATTACAATTCATGATGAGAAAATTAGCTATTCCAGTGCTATGGAAGTTCGTCAAACACAAGGGTTCTAAATGAATTGTATGTTGTCAATTAGATCCAGCAAGAGATGTATTATGTTTAAGACGAACATTACCAAGTCCAAGTAGTAGTGCAGTACCAGCAGATCATG GAGCACATTTAAGTTTAAGAGGATTACCAGTATGTGCATTTAGTAGAAGAACATTAGGATTAAGTGCAATGAGTACAACAGATTTAGAAGCCCTGCAGGltaaattcaaagatgtatatagacacaaaaaaaaaaaaaaaataataaaaaaataaaaatat atatatatatttatalatttatgtatcaltgtttaaaaaaatlaacaaatlaacctlcttatgtttttacaattlacatactgattctltttctttttcttttlctttltccgcatlaaalalaatgtatcaaaatgattattttaaltatatgaaaataaaatatalclg taaaaataataatgtgtaaaaaaaataaaactatttatatacattatatgtgataattatatattatatgtatatatat taattatttttattttattgattaaaatataatataatgttttatttgtatatgaatatatattgtcaatgctttatatatattt aaaatatatgaaggaatttatttttatatcttgtctatatatatatatatatatatatatatatatgtatatatattttttatt ctaacaaataaatctagtttctttatatatataacttatcaacattaaaataaataaaataataattgtgaatata taatatcaaatgttaatgctttttatatcccttataaaaacatattcgaaaaatatacatatatatatatatatatata tatatatatatatatatatatatgtttgaaccgttaagtaaataatatggatagtaatattctaatacttttatgaattccct cgtaattttaataatgccacagcttaatttgaaataaatPf_Mei2_P tttattttatatgaaccatttaagacatatatatatatatatatatatatatatatgtacatattatacacatatctaaat PCS P prom gttatgaatatacgtcaaattttctgttacataaaaaaatccgcataatataatataatataatatataaatctatac oter PFCS atatattattttatatagtattataatttaatttttttttacatgtagatgtacgaatatatatttatataaaaaaatat PPEXEL_ atttttaaatattttatttttttgtttgacactgtttattatgtaaatataattaatattatatatttgtttattcaaaataaa HBxAg aaattttatgttctttatatatatatgataaaagaaatgaaatatatttaactattgaaaaatattatgtgtaat gatcttcaccgtactcacacatatatatatatatatatatatatatagagagagagagagaatacctaaaaatataa acgcaataaaatacgagttctatattgaaacggcagaaatgctttattttctttattttttttcttttatatatttt taaatatatatatatatatatatatatatacatatatatatatgagtctttaccattttattccctatattattcatatgtttat atatgatcaagtaagaaaagtacaaaaaaagtaaaaaataatataatataaatatataaaataaaaagaaataat aatgttcgatgataataatttaatagtttatggatatatacaaccttatataagtaccctataaaaatatctgt tcatatcatatatatatatatatatatttacacatattattttttacacatatttattttgtacacatatatattttt gttgaaagaggcgcgCTATATGTTTGAGCTTATTTCAATTGTTGTGTTTTAT TTTAATTATTTTTAAGTATACATTATAAGTAGGTGTAATATTAACAT AAGTACTTGCTTTTTCCTTTTAATATTATTTTATAAAGCTTTTATAA TATATTTTTGTTTTATTTCATATTTATAGTTAGTAAAATATAATTTCCTTTTTAAAGCACGTGATAAAGTAATTGTATGTAAATTAAAATTTTAATTGTTAAAATAATAAAAAGTGAATTTATAAAAAAAAGCACGAA TGAAACTATTTAATTTTCTTTTGGGCATTTTATTATTATTATAATTTT TTAAATAATATGTTGCATTTATCATGCCTGGTGTGTATTTTATAACT ATTATGTAAGAACATACCTAAAAGATGAAATTAATAAAAATGCTA TGATCAGGAAGCATGAGCACATATATATCATATGTTTAATCAAACA TTTTATGGGATTATTGTAAATATAACATGCACATTTTGTATAAGTTC CTTAATTTTTTTTTTTTTTTTTTTGGGGGGGGAGGGGTAAAGGGGG GCTTAATTATAAAACAGAAATTATTCTTATCTTACATGCACATATA AAAAAATGGATTGGTGGTAAACCAAAAAAATAAATTCACTATATGTTCTTAAGGAAGCATATAATGTTTTCCTTTTTTTTTACATGCAGATACGATTTGGATATATATTTTTTTTTTTGTAACTTTCTATCATACTTGTCGCTATGGAAGTTCGTCAAACACAAGGGTTCTAAATGAATTAAATTGGCCAGAAGCAAATCAAGTAGGAGCAGGAGCATTTGGACCAGGATCAAATAGACAAAGTGGAAGACAACCAACACCAATAAGTCCACCATACCAAATATGGAAAATACAACAAGTGGATTTTTAGGACCATTATTCCACAAAGTTTAGATAGTTGGTGGACAAGTTTAAATTTTTTAGGAG GAGCACCAACATGTCCAGGACAAAATAGTCAAAGTCCAACAAGTAGATGTGTTTAAGAAGATTTATAATATTTTTATTTATATTATTATTATGTTTAATATTTTTATTAGTATTATTAGATTATCAAGGAATGTTACCAGTATGTCCATTATTACCAGGAACAAGTACAACAAGTACAGGACCA TGTAAAACATGTACAATACCAGCACAAGGAACAAGTATGTTTCCA AGTTGTTGTTGTACAAAACCAAGTGATGGAAATTGTACATGTATAC CAATACCAAGTAGTTGGGCATTTGCAAGATTTTTATGGGAATGGGC AAGTGTAAGATTTAGTTGGTTAAGTTTATTAGTACCATTTGTACAA TGGTTTGTAGGATTAAGTCCAACAGTATGGTTAAGTGTAATATGGA TGATGTGGTATTGGGGACCAAGTTTATATAATATATTAAGTCCATT TTTACCATTATTACCAATATTTTTTTGTTTATGGGTATATATATAAC TCGAGcccgggC CT GC AGGtggggaaaagtaaaatcgcgataaaaatcaaaaataaaaattaa aaataaaatcaatcattttaaagaaaactactatagtgtgtttatatatttttttttttttttttacaaaataggttgtca cattatcttaaaaggatttaaacatatatatatatatatatatatatatatatatacatatacatatacacatccccatac acatgttcctttttaaatctttcaaaataaaatgtgttgtctaccttttttttcaatgtatcattttttagtttatactatataat aattttttttcttttaattatcaaaaatggcttcagtgaattatatacagactcaaagaacatatatacaatataataat catttataaaaagaaaaaaaaaaaaaaaaaaaaaaaaaaaaaagtcttgaaaagacatatatatgcaatagtt aaaataaataaaaaaaaagactcatattattaagtatatcatgttaaatataaaatatatttttttttattttatttcttat atgtttttttttttttttttttgacacacataataaaatacaaaaaatatatataatagaatttttttttttttttttttttttgcatt cttccaaaaaaaaaatatatatatatataatatatatatatatatattatcatcatgtagttcgtttaaattatttcccttg aagggtagaacaaacaaatgtactatgtccgtctttaatatcatttgattaacaatatattataaaaaaaaaag agcaatttaaaaaataaatatatggataaatatataaataatttaaatttaaaataactataaaatggttttttttttttt tttttatatatatataataaatattttccgtacttaataaaatcacatatatattPy_Mei2_ ttttattttttatatgaaccatttaagacatatatatatatatatatatatatatatatgtacatattatacacatatctaaat UIS4 gtttatgaatatacgtcaaatttttctgttacataaaaaaatccgcattaatataatattaatataatatataaatctatac promoter? atatattatttttatatagtattataatttaattttttttttacatgtagatgtacgaatatatatttatataaaaaaatat YCSPPEX attttttaaatattttattttttttgtttgacactgtttattattgtaaatataataatattatatatttgtttattcaaaataaa EL HBcAg aaatttttatgtttcttttattatatatatgattaaaagaaattgaaatatatttttaactattgaaaaatattattgtgtaatt gattcttcaccgtactcacacatatatatatatatatatatatatatagagagagagagagaatacctaaaaatataa acgcaataaaatacgagttctatattgaaacggcagaaatgcttttatttttcttttattttttttcttatatatttttt taaatatatatatatatatatatatatatacatatatatatatgagtctttaccattattccctatatatcatatgttat atatgatcaagtaagaaaagtacaaaaaaagtaaaaaataatataatataaatattataaaataaaaagaaataat aattgttcgatgataataattttaattagtttatggattattatacaacctttatataagtacccttataaaaatattcttgt tcatatcatatatatatatatatatattttacacatatttattttttacacatattatttttgtacacatatatatttt gtgaaagaggcgcgCTATATGTTTGAGCTTATTTCAATTGTTGTGTTTTAT TTTAATTATTTTTAAGTATACATTATAAGTAGGTGTAATATTAACATAAGTACTTGCTTTTTCCTTTTAATATTATTTTATAAAGCTTTTATAACTTTTTAAAGCACGTGATAAAGTAATTGTATGTAAATTAAAATTTTCTTAATTTTTTTTTTTTTTTTTTTGGGGGGGGAGGGGTAAAGGGGG GCTTAATTATAAAACAGAAATTATTCTTATCTTACATGCACATATACGATTTGGATATATATTTTTTTTTTTGTAACTTTCTATCATACTTGTC ATAAATTCTGAATTATCAAATAACTCAAATATATTTCATAATATCAGCTATGGAAGTTCGTCAAACACAAGGGTTCTAAATGAATTAAATTATCACTTGGAGAAAATGATGATCCGCGGccgcggatggatatagatccatataaag aatttggagcaagtgtagaatattaagttttttaccaagtgatttttttccaagtataagagatttatagatacagca agtgcattatatagagaagcattagaaagtccagaacatgtagtccacatcatacagcattaagacaagcaatagataaaaataagacaatatatggttcatataagtgttaacatttgg aagagaaacagtattagaatattagtaagttttggagtatggataagaacaccaccagcatatagaccaccaaaaccaagtccaagaagaagaagaagtcaaagtccaagaagaagaagaagtcaaagtagagaaagtcaatgtT A ACT C GAGcccgggCCTGC AGGttggggaaaagtaaaatcgcgataaaaatcaaaaattaaa aattaaaaattaaaatcaatcatttaaagaaaactactatagtgtgtttattatattttttttttttttttacaaaatagg tgtcacattattctaaaaggattaaacatatatatatatatatatatatatatatatatacatatacatatacacatcc ccatacacatgtcctttaaatctttcaaaataaaatgtgtgtctacctttttttcaatgtatcatttttagtttatact atataataatttttttcttttaatatcaaaaatggcttcagtgaatatatacagactcaaagaacatatatacaata taataatcattataaaaagaaaaaaaaaaaaaaaaaaaaaaaaaaaaaagtcttgaaaagacatatatattgca atagtttaaaataaataaaaaaaaagactcatattattaagtatattcatgtaaatataaaatatatttttttttattttat tcttatatgttttttttttttttttgacacacataataaaatacaaaaaatatatataatagaatttttttttttttttttt tgcattctccaaaaaaaaaatatatatatatataatatatatatatatatattatcatcatgtagtcgtttaaatattt cccttgaagggtagaacaaacaaatgtactatgtccgttcttttaatattcattgataacaatatattataaaaaa aaaagagcaatttaaaaaataaatatatggataaatatataaataatttaaattttaaaataactataaaatggttttttt tttttttttttatatatatataataaatatttccgtacttaataaaatcacatatatattPfSPZ- CACTTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGAT LARC2 (P. AACAATTTCACACAGGAAACAGCTATGACCATGATTACGCCAAGC Falciparum TTATACTAGTTTTTATTTTTTATATGAACCATTTAAGACATATATAT LINUP ATATATATATATATATATATATGTACATATTATACACATATCTAAA KO)- TGTTTATGAATATACGTCAAATTTTTCTGTTACATAAAAAAATCCG VECTOR CATTAATATAATATTAATATAATATATAAATCTATACATATATTTA TTTTTATTATAGTATTATAATTTTAATTTTTTTTTTTACATGTAGATT GTTACGAATATATATTTATATAAAAAAATATATTTTTTAAATATTTT ATTTTTTTTGTTTGACACTGTTTATTATTGTAAATATAATTAATATT TATATATTTGTTTATTCAAAATAAAAAATTTTTATGTTTCTTTTATT ATATATATGATTAAAAGAAATTGAAATATATTTTTAACTATTGAAA AATATTATTGTGTAATTGATTCTTCACCGTACTCACACATATATATA TATATATATATATATATAGAGAGAGAGAGAGAATACCTAAAAATA TAAACGCAATAAAATACGAGTTCTATATTTGAAACGGCAGAAATGCTTTTTATTTTTCTTTTTATTTTTTTTTTCTTTTATATATTTTTTTAAATATATATATATATATATATATATATACATATATATATATGAGTCTTT ACCATTTTATTCCCTATTATTATTCATATGTTTATATATGATCAAGT AAGAAAAGTACAAAAAAAGTAAAAAATAATATAATATAAATATTA TAAAATAAAAAGAAATAATAATTGTTCGATGATAATAATTTTAATT AGTTTATGGATTATTATACAACCTTTATATAAGTACCCTTATAAAAATATTCTTGTTCATATCATTATATATATATATATATATTTTACACATGAAAAGACATATATATTGCAATAGTTTAAAATAAATAAAAAAAAA GACTCATATTATTAAGTATATTCATGTTAAATATAAAATATATTTTT ATAATAAAATACAAAAAATATATTATAATAGAATTTTTTTTTTTTTTACTTTCTGATTTCCTTTTGTCAATTACAATATTGTATACGATATAGT GCAACTATAAAATTATTTTGTTTCTCCTCTTTCTTATTCTGATTTTTTCATTTAACTATATACATATATATATATATATATATATATATATATATCTGTCGAGGGCACCAGTAACAATTCTGACCTCTGGGAAGGCGGCACATACCTTTCAGATATATCCTCTGGTAATTTTTCGTAGAATAACTTT GGTAAAGCAGTCTCCTCGTCCCTTTGAATTAAAATTTTACCGATAC GCACAGACCTACAACAGTCTCTTAATCCTTGCTCCATCGATTCACCCAGGTAGATGGTTCAAACCTTCTTCAACCAACAATCTGATGATTCTGAGACAGCGTCGTATACAAAGTGGTATCTTTGTACACTTTGCCCTC GCGGATCCCTTTTGAAATCTCATGTTGTGACCACGACCGAGAACAC CTCTTTGTAACCTAAGGCCGCCTCCTCATAGGCAATGTCCATACCC GAATATATTTTTTTTAATTAATATAAGAAATTTTTATTGAAATGTAAGAAATATATGTATAATATTAAAAAAAAAATTTATTATTAATTTTTTAAAAAAAAAAAAAAAAAAAAAATTCACCTATATATTATATATATT CTTCGGTCATATACTAGAAGGTACCTTTTAACGGTTCACCCCTCTT GGTAAATGAATAATATTTATATATTAATATTTTATATATGCCCAAA GCTTCAAAAAAAACTTATAATCTTTATATAAATATAAAAAAAATG CTTAAATATATATTATTATAATTCTTAAAATTATATTGAAATAAAAGAATCGACCTTTAAAGGGTAGAATTAATTTAGTTCTCAGCAGAGAGATGATGCCTTAAAACTTACTGAACAACCAGAATTAGCAAATAAA GTAGACATGGTCTGGATAGTTGGTGGCAGTTCTGTTTATAAGGAAG CCATGAATCACCCAGGCCATCTTAAACTATTTGTGACAAGGATCAT GCAAGACTTTGAAAGTGACACGTTTTTTCCAGAAATTGATTTGGAGATGTCGAGGAGAATCCTGGCCCAGAATCGATGGACTATAAGGACCGAGTCCCAGCAGCCGACAAGAAGTACAGCATCGGCCTGGACATCG GCACCAACTCTGTGGGCTGGGCCGTGATCACCGACGAGTACAAGGGCATCAAGAAGAACCTGATCGGAGCCCTGCTGTTCGACAGCGGCGGAAGAGTCCTTCCTGGTGGAAGAGGATAAGAAGCACGAGCGGCAC CCCATCTTCGGCAACATCGTGGACGAGGTGGCCTACCACGAGAAGGACAAGGCCGACCTGCGGCTGATCTATCTGGCCCTGGCCCACATGGACAACAGCGACGTGGACAAGCTGTTCATCCAGCTGGTGCAGACCGACGCCAAGGCCATCCTGTCTGCCAGACTGAGCAAGAGCAGACGG CTGGAAAATCTGATCGCCCAGCTGCCCGGCGAGAAGAAGAATGGCCTGTTCGGAAACCTGATTGCCCTGAGCCTGGGCCTGACCCCCAACTGCAAGGACACCTACGACGACGACCTGGACAACCTGCTGGCCCAGA CGACGCCATCCTGCTGAGCGACATCCTGAGAGTGAACACCGAGAT CACCAAGGCCCCCCTGAGCGCCTCTATGATCAAGAGATACGACGA GCACCACCAGGACCTGACCCTGCTGAAAGCTCTCGTGCGGCAGCA GCTGCCTGAGAAGTACAAAGAGATTTTCTTCGACCAGAGCAAGAA CGGCTACGCCGGCTACATTGACGGCGGAGCCAGCCAGGAAGAGTT CTACAAGTTCATCAAGCCCATCCTGGAAAAGATGGACGGCACCGA GGAACTGCTCGTGAAGCTGAACAGAGAGGACCTGCTGCGGAAGCA GCGGACCTTCGACAACGGCAGCATCCCCCACCAGATCCACCTGGG CTGAAGGACAACCGGGAAAAGATCGAGAAGATCCTGACCTTCCGC GCCTGGATGACCAGAAAGAGCGAGGAAACCATCACCCCCTGGAAC GAGCGGATGACCAACTTCGATAAGAACCTGCCCAACGAGAAGGTG CTGCCCAAGCACAGCCTGCTGTACGAGTACTTCACCGTGTATAACG GCCTTCCTGAGCGGCGAGCAGAAAAAGGCCATCGTGGACCTGCTG GAAAATTATCAAGGACAAGGACTTCCTGGACAATGAGGAAAACGA GGACATTCTGGAAGATATCGTGCTGACCCTGACACTGTTTGAGGAC GACGACAAAGTGATGAAGCAGCTGAAGCGGCGGAGATACACCGG CTGGGGCAGGCTGAGCCGGAAGCTGATCAACGGCATCCGGGACAA GCAGTCCGGCAAGACAATCCTGGATTTCCTGAAGTCCGACGGCTTC GCCAACAGAAACTTCATGCAGCTGATCCACGACGACAGCCTGACCGCCAGAGAGAACCAGACCACCCAGAAGGGACAGAAGAACAGCCG CGAGAGAATGAAGCGGATCGAAGAGGGCATCAAAGAGCTGGGCA GCCAGATCCTGAAAGAACACCCCGTGGAAAACACCCAGCTGCAGACAACAAGGTGCTGACCAGAAGCGACAAGAACCGGGGCAAGAGCG GGCGGCAGCTGCTGAACGCCAAGCTGATTACCCAGAGAAAGTTCGCAAAGCACGTGGCACAGATCCTGGACTCCCGGATGAACACTAAGTCAAAGTGCGCGAGATCAACAACTACCACCACGCCCACGACGCCTA CCTGAACGCCGTCGTGGGAACCGCCCTGATCAAAAAGTACCCTAA GCTGGAAAGCGAGTTCGTGTACGGCGACTACAAGGTGTACGACGT GCGGAAGATGATCGCCAAGAGCGAGCAGGAAATCGGCAAGGCTA CCGCCAAGTACTTCTTCTACAGCAACATCATGAACTTTTTCAAGAC CGAGATTACCCTGGCCAACGGCGAGATCCGGAAGCGGCCTCTGAT CGAGACAAACGGCGAAACCGGGGAGATCGTGTGGGATAAGGGCC GGGATTTTGCCACCGTGCGGAAAGTGCTGAGCATGCCCCAAGTGACCGTGGCCTATTCTGTGCTGGTGGTGGCCAAAGTGGAAAAGGGCACTGCCTAAGTACTCCCTGTTCGAGCTGGAAAACGGCCGGAAGAGA CTGCCCTCCAAATATGTGAACTTCCTGTACCTGGCCAGCCACTATGGAGAGCAGGCCGAGAATATCATCCACCTGTTTACCCTGACCAATCT GGGAGCCCCTGCCGCCTTCAAGTACTTTGACACCACCATCGACCGG CACCAGAGCATCACCGGCCTGTACGAGACACGGATCGACCTGTCT CAGCTGGGAGGCGACAAAAGGCCGGCGGCCACGAAAAAGGCCGG CCAGGCAAAAAAGAAAAAGTAAACTAATAAAAAAAAAAAACATT CATATATTTATATATATATATATATATATATATATATTCATGTGTAA GTTATATAAATAAACAAGCTATTTATGGATCACACAAATATAATTA GTAATTATTTATTTTTTTTTTCCTCATAAAATTATAATAAACTTTTGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCA CCGAGTCGGTGCTTTTTTTGGACGTCAGGTGGCACTTTTCGGGGAAGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAA CCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGCACCACGATGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGC GTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGC CCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTAT GGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCA GTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACT CAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGG GGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCG AACTGAGATACCTACAGCGTGAGCATTGAGAAAGCGCCACGCTTC CCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTC GGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTG GTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTC GATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACG CCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACG ACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCC AATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGC AGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGC AACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCCCAGGCTT LApcl3- CACTTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGAT EFlalnterg AACAATTTCACACAGGAAACAGCTATGACCATGATTACGCCAAGC enic_yfcu_ ETATACTAGTATAGCTAGCTATGTCGACTATCCGCGGCCGCAATAT Mei2HA TAGATGCATCTTGTGCTTACATATAACATTTGCATGTGTGGAAATA PLASMID TTCTCTTTCCATAAAAAGATACAACTTCTCTTGTTTTTGTTTTATTT SEQUENC ATTTTGATTATTGAGAAATAATATAAGAATATTTAACCAATGAATT E AAATTAAAAATGGTATTCTTTTTAAAAATGAAAAAAAAAAAAAAA AAACGTCAATGTAAGAACAAAAAAGTGACAAATTCATTTGGTATT LTTTATATAATATATTTTTTTTAATTTATTTATTTATTCCTTTATATA EATATATATATATATATATATATATATACATACATACATATATATA EAATTTATATATAATTTATATATATTATATTAAATGATATGTTTATA AGAAAAAATAAAATTATGTGAATATTTTAATTGTTATAATTGATTA LAAATTATTTGTGTTTTTATTTTGTATAATTGTATGCTCCAACATTT AATGTGTATGCTAAATATCGATGGAATATTATTAAATATATAATTC ETAAAAATTGAGATTTTACATATTTTTTTTTATTCCCGTTTTTAAGC EGTTTCATAAAAAAAAAAAAAATTCATAAAGATATGAAATCCATA TTTTTTTAAATATTTGGAAAAAGGAATATAACATGTAATTATATAA EATATATATATATATATATATATATATAATTTGGACTAATCATATTG ATATAAAAAAATATTTTTATAATAAAAAATAATAAATAAATAAAT AATAGAGGAGAGACATAACCAAAAAGTGTTGTAAATAATACATTA ATATATATATATATATAGATATATATATATTTTTTTTTTTTTTTTTTT CTCCTTTTATCAAAATGCATAAGAGGCGCGCCCTTAAGGCCTGCAG GTTAAATTCAAAGATGTATATAGACACAAAAAAAAAAAAAAAATA ATAAAAAAATAAAAATATATATATATATTTATATATTTATGTATCA ETGTTTAAAAAAATTAACAAATTAACCTTCTTATGTTTTTACAATTT ACATACTGATTATATATATATACGTGTATAATTTACTATTCATCTTA ECTTATATATTATCTTCTTTTATTTATTTATTTATTTATTTGTTTTTTTrTTTTTTTCTTTTTCTTTTTCTTTTTCTTTTTCCGCATTAAATATAATGCTTATAAAAACATATTTCGAAAAATATACATATATATATATATATAGCAACTATAAAATTATTTTGTTTCTCCTCTTTCTTATTCTGATTTTTTATAATCTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTATATATA CATTTAACTATATACATATATATATATATATATATATATATATATATCTGTCGAGGGCACCAGTAACAATTCTGACCTCTGGGAAGGCGGCACATACCTTTCAGATATATCCTCTGGTAATTTTTCGTAGAATAACTTT GGTAAAGCAGTCTCCTCGTCCCTTTGAATTAAAATTTTACCGATAC GCACAGACCTACAACAGTCTCTTAATCCTTGCTCCATCGATTCACC CAGGTAGATGGTTCAAACCTTCTTCAACCAACAATCTGATGATTCTGAGACAGCGTCGTATACAAAGTGGTATCTTTGTACACTTTGCCCTC GCGGATCCCTTTTGAAATCTCATGTTGTGACCACGACCGAGAACAC CTCTTTGTAACCTAAGGCCGCCTCCTCATAGGCAATGTCCATACCC GAATATATTTTTTTTAATTAATATAAGAAATTTTTATTGAAATGTAAAACATATATATATATATATATATATATATATATATATATTATATATT GAAATATATGTATAATATTAAAAAAAAAATTTATTATTAATTTTTTCTTCGGTCATATACTAGAAGGTACCTTTTAACGGTTCACCCCTCTTGGTAAATGAATAATATTTATATATTAATATTTTATATATGCCCAAAGCTTCAAAAAAAACTTATAATCTTTATATAAATATAAAAAAAATGCTTAAATATATATTATTATAATTCTTAAAATTATATTGAAATAAAAAGAACGGGGACCTGCCCTGGCCACCGCTCAGGAACGAATTTAGATGAATCGACCTTTAAAGGGTAGAATTAATTTAGTTCTCAGCAGAGAGATGATGCCTTAAAACTTACTGAACAACCAGAATTAGCAAATAAAGTAGACATGGTCTGGATAGTTGGTGGCAGTTCTGTTTATAAGGAAGCCATGAATCACCCAGGCCATCTTAAACTATTTGTGACAAGGATCAT GCAAGACTTTGAAAGTGACACGTTTTTTCCAGAAATTGATTTGGAGGAGTCCCAGCAGCCGACAAGAAGTACAGCATCGGCCTGGACATCG GCACCAACTCTGTGGGCTGGGCCGTGATCACCGACGAGTACAAGGGCATCAAGAAGAACCTGATCGGAGCCCTGCTGTTCGACAGCGGCGAGCAACGAGATGGCCAAGGTGGACGACAGCTTCTTCCACAGACTG GAAGAGTCCTTCCTGGTGGAAGAGGATAAGAAGCACGAGCGGCAC CCCATCTTCGGCAACATCGTGGACGAGGTGGCCTACCACGAGAAGGACAAGGCCGACCTGCGGCTGATCTATCTGGCCCTGGCCCACATGGACAACAGCGACGTGGACAAGCTGTTCATCCAGCTGGTGCAGACCGACGCCAAGGCCATCCTGTCTGCCAGACTGAGCAAGAGCAGACGG CTGGAAAATCTGATCGCCCAGCTGCCCGGCGAGAAGAAGAATGGC CTGTTCGGAAACCTGATTGCCCTGAGCCTGGGCCTGACCCCCAACTGCAAGGACACCTACGACGACGACCTGGACAACCTGCTGGCCCAGACGACGCCATCCTGCTGAGCGACATCCTGAGAGTGAACACCGAGAT CACCAAGGCCCCCCTGAGCGCCTCTATGATCAAGAGATACGACGA GCACCACCAGGACCTGACCCTGCTGAAAGCTCTCGTGCGGCAGCA GCTGCCTGAGAAGTACAAAGAGATTTTCTTCGACCAGAGCAAGAA CGGCTACGCCGGCTACATTGACGGCGGAGCCAGCCAGGAAGAGTTCTACAAGTTCATCAAGCCCATCCTGGAAAAGATGGACGGCACCGAGGAACTGCTCGTGAAGCTGAACAGAGAGGACCTGCTGCGGAAGCA GCGGACCTTCGACAACGGCAGCATCCCCCACCAGATCCACCTGGG CTGAAGGACAACCGGGAAAAGATCGAGAAGATCCTGACCTTCCGC GCCTGGATGACCAGAAAGAGCGAGGAAACCATCACCCCCTGGAAC GAGCGGATGACCAACTTCGATAAGAACCTGCCCAACGAGAAGGTG CTGCCCAAGCACAGCCTGCTGTACGAGTACTTCACCGTGTATAACG GCCTTCCTGAGCGGCGAGCAGAAAAAGGCCATCGTGGACCTGCTGGAAAATTATCAAGGACAAGGACTTCCTGGACAATGAGGAAAACGA GGACATTCTGGAAGATATCGTGCTGACCCTGACACTGTTTGAGGAC GACGACAAAGTGATGAAGCAGCTGAAGCGGCGGAGATACACCGG CTGGGGCAGGCTGAGCCGGAAGCTGATCAACGGCATCCGGGACAA GCAGTCCGGCAAGACAATCCTGGATTTCCTGAAGTCCGACGGCTTC GCCAACAGAAACTTCATGCAGCTGATCCACGACGACAGCCTGACCGCCAGAGAGAACCAGACCACCCAGAAGGGACAGAAGAACAGCCG CGAGAGAATGAAGCGGATCGAAGAGGGCATCAAAGAGCTGGGCA GCCAGATCCTGAAAGAACACCCCGTGGAAAACACCCAGCTGCAGACAACAAGGTGCTGACCAGAAGCGACAAGAACCGGGGCAAGAGCGGGCGGCAGCTGCTGAACGCCAAGCTGATTACCCAGAGAAAGTTCGCAAAGCACGTGGCACAGATCCTGGACTCCCGGATGAACACTAAGTCAAAGTGCGCGAGATCAACAACTACCACCACGCCCACGACGCCTA CCTGAACGCCGTCGTGGGAACCGCCCTGATCAAAAAGTACCCTAA GCTGGAAAGCGAGTTCGTGTACGGCGACTACAAGGTGTACGACGT GCGGAAGATGATCGCCAAGAGCGAGCAGGAAATCGGCAAGGCTA CCGCCAAGTACTTCTTCTACAGCAACATCATGAACTTTTTCAAGAC CGAGATTACCCTGGCCAACGGCGAGATCCGGAAGCGGCCTCTGAT CGAGACAAACGGCGAAACCGGGGAGATCGTGTGGGATAAGGGCC GGGATTTTGCCACCGTGCGGAAAGTGCTGAGCATGCCCCAAGTGAAGTCTATCCTGCCCAAGAGGAACAGCGATAAGCTGATCGCCAGAACCGTGGCCTATTCTGTGCTGGTGGTGGCCAAAGTGGAAAAGGGCACTGCCTAAGTACTCCCTGTTCGAGCTGGAAAACGGCCGGAAGAGACTGCCCTCCAAATATGTGAACTTCCTGTACCTGGCCAGCCACTATGGAGAGCAGGCCGAGAATATCATCCACCTGTTTACCCTGACCAATCT GGGAGCCCCTGCCGCCTTCAAGTACTTTGACACCACCATCGACCGGCACCAGAGCATCACCGGCCTGTACGAGACACGGATCGACCTGTCT CAGCTGGGAGGCGACAAAAGGCCGGCGGCCACGAAAAAGGCCGG CCAGGCAAAAAAGAAAAAGTAAACTAATAAAAAAAAAAAACATTCATATATTTATATATATATATATATATATATATATATTCATGTGTAA GTTATATAAATAAACAAGCTATTTATGGATCACACAAATATAATTA GTAATTATTTATTTTTTTTTTCCTCATAAAATTATAATAAACTTTTGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCA CCGAGTCGGTGCTTTTTTTGGACGTCAGGTGGCACTTTTCGGGGAAGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAA CCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGCACCACGATGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGAC TGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCC CTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGC GTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGC CCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTAT GGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGAT TAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAG ATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGA TCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCG TTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTT GAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAA ACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCA ACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAA ATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAA CTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCA GTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACT CAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGG GGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCG AACTGAGATACCTACAGCGTGAGCATTGAGAAAGCGCCACGCTTC CCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTC GGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTG GTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTC GATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACG CCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTT GCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGrATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACG ACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCC AATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGC AGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGC AACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCCCAGGCTT TAPySPZLAR CCGTCGTTTTACAACGTCGTGACTGGGAAAACCCTGGCGTTACCCA C2 (P. ACTTAATCGCCTTGCAGCACATCCCCCTTTCGCCAGCTGGCGTAATyoelii mei- AGCGAAGAGGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGC2 / LINUP CTGAATGGCGAATGGCGCCACTATCCAACTACACTCATAATGCTTA KO)CCAAAAAAAATATATATAAATATATGTAATATATAATATATATATA GTATGTATGTATATATGTATGTATGTATGTATAGTTTATATTTATTT GGCAAGTTTTTCTGTAAAAATGTTTAATTAAAAAAAAAAATATATAGAAAAAGTGGCACCGAGTCGGTGCTTTTTTTGGACGTCAGGTGGC ATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACAT CAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGC GGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGA GAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTAC GGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCAT GAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGG GTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGG CAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCA CTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCT CTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGC CTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAA GCGGAAGAGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGG CCGATTCATTAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAA GCGGGCAGTGAGCGCAACGCAATTAATGTGAGTTAGCTCACTCATGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCTATGCTCATAAATATATTTATTTCATATAATTGCACATATTTAAATTATTAGTTATATTTTTCTTAAACAGAGAAAGTATACATATATATATGTTTGCCCAAAGAACTAATTAAAACGCATGCTTATTATTGTCATCGTAAAA CATTTAAATAGTATATTATGTAAAGAGAATATTTTAAAAAAAAAA GTATTTTCAGAATTTTCCCTATTTTCTGCATTTTCTGCATTCTCTGCA CCACGTTTGATCCAAATCAATGCTATTATATATTTTTATTACTTCCC GTTTACATATCTCAATTAGTCAATTTTTTTTAAGTAGTTGGCTAGCTCATTTACTTTTCTCTAATTTGTGAACCCTATGCCTATACATGCATGC GATAAACCTTTTCTTTAAAATGGCTTGTGCCACTAAAACTTATATTT AGGGGCTAAAAAAACATTTATATAAAATTCAAATAAAAATATCAT GATATATATTAAAATTGTCTTGCAAAAAAAAAAAAAAAAAAAAAACCACTGAAATACCATTAAATTTTCAAAAAAATACTATGCATATAATGTTATACATATAAACATAAAACGCCATGTAAATCAAAAAATATATCGGCAAGAACGGGGACCTGCCCTGGCCACCGCTCAGGAACGAATT GAGAAGAATCGACCTTTAAAGGGTAGAATTAATTTAGTTCTCAGC AGAGAACTCAAGGAACCTCCACAAGGAGCTCATTTTCTTTCCAGAGGATCATGCAAGACTTTGAAAGTGACACGTTTTTTCCAGAAATTGA GATGTCCAGGAGGAGAAAGGCATTAAGTACAAATTTGAAGTATAT GAGAAGAATGATGGTTCGGGAGAGGGCAGAGGATCCCTGCTAACA GACGATGACGATAAGATGGCCCCAAAGAAGAAGCGGAAGGTCGG GGACATCGGCACCAACTCTGTGGGCTGGGCCGTGATCACCGACGA GTACAAGGTGCCCAGCAAGAAATTCAAGGTGCTGGGCAACACCGA CCGGCACAGCATCAAGAAGAACCTGATCGGAGCCCTGCTGTTCGA CAGCGGCGAAACAGCCGAGGCCACCCGGCTGAAGAGAACCGCCA GAAGAAGATACACCAGACGGAAGAACCGGATCTGCTATCTGCAAGCCCACATGATCAAGTTCCGGGGCCACTTCCTGATCGAGGGCGACCT GAACCCCGACAACAGCGACGTGGACAAGCTGTTCATCCAGCTGGT GCAGACCTACAACCAGCTGTTCGAGGAAAACCCCATCAACGCCAG CGGCGTGGACGCCAAGGCCATCCTGTCTGCCAGACTGAGCAAGAG CAGACGGCTGGAAAATCTGATCGCCCAGCTGCCCGGCGAGAAGAA GAATGGCCTGTTCGGAAACCTGATTGCCCTGAGCCTGGGCCTGACC CCCAACTTCAAGAGCAACTTCGACCTGGCCGAGGATGCCAAACTG CAGCTGAGCAAGGACACCTACGACGACGACCTGGACAACCTGCTG GCCCAGATCGGCGACCAGTACGCCGACCTGTTTCTGGCCGCCAAGCGGCAGCAGCTGCCTGAGAAGTACAAAGAGATTTTCTTCGACCAGGAAGAGTTCTACAAGTTCATCAAGCCCATCCTGGAAAAGATGGAC GGCACCGAGGAACTGCTCGTGAAGCTGAACAGAGAGGACCTGCTG CGGAAGCAGCGGACCTTCGACAACGGCAGCATCCCCCACCAGATC CACCTGGGAGAGCTGCACGCCATTCTGCGGCGGCAGGAAGATTTTGCAGATTCGCCTGGATGACCAGAAAGAGCGAGGAAACCATCACCC CCTGGAACTTCGAGGAAGTGGTGGACAAGGGCGCTTCCGCCCAGA GCTTCATCGAGCGGATGACCAACTTCGATAAGAACCTGCCCAACGGAAAGCCCGCCTTCCTGAGCGGCGAGCAGAAAAAGGCCATCGTGGCGATCTGCTGAAAATTATCAAGGACAAGGACTTCCTGGACAATGA GGAAAACGAGGACATTCTGGAAGATATCGTGCTGACCCTGACACTGTTTGAGGACAGAGAGATGATCGAGGAACGGCTGAAAACCTATGCCCACCTGTTCGACGACAAAGTGATGAAGCAGCTGAAGCGGCGGAGCCGGGACAAGCAGTCCGGCAAGACAATCCTGGATTTCCTGAAGTC CGACGGCTTCGCCAACAGAAACTTCATGCAGCTGATCCACGACGA CAGCCTGACCTTTAAAGAGGACATCCAGAAAGCCCAGGTGTCCGG CCAGGGCGATAGCCTGCACGAGCACATTGCCAATCTGGCCGGCAG CCCCGCCATTAAGAAGGGCATCCTGCAGACAGTGAAGGTGGTGGA CGAGCTCGTGAAAGTGATGGGCCGGCACAAGCCCGAGAACATCGT GATCGAAATGGCCAGAGAGAACCAGACCACCCAGAAGGGACAGAGAGCTGGGCAGCCAGATCCTGAAAGAACACCCCGTGGAAAACACC CAGCTGCAGAACGAGAAGCTGTACCTGTACTACCTGCAGAATGGG CGGGATATGTACGTGGACCAGGAACTGGACATCAACCGGCTGTCCACTCCATCGACAACAAGGTGCTGACCAGAAGCGACAAGAACCGGG GC AAGAGC GAC AAC GTGCCCTCC GAAGAGGTCGTGAAGAAGATGAGAAAGTTCGACAATCTGACCAAGGCCGAGAGAGGCGGCCTGAGCGGGCAGATCACAAAGCACGTGGCACAGATCCTGGACTCCCGGATGACCAGTTTTACAAAGTGCGCGAGATCAACAACTACCACCACGCCCA CGACGCCTACCTGAACGCCGTCGTGGGAACCGCCCTGATCAAAAA GTACCCTAAGCTGGAAAGCGAGTTCGTGTACGGCGACTACAAGGT GTACGACGTGCGGAAGATGATCGCCAAGAGCGAGCAGGAAATCG GCAAGGCTACCGCCAAGTACTTCTTCTACAGCAACATCATGAACTTGCCTCTGATCGAGACAAACGGCGAAACCGGGGAGATCGTGTGGGACCAAGTGAATATCGTGAAAAAGACCGAGGTGCAGACAGGCGGCTT CAGCAAAGAGTCTATCCTGCCCAAGAGGAACAGCGATAAGCTGAT CGCCAGAAAGAAGGACTGGGACCCTAAGAAGTACGGCGGCTTCGAAAGGGCAAGTCCAAGAAACTGAAGAGTGTGAAAGAGCTGCTGGG GATCACCATCATGGAAAGAAGCAGCTTCGAGAAGAATCCCATCGA CTTTCTGGAAGCCAAGGGCTACAAAGAAGTGAAAAAGGACCTGAT CATCAAGCTGCCTAAGTACTCCCTGTTCGAGCTGGAAAACGGCCG GAAGAGAATGCTGGCCTCTGCCGGCGAACTGCAGAAGGGAAACGA ACTGGCCCTGCCCTCCAAATATGTGAACTTCCTGTACCTGGCCAGC CACTATGAGAAGCTGAAGGGCTCCCCCGAGGATAATGAGCAGAAA CAGCTGTTTGTGGAACAGCACAAGCACTACCTGGACGAGATCATC GAGCAGATCAGCGAGTTCTCCAAGAGAGTGATCCTGGCCGACGCT AATCTGGACAAAGTGCTGTCCGCCTACAACAAGCACCGGGATAAG CCCATCAGAGAGCAGGCCGAGAATATCATCCACCTGTTTACCCTGA CCAATCTGGGAGCCCCTGCCGCCTTCAAGTACTTTGACACCACCAT CGACCGGAAGAGGTACACCAGCACCAAAGAGGTGCTGGACGCCAC CCTGATCCACCAGAGCATCACCGGCCTGTACGAGACACGGATCGA CCTGTCTCAGCTGGGAGGCGACAAAAGGCCGGCGGCCACGAAAAA GGCCGGCCAGGCAAAAAAGAAAAAGTAAGAATTACCGGCCGGTG ATCCCGTTTTTCTTACTTATATATTTATACCAATTGATTGTATTTAT AACTGTAAAAATGTGTATGTTGTGTGCATATTTTTTTTTGTGCATGC ACATGCATGTAAATAGCTAAAATTATGAACATTTTATTTTTTGTTC AGAAAAAAAAAACTTTACACACATAAAATGGCTAGTATGAATAGC CATATTTTATATAAATTAAATCCTATGAATTTATGACCATATTAAA AATTTAGATATTTATGGAACATAATATGTTTGAAACAATAAGACAA AATTATTATTATTATTATTATTTTTACTGTTATAATTATGTTGTCTCT TCAATGATTCATAAATAGTTGGACTTGATTTTTAAAATGTTTATAA EATGATTAGCATAGTTAAATAAAAAAAGTTGAAAAATTAAAAAAA AACATATAAACACAAATGATGTTTTTTCCTTCAATTTCGGP230p Ebo GAAGATTTTATCATTATTCGAGTCAGAGTACATCAAACTTATAAAA la Virus AAATATTAGGAATAAGTAGTGATAAAAATGATAAACCATATTTTG VP35 AAGAAATTATTAGTGAAGAAAAAGATATATATGAATTTGAAGGAT ATTTAGAAAAAGTAATTGGAATCAAATTAGAAGGATATGAATTAG ATCCACCAAATTGTTTTAAAAGTGTATATGAAGATGATAAAAGAA TATATTTAGAAGTAGAATATCATTATTCAAAATGTATTAACTTAGA CAGAAAAAATTATAAATTAAGATTTTATTTTTTATCACAATATTTTGCTGATTATGAATTAAAGTTTTCATGTAATATAATTAATATAAAAACTCAAAAAAAAAAAACTGTTACATTTGGTGATGGAATGGCAACAT CTGATCACATTTTAAAAATAGTTGATGATGATAGTAAAACGATTAAAAAATAATGAAAAAACATAAATTATATGGATATATACATATATAAGAAAATCAGTTATTTAAATTTTAACTGAAGAAATTAAATAAGTATACATTTTAGTTGTAGCGTCACTTTTATTAGTTGATTCTCTACTTCCAG GATATGGACAAAATAAAAGTGTCCAAGCCCAAAGAAACTTAAACGGTCAATTGAATTCTCTAGCACTCGAAGCTTATTGTCTTCAATGTAA GGGCCATACTGCGGCCACGACTCAAAACGACAGAATGCCAGGCCC CCTGTAAGCGACATCTTCTGTGATATTGAGAACAATCCAGGATTAT GCTACGCATCCCAAATGCAACAAACGAAGCCAAACCCGAAGACGC GCAACAGTCAAACCCAAACGGACCCAATTTGCAATCATAGTTTTGCTCATTGAACAGGGTTTGTGCTGAGATGGTTGCAAAATATGATCTT CTGGTGATGACAACCGGTCGGGCAACAGCAACCGCTGCGGCAACT GAGGCTTATTGGGCCGAACATGGTCAACCACCACCTGGACCATCA CTTTATGAAGAAAGTGCGATTCGGGGTAAGATTGAATCTAGAGAT GAGACCGTCCCTCAAAGTGTTAGGGAGGCATTCAACAATCTAAAC CGGCAAAGGATTTGAGAAACATTATGTATGATCACTTGCCTGGTTTGCCTGGCTGAAGGAGACTCTCCTCAATGTGCCCTAATTCAAATTACGTCCAGTCCCACCATCGCCCAAGATTGATCGAGGTTGGGTATGTGTACTGCTGAACTATAGGGTACGTTACATTAATGATACACTTGTGAGTCAAAAGTGATGAAGATTAAGAAAAACATTATGAGGGTAATTCAGAACATTATAACAAAATCACATTTTAACCATTATTTTGCTTTATGTGTA TAAAAAACTTTTACAATATATTTTTTTATTTTGAACGTATATAAAA ATATTTCCTTTAATTTGTAGTATGCAAATAATTTATAAAAATATATT GTGTCTAAAAGAAATGAAAACGGATTCTAGCATATAAACAAATCT GGCTTCGAAAAGAAATAGAAGAAGAAAAAAAATTATATATAATAT ATTTGATAATTATATTTGTGAGAGTGTGTATTATTTTTGTTCGAAAATTTTTTTTATGTATTTTTGAGAGTAATTTTTAGTGTTTGTTTTTTTTGAATAATTTGTACGTTTTTTAGGATAGAGAGTGTTATACTTCATAAT GTATTATTTGAACGATTAAAAAATATTTTTTGTAACAGTGAAATAA AAATATGAATGAAAACAGCCGTATATGCAAACCTGGACTGATTAT AGAAGAGCAATTATTTTATTACATGTTCGAACAGATTTAAAAGATA GACCAGTTTCATTTTGTGATTTTAGAAAAGGTGAATTATATAATTA TTTAAATGCTTATAGTGAAGGAGATCTATGTATAATAATTTCCAAA TCAAATACAAGTTTTGGTTTTAGATGCCCCGAAAATACAAAAAAA ATGCCAGAAAAATGTTTTACTCAAGTATATGAAAAAGGCTATCTAC ATGATTCCTATAAAATTAATACTAAAAATATTATTAACTATTCATT TGAAAATCCAGAATATGCATTAGCTGGTTTTAATTATACATTAACA AAATCATATCAATTTGAATGTCATTGTGTAGATAAAGAAACAGAA CAAATTGTAAAAACTGTTTTAGTAAAATATGTAAATGAAGATGAA ATATATGATTATAATGATTTACCATTGGTTAATCATAAATCTATTGT TGCACATCCAAATAAAACACATCTATGTGACTTTATGACATCTGAT AATATGTTATCACCTAAAAAAGAAGATTCAGTAAATTATGTTTGTA ATGTATTTCCAAAACCATTAGAATATGTAGCATTACATTGTCCAAC CAATATAGTAGATGTAGAAAATGAAGATGATATTTCAAGACTATC AAAGGAAATGCATAAAGAAGAAATAAAAGCGGAATTAAGAATCA AACTTCP230p SA GAAGATTTTATCATTATTCGAGTCAGAGTACATCAAACTTATAAAA RS CoV-2 AAATATTAGGAATAAGTAGTGATAAAAATGATAAACCATATTTTG NSP13 / NS AAGAAATTATTAGTGAAGAAAAAGATATATATGAATTTGAAGGAT P15 ATTTAGAAAAAGTAATTGGAATCAAATTAGAAGGATATGAATTAG ATCCACCAAATTGTTTTAAAAGTGTATATGAAGATGATAAAAGAA TATATTTAGAAGTAGAATATCATTATTCAAAATGTATTAACTTAGA CAGAAAAAATTATAAATTAAGATTTTATTTTTTATCACAATATTTTGCTGATTATGAATTAAAGTTTTCATGTAATATAATTAATATAAAAA -Ill-CTCAAAAAAAAAAAACTGTTACATTTGGTGATGGAATGGCAACAT CTGATCACATTTTAAAAATAGTTGATGATGATAGTAAAACGATTAAAAAATAATGAAAAAACATAAATTATATGGATATATACATATATAAGAAAATCAGTTATTTAAATTTTAACTGAAGAAATTAAATAAGTATACATTTTAGTTGTAGCGTCACTTTTATTAGTTGATTCTCTACTTCCAG GATATGGACAAAATAAAAGTGTCCAAGCCCAAAGAAACTTAAACGGGCACGTCAACATCTTAAAGATGGCACTTGTGGCTTAGTAGAAGTT GAAAAAGGCGTTTTGCCTCAACTTGAACAGCCCTATGTGTTCATCA GCTGGTAGCAGAACTCGAAGGCATTCAGTACGGTCGTAGTGGTGA GACACTTGGTGTCCTTGTCCCTCATGTGGGCGAAATACCAGTGGCTGGAGGGGCATACACTCGCTATGTCGATAACAACTTCTGTGGCCCTG ATGGCTACCCTCTTGAGTGCATTAAAGACCTTCTAGCACGTGCTGGGTCCAAATTTTGTATTTCCCTTAAATTCCATAATCAAGACTATTCAA CCAAGGGTTGAAAAGAAAAAGCTTGATGGCTTTATGGGTAGAATT CGATCTGTCTATCCAGTTGCGTCACCAAATGAATGCAACCAAATGT GCCTTTCAACTCTCATGAAGTGTGATCATTGTGGTGAAACTTCATG GCAGACGGGCGATTTTGTTAAAGCCACTTGCGAATTTTGTGGCACT GAGAATTTGACTAAAGAAGGTGCCACTACTTGTGGTTACTTACCCC CTTGAAAACCATTCTTCGTAAGGGTGGTCGCACTATTGCCTTTGGA GGTTCCACGTGCTAGCGCTAACATAGGTTGTAACCATACAGGTGTT GTTGGAGAAGGTTCCGAAGGTCTTAATGACAACCTTCTTGAAATACGTGCTTTTGTGGAAACTGTGAAAGGTTTGGATTATAAAGCATTCAAGAGTCCTCTTTATGCATTTGCATCAGAGGCTGCTCGTGTTGTACGACTAACAATCTAGTTGTAATGGCCTACATTACAGGTGGTGTTGTTCA GTTGACTTCGCAGTGGCTAACTAACATCTTTGGCACTGTTTATGAAGGTGTAGAGTTTCTTAGAGACGGTTGGGAAATTGTTAAATTTATCT CAACCTGTGCTTGTGAAATTGTCGGTGGACAAATTGTCACCTGTGCGGGATTGTACAGAAAGTGTGTTAAATCCAGAGAAGAAACTGGCCTGAAACACTTCCCACAGAAGTGTTAACAGAGGAAGTTGTCTTGAAAGCTCGAAATCAAAGACACAGAAAAGTACTGTGCCCTTGCACCTAACAGAAGTAAATGAGTTCGCCTGTGTTGTGGCAGATGCTGTCATAAAGTGAGTTTAAATTGGCTTCACATATGTATTGTTCTTTCTACCCTCCA GATGAGGATGAAGAAGAAGGTGATTGTGAAGAAGAAGAGTTTGA GCCATCAACTCAATATGAGTATGGTACTGAAGATGATTACCAAGGGAAGAGCAAGAAGAAGATTGGTTAGATGATGATAGTCAACAAACT GTTGGTCAACAAGACGGCAGTGAGGACAATCAGACAACTACTATTCAAACAATTGTTGAGGTTCAACCTCAATTAGAGATGGAACTTACACCAGTTGTTCAGACTATTGAAGTGAATAGTTTTAGTGGTTATTTAAAGTGCTGACCCTATACATTCTTTAAGAGTTTGTGTAGATACTGTTCG CACAAATGTCTACTTAGCTGTCTTTGATAAAAATCTCTATGACAAA CTTGTTTCAAGCTTTTTGGAAATGAAGAGTGAAAAGCAAGTTGAAC CTGAAAGTAAACCTTCAGTTGAACAGAGAAAACAAGATGATAAGA AAATCAAAGCTTGTGTTGAAGAAGTTACAACAACTCTGGAAGAAA CTAAGTTCCTCACAGAAAACTTGTTACTTTATATTGACATTAATGG CAATCTTCATCCAGATTCTGCCACTCTTGTTAGTGACATTGACATC CATATATAGTGGGTGATGTTGTTCAAGAGGGTGTTTTAACTGCTGT GGTTATACCTACTAAAAAGGCTGGTGGCACTACTGAAATGCTAGC GAAAGCTTTGAGAAAAGTGCCAACAGACAATTATATAACCACTTA CCCGGGTCAGGGTTTAAATGGTTACACTGTAGAGGAGGCAAAGACCTGTCTGTGTGGAAACTAAAGCCATAGTTTCAACTATACAGCGTAAGCTATGTAACACATGGCTTAAATTTGGAAGAAGCTGCTCGGTATAT GAGATCTCTCAAAGTGCCAGCTACAGTTTCTGTTTCTTCACCTGAT GCTGTTACAGCGTATAATGGTTATCTTACTTCTTCTTCTAAAACACCGATTGGTCCTATTCTGGACAATCTACACAACTAGGTATAGAATTTCCTTCTTTCTTTGAGAGAAGTGAGGACTATTAAGGTGTTTACAACAG CATATGGACAACAGTTTGGTCCAACTTATTTGGATGGAGCTGATGT GTTTTACCTAATGATGACACTCTACGTGTTGAGGCTTTTGAGTACT GATGTTAGAGAAACAATGAGTTACTTGTTTCAACATGCCAATTTAG ATTCTTGCAAAAGAGTCTTGAACGTGGTGTGTAAAACTTGTGGACA CACACTTTCTTATGAACAATTTAAGAAAGGTGTTCAGATACCTTGT CCTTTTGTTATGATGTCAGCACCACCTGCTCAGTATGAACTTAAGC GACGGTGCTTTACTTACAAAGTCCTCAGAATACAAAGGTCCTATTA CGGATGTTTTCTACAAAGAAAACAGTTACACAACAACCATAAAAC CAGTTACTTATAAATTGGATGGTGTTGTTTGTACAGAAATTGACCC CAACCAATTGATCTTGTACCAAACCAACCATATCCAAACGCAAGCTCACAGATCTAATGGCTGCTTATGTAGACAATTCTAGTCTTACTATTCTACTCATGGTTTAGCTGCTGTTAATAGTGTCCCTTGGGATACTATACTGTACTGGTTACAGAGAAGGCTATTTGAACTCTACTAATGTCACGTGGTTTAGATTCTTTAGACACCTATCCTTCTTTAGAAACTATACAGTACTTGGATTGGCTGCAATCATGCAATTGTTTTTCAGCTATTTTGCGTCCTTTTATGTCTATGCTAATGGAGGTAAAGGCTTTTGCAAACTA CACAATTGGAATTGTGTTAATTGTGATACATTCTGTGCTGGTAGTA CATTTATTAGTGATGAAGTTGCGAGAGACTTGTCACTACAGTTTAAGTTACAGTGAAGAATGGTTCCATCCATCTTTACTTTGATAAAGCTG GTCAAAAGACTTATGAAAGACATTCTCTCTCTCATTTTGTTAACTTGTTATAGTTTTTGATGGTAAATCAAAATGTGAAGAATCATCTGCAAGTTACTAGATCAGGCATTAGTGTCTGATGTTGGTGATAGTGCGGAA GTTGCAGTTAAAATGTTTGATGCTTACGTTAATACGTTTTCATCAA CTTTTAACGTACCAATGGAAAAACTCAAAACACTAGTTGCAACTGCCATAGAAGTTACTGGCGATAGTTGTAATAACTATATGCTCACCTATGTAACAACAAAGATAGCACTTAAGGGTGGTAAAATTGTTAATAATCTGACTTTTCAAGTGAAATCATAGGATACAAGGCTATTGATGGTGGCATGCTGATTTTGACACATGGTTTAGCCAGCGTGGTGGTAGTTATA CTAATGACAAAGCTTGCCCATTGATTGCTGCAGTCATAACAAGAGCTTTGCAACATCAGCTTGTGTTTTGGCTGCTGAATGTACAATTTTTAAGGCACGGCACTTGTGAAAGATCAGAAGCTGGTGTTTGTGTATCTA CTAGTGGTAGATGGGTACTTAACAATGATTATTACAGATCTTTACCCTATAGTAGCTGGTGGTATTGTAGCTATCGTAGTAACATGCCTTGC CTACTATTTTATGAGGTTTAGAAGAGCTTTTGGTGAATACAGTCATGTAGTTGCCTTTAATACTTTACTATTCCTTATGTCATTCACTGTACTCTGTTTAACACCAGTTTACTCATTCTTACCTGGTGTTTATTCTGTTAGTTCTTTAGTAATTACCTAAAGAGACGTGTAGTCTTTAATGGTGTTGTCATCTCGCAAAGGCTCTCAATGACTTCAGTAACTCAGGTTCTGAGTTGTATGGTACAAGTAACTTGTGGTACAACTACACTTAACGGTCTGGTTATTGGACATTCTATGCAAAATTGTGTACTTAAGCTTAAGGTT GATACAGCCAATCCTAAGACACCTAAGTATAAGTTTGTTCGCATTCGGTTCATTCCTTAATGGTTCATGTGGTAGTGTTGGTTTTAACATAGCTTTTGTTGACAGGCAAACAGCACAAGCAGCTGGTACGGACACAA CTATTACAGTTAATGTTTTAGCTTGGTTGTACGCTGCTGTTATAAAT GGAGACAGGTGGTTTCTCAATCGATTTACCACAACTCTTAATGACTCCATGTTGACATACTAGGACCTCTTTCTGCTCAAACTGGAATTGCC GTTTTAGATATGTGTGCTTCATTAAAAGAATTACTGCAAAATGGTACTCACAATTTTGACTTCACTTTTAGTTTTAGTCCAGAGTACTCAATGGTCTTTGTTCTTTTTTTTGTATGAAAATGCCTTTTTACCTTTTGCTATGGGTATTATTGCTATGTCTGCTTTTGCAATGATGTTTGTCAAACATA CTAAAAGACTGTGTTATGTATGCATCAGCTGTAGTGTTACTAATCC GGACACTTATGAATGTCTTGACACTCGTTTATAAAGTTTATTATGGGCTATTTTTGTACTTGTTACTTTGGCCTCTTTTGTTTACTCAACCGCT GAGTTTAGATATATGAATTCACAGGGACTACTCCCACCCAAGAAT AGCATAGATGCCTTCAAACTCAACATTAAATTGTTGGGTGTTGGTG GCAAACCTTGTATCAAAGTAGCCACTGTACAGTCTAAAATGTCAG CAGAGTAGAATCATCATCTAAATTGTGGGCTCAATGTGTCCAGTTA CACAATGACATTCTCTTAGCTAAAGATACTACTGAAGCCTTTGAAA GACATAAACAAGCTTTGTGAAGAAATGCTGGACAACAGGGCAACC CTTTTGCTACTGCTCAAGAAGCTTATGAGCAGGCTGTTGCTAATGG GATCTGAGGACAAGAGGGCAAAAGTTACTAGTGCTATGCAGACAA CATTATCAACAATGCAAGAGATGGTTGTGTTCCCTTGAACATAATA CCTCTTACAACAGCAGCCAAACTAATGGTTGTCATACCAGACTATAGCTTACTACAACACAACAAAGGGAGGTAGGTTTGTACTTGCACTGT GTTACAGACACACCTAAAGGTCCTAAAGTGAAGTATTTATACTTTA CTAAAGCTTACAAAGATTATCTAGCTAGTGGGGGACAACCAATCA CTAATTGTGTTAAGATGTTGTGTACACACACTGGTACTGGTCAGGC CTACAACTTGTGCTAATGACCCTGTGGGTTTTACACTTAAAAACAC GATCAACTCCGCGAACCCATGCTTCAGTCAGCTGATGCACAATCGT GGCACAGGCACTAGTACTGATGTCGTATACAGGGCTTTTGACATCTCTTTAAGGCATTTTGATGAAGGTAATTGTGACACATTAAAAGAAAT GACTGGTATGATTTTGTAGAAAACCCAGATATATTACGCGTATACG C C AACTTAGGTGAACGTGTAC GC C AAGCTTTGTTAAAAAC AGTACGCAGAGTCACATGTTGACACTGACTTAACAAAGCCTTACATTAAGTGGGATTTGTTAAAATATGACTTCACGGAAGAGAGGTTAAAACTCTTGTTAACTGTTTGGATGACAGATGCATTCTGCATTGTGCAAACTTTAGTGAGAAAAATATTTGTTGATGGTGTTCCATTTGTAGTTTCAACTG GATACCACTTCAGAGAGCTAGGTGTTGTACATAATCAGGATGTAACTTTTCAAACTGTCAAACCCGGTAATTTTAACAAAGACTTCTATGA CTTTGCTGTGTCTAAGGGTTTCTTTAAGGAAGGAAGTTCTGTTGAAACGATGGTGGCTGTATTAATGCTAACCAAGTCATCGTCAACAACCTGCATATACAAAACGTAATGTCATCCCTACTATAACTCAAATGAATCGTGTCTCTATCTGTAGTACTATGACCAATAGACAGTTTCATCAAAAGGAACAAGCAAATTCTATGGTGGTTGGCACAACATGTTAAAAACT GTTTATAGTGATGTAGAAAACCCTCACCTTATGGGTTGGGATTATC CTAAATGTGATAGAGCCATGCCTAACATGCTTAGAATTATGGCCTCCGTTTCTATAGATTAGCTAATGAGTGTGCTCAAGTATTGAGTGAAAATCAGGAGATGCCACAACTGCTTATGCTAATAGTGTTTTTAACATTGTAACAAAATTGCCGATAAGTATGTCCGCAATTTACAACACAGACGAATGAGTTTTACGCATATTTGCGTAAACATTTCTCAATGATGATA CTCTCTGACGATGCTGTTGTGTGTTTCAATAGCACTTATGCATCTCACAAAACAATGTTTTTATGTCTGAAGCAAAATGTTGGACTGAGACTG CAGATGGTACACTTATGATTGAACGGTTCGTGTCTTTAGCTATAGA CAGGACACATGTTAGACATGTATTCTGTTATGCTTACTAATGATAA CACTTCAAGGTATTGGGAACCTGAGTTTTATGAGGCTATGTACACA CCGCATACAGTCTTACAGGCTGTTGGGGCTTGTGTTCTTTGCAATT CACAGACTTCATTAAGATGTGGTGCTTGCATACGTAGACCATTCTTGACAAGTTTTTGGTTTATATAAAAATACATGTGTTGGTAGCGATAA GGTGATTACATTTTAGCTAACACCTGTACTGAAAGACTCAAGCTTT CTTATGGTATTGCTACTGTACGTGAAGTGCTGTCTGACAGAGAATTCTAGTGCCACAAGAGCACTATGTTAGAATTACTGGCTTATACCCAA CACTCAATATCTCAGATGAGTTTTCTAGCAATGTTGCAAATTATCA AAAGGTTGGTATGCAAAAGTATTCTACACTCCAGGGACCACCTGG CTGCTCGCATAGTGTATACAGCTTGCTCTCATGCCGCTGTTGATGCCTAAGCACTATGTGTACATTGGCGACCCTGCTCAATTACCTGCACCGGAACTTGTCGGCGTTGTCCTGCTGAAATTGTTGACACTGTGAGTG CTTTGGTTTATGATAATAAGCTTAAAGCACATAAAGACAAATCAGCCTTACACGTAACCCTGCTTGGAGAAAAGCTGTCTTTATTTCACCTTGTCTGATAGAGACCTTTATGACAAGTTGCAATTTACAAGTCTTGAA ATTCCACGTAGGAATGTGGCAACTTTACAAGCTGAAAATGTAACA GGACTCTTTAAAGATTGTAGTAAGGTAATCACTGGGTTACATCCTA CACAGGCACCTACACACCTCAGTGTTGACACTAAATTCAAAACTGCTAATAATACAGATTTTTCCAGAGTTAGTGCTAAACCACCGCCTGGCTTAAAAATCTCTCTGACAGAGTCGTATTTGTCTTATGGGCACATG GCTTTGAGTTGACATCTATGAAGTATTTTGTGAAAATAGGACCTGA GCGCACCTGTTGTCTATGTGATAGACGTGCCACATGCTTTTCCACT GCTTCAGACACTTATGCCTGTTGGCATCATTCTATTGGATTTGATTA CGTCTATAATCCGTTTATGATTGATGTTCAACAATGGGGTTTTACA GGTAACCTACAAAGCAACCATGATCTGTATTGTCAAGTCCATGGTACCTATAATTGGTGATGAACTGAAGATTAATGCGGCTTGTAGAAAG GTTCAACACATGGTTGTTAAAGCTGCATTATTAGCAGACAAATTCC CAGTTCTTCACGACATTGGTAACCCTAAAGCTATTAAGTGTGTACC GACAAAGCTTATAAAATAGAAGAATTATTCTATTCTTATGCCACAC GAGTGCTATCTAACCTTAACTTGCCTGGTTGTGATGGTGGCAGTTT GTATGTAAATAAACATGCATTCCACACACCAGCTTTTGATAAAAGT GCTTTTGTTAATTTAAAACAATTACCATTTTTCTATTACTCTGACAG GTACCACTAAAGTCTGCTACGTGTATAACACGTTGCAATTTAGGTG GTGCTGTCTGTAGACATCATGCTAATGAGTACAGATTGTATCTCGA AAACAATTTGATACTTATAACCTCTGGAACACTTTTACAAGACTTC GTTTACACAAAAGTTGATGGTGTTGATGTAGAATTGTTTGAAAATA CAACATTAAACCAGTACCAGAGGTGAAAATACTCAATAATTTGGG GCTCCAGCACATATATCTACTATTGGTGTTTGTTCTATGACTGACAT GTAATGGTGTTCTTATTACAGAAGGTAGTGTTAAAGGTTTACAACC GGAGAAGCCGTAAAAACACAGTTCAATTATTATAAGAAAGTTGAT GGTGTTGTCCAACAATTACCTGAAACTTACTTTACTCAGAGTAGAA CTATGCCTTCGAACATATCGTTTATGGAGATTTTAGTCATAGTCAGGTGTGTTCTGTTATTGATTTATTACTTGATGATTTTGTTGAAATAATCTAAAGGCATAATGATGAATGTCGCAAAATATACTCAACTGTGTCGCTGTTTTAAGACAGTGGTTGCCTACGGGTACGCTGCTTGTCGATT CAGATCTTAATGACTTTGTCTCTGATGCAGATTCAACTTTGATTGGT GATTGTGCAACTGTACATACAGCTAATAAATGGGATCTCATTATTA GTGATATGTACGACCCTAAGACTAAAAATGTTACAAAAGAAAATG AAAGCTAGCTCTTGGAGGTTCCGTGGCTATAAAGATAACAGAACACCAATTCAGTTGTCTTCCTATTCTTTATTTGACATGAGTAAATTTCC CCTTAAATTAAGGGGTACTGCTGTTATGTCTTTAAAAGAAGGTCAACAACTAACATTATGAGGGTAATTCAGAAAGAGAACAAATTGTTTTATTTTTTTATTTTGAACGTATATAAAAATATTTCCTTTAATTTGTAGGAGAGTGTGTATTATTTTTGTTCGAAAATTTTTTTTATGTATTTTTGAAAATATTTTTTGTAACAGTGAAATAAAAATATGAATGAAAACAG CCGTATATGCAAACCTGGACTGATTATAGAAGAGCAATTATTTTAT TACATGTTCGAACAGATTTAAAAGATAGACCAGTTTCATTTTGTGA TTTTAGAAAAGGTGAATTATATAATTATTTAAATGCTTATAGTGAA GGAGATCTATGTATAATAATTTCCAAATCAAATACAAGTTTTGGTT TTAGATGCCCCGAAAATACAAAAAAAATGCCAGAAAAATGTTTTA CTCAAGTATATGAAAAAGGCTATCTACATGATTCCTATAAAATTAArACTAAAAATATTATTAACTATTCATTTGAAAATCCAGAATATGCA TTAGCTGGTTTTAATTATACATTAACAAAATCATATCAATTTGAAT GTCATTGTGTAGATAAAGAAACAGAACAAATTGTAAAAACTGTTT TAGTAAAATATGTAAATGAAGATGAAATATATGATTATAATGATTT ACCATTGGTTAATCATAAATCTATTGTTGCACATCCAAATAAAACA CATCTATGTGACTTTATGACATCTGATAATATGTTATCACCTAAAA AAGAAGATTCAGTAAATTATGTTTGTAATGTATTTCCAAAACCATT AGAATATGTAGCATTACATTGTCCAACCAATATAGTAGATGTAGA AAATGAAGATGATATTTCAAGACTATCAAAGGAAATGCATAAAGA AGAAATAAAAGCGGAATTAAGAATCAAACTTCP230p_Var GAAGATTTTATCATTATTCGAGTCAGAGTACATCAAACTTATAAAA icella AAATATTAGGAATAAGTAGTGATAAAAATGATAAACCATATTTTG Zoster virus AAGAAATTATTAGTGAAGAAAAAGATATATATGAATTTGAAGGAT ORF62 ATTTAGAAAAAGTAATTGGAATCAAATTAGAAGGATATGAATTAG (IE6) ATCCACCAAATTGTTTTAAAAGTGTATATGAAGATGATAAAAGAA TATATTTAGAAGTAGAATATCATTATTCAAAATGTATTAACTTAGA CAGAAAAAATTATAAATTAAGATTTTATTTTTTATCACAATATTTT GCTGATTATGAATTAAAGTTTTCATGTAATATAATTAATATAAAAA CTCAAAAAAAAAAAACTGTTACATTTGGTGATGGAATGGCAACAT CTGATCACATTTTAAAAATAGTTGATGATGATAGTAAAACGATTAA ATATTTTAACGATATACCTTATCAGATGTGTAATTTTGATTATAATT TAAGCAAATTAAGTGAAATACAAATATGTGAAAAAACAATAAATG AATTTAGTTTATTCATGTATAATTGTGAGCAAATAACAGATAATAG AATCGTATATGGTAAAGAACCTATTAATACCATAAAATATTTAAGT AATGTATTTCCTATAAATAAATTTACAGATTTATTTTTTAATACAAA AGATATAGATATACCAGAAATAAATGAACAATTTAAAGGTTTTAAATTTTTTATGACCTCATTTATAAATCATGGATCATATCCACTAACCTGAAAATCAGTTATTTAAATTTTAACTGAAGAAATTAAATAAGTATACATTTTAGTTGTAGCGTCACTTTTATTAGTTGATTCTCTACTTCCAG GATATGGACAAAATAAAAGTGTCCAAGCCCAAAGAAACTTAAACGCCCCAACGCGCGGGGTCGCCTGATACTTTGGAGTTAATGGGATCGTTTCAATTCCCTCCGTATCG GGACTTCAACCAGAACCCAGAACGGAAGATGTTGGCGAAGAGCTACTTCCACACCCGATTCTCGGAGGC CGGCGCGCGAGAACCAACAGGAGCCGATCGCTCCCTTGAGACAGT CTCTCTCGGAACGAAGCTTGCTAGGCCCCTGTATCCCCCGCGTCTCCTGTTGGAGACGCCGCCGGGAACGAGGTCCATCGAGACAGACAAACTCAGCATGCATCGGGGGACGGAGTAGTTCCCCGGCGCGGAAACCCGGTGGACCTGTACCCGGCCCGGTTGAGCAGTTCCCGCTAAGGGGGCAAAGGCGGACCTGCCGTTTGAGACCGAT GATACCCGCCCAAGGAAACATGATG CCCGGGGTATAACACCTCGCGTCCCTGGACGTTCGTCGGGGGGCAGGGAAGATCCCACGCACCAGACCCGATTGAGGATGACAGCCCAGT GGAGAAAAAGCCAAAGAGTCGTGAGGGATGAAGACGATGAACCCCGGC GCGTTTCGGTGGGAAGTGAAACTACAGGCAGCAGGTCCGGACGCGGGATGATTCGGACTCAAATGATGGTGGGTCGACGAAACAAAATAT CCAACCGGGATATCGATCCATCAGC GGTCCCGATCCGAGGATTCGTAAGACCAAACGTCTTGCGGGGGAA CCGGGGCGCCAGAGACAGAAATCATGCCGCTCATGATGCCCGACGGAAG CCCTTGGCCCGGATCGGCACCCCTCCCATCCAACAGGGTGCGGTTTGGACCGTCCGGGGAGACCAGAGAGGGTCACTGGGAGGATGAGGCTGTGAGAGCGGCGCGGGCTCGTTAC GAGGCCTCAACGGAACCCGTGCCGCCAGAGACCCTATAGCATGGCTCCAGAACCCCAAGCTGACCGGTGT CAACTCGGCCCTGAACCAGTTCTAC CAAAAGCTGTTGCCACCGGGACGGGCGGGTACCGCCGTTACGGGGCCGTCGATACGACCGGGCCCAAAAACACTTTATCCTACAGAGTCTC CGCAGAGCCTTTGCCAGCATGGCAGGTCACCCTTCTCCAACAACCCCGG CCACACAGGCTCCCGACCCTCAGCCGTCGGCCGCCGCACGCTCTCTCCGTGACCATCACCTCGGGCCCTGTGGTGGATCCCCCCGCCGTAATCCCAAACGGGGGATTTCGGCGTATTCCCCGGGGGGCCCTGCATAC CCCGGTCCCGTCGGACCAGGCTCGCCCATTGTTTCCCACGGCCTGGCGCC CTGCGCTAAGCTTTGATCCCGGCGCCTTGGCGGAAATCGCCGCTCG GCGTCCGGGCGGAGGAGACCGACG GTTTGGTCCACCCAGCGGAGTGGAGGCGCTGCGACGGAGGTGCGC CTGGATGCGGCAGATCCCAGACCCG GAGGATGTGAGGCTTCTGATCATCTACGATCCGTTGCCCGGAGAG GACATCAACGGCCCCCTCGAGAGCA CCCTCGCGACAGATCCGGGACCGTCATGGAGTCCATCCCGAGGGGGACTGTCTGTGGTCCTGGCAGCCCTGAGTAACCGGTTGTGCCTGCCGAGCACTCATGCCTGGGCCGGGAA CTGGACCGGCCCGCCGGACGTGTCC GCTTTGAACGCCCGGGGCGTTTTATTACTGTCGACCCGAGACCTGG CCTTTGCCGGGGCCGTCGAGTATCCGCGGTGGCCCTCGAGAGGTGGCC CAGGGATGGACCCGCTTTGTCTCAGTATCACGTGTACGTCCGGGCC CCGGCGCGACCGGACGCCCAGGCC GTCGTCCGATGGCCAGACTCGGCGGTCACAGAAGGACTCGCCCGG GCCGTGTTTGCATCGTCGCGCACCTGTACCCGGGCGAACAACCCCTGTGGGCCCCAAGACCCGCGTCCCCCTGGCAAGGACCTCGCGCGACAGTCTC GGGGTCTGGGGCTCGGGGCAGCCGACTTTGTGGACGAGGCGGCACGGGCCTGGGTGCCGCGCTTCGACCCGTCTTCCTTCCCGAGGGACGGGCCGGCGACGTACCCACCTGGGCGAGGGTGTTTTGCCGCCACGCC CTGCTGGAACCCGACCCTGCCGCAGGCCGCGGAGACGGTGTTGGAGGGGCGGCCGTCAGAAACCCAGGCGGGGC GACAGCGGCGCACCGCAGACGACAGAGAACACGCTTTGGAGCTGGCGCGTGGGACAGCGAGGAGGGGGGCGGGGACGACGGGGACGCAC CGGGGTCATCCTTTGGGGTGAGCATC GTGTCGGTGGCCCCGGGTGTGCTGCGAGACCGCCGGGTGGGTTTGCGCCCGGCGGTCAAGGTGGAGCTGTGGCGCGGGGGGAGGAGCCCCCCGCA GAGTCGGGGGTGACGGAGTCCCCTCCTTTTCTCGTGAGCGCCACTG GCGCGCGGACTGTTTGTTGTTAATGTTATAGAGAAAAGAAGGAAAAACATTATAACAAAATCACATTTTCAAATAATTTATAAAAATATATTGTGTCTAAAAGAAATGAAAACG GATTCTAGCATATAAACAAATCTGGCTTCGAAAAGAAATAGAAGAGTGTGTATTATTTTTGTTCGAAAATTTTTTTTATGTATTTTTGAGAGGATCTATGTATAATAATTTCCAAATCAAATACAAGTTTTGGTTTTA GATGCCCCGAAAATACAAAAAAAATGCCAGAAAAATGTTTTACTCGCTGGTTTTAATTATACATTAACAAAATCATATCAATTTGAATGTCCTATGTGACTTTATGACATCTGATAATATGTTATCACCTAAAAAAGGAAGATGATATTTCAAGACTATCAAAGGAAATGCATAAAGAAGAAP230p Bor GAAGATTTTATCATTATTCGAGTCAGAGTACATCAAACTTATAAAA na Virus P AAATATTAGGAATAAGTAGTGATAAAAATGATAAACCATATTTTG protein ATTTAGAAAAAGTAATTGGAATCAAATTAGAAGGATATGAATTAG CAGAAAAAATTATAAATTAAGATTTTATTTTTTATCACAATATTTT GCTGATTATGAATTAAAGTTTTCATGTAATATAATTAATATAAAAA CTCAAAAAAAAAAAACTGTTACATTTGGTGATGGAATGGCAACAT CTGATCACATTTTAAAAATAGTTGATGATGATAGTAAAACGATTAA AATGTATTTCCTATAAATAAATTTACAGATTTATTTTTTAATACAAAGAAAATCAGTTATTTAAATTTTAACTGAAGAAATTAAATAAGTATACATTTTAGTTGTAGCGTCACTTTTATTAGTTGATTCTCTACTTCCAG GATATGGACAAAATAAAAGTGTCCAAGCCCAAAGAAACTTAAACGCCATGGAGGA TATGAACCTC CCGCGAGCCTCCCCAAGCTCCCTGGGAAATGCATGAGAAAGACATCAGGCAGAACGCAGTGGCATTGTTAGACCA GTCACGGCGCGATATGTTTCACACAGTAACGCCTAGCCTTGTGTTT CTA TGTTTGCTAA TCCCAGGACT GCACGCTGCGTTTGTTCACGGAGGGGTGCC TCGTGAATCC CGCCTGTTAC GCGTGGAGAACAGACTGTTG GAAGTTTTAC GGGGAAAAGA CGACGCAACG TCTCTTCTAT CTTCAGCCAT TACTAATAGG GGTTGTGATAGGATCGTCGT CTAAGATCAA AGCAGGAGCC GAGCAAATCA AGAAAAGGTT TAAAACTATGATGGCAGCCT ATCCCATGGT GAGACTGCTA CACTACTCCA GATGTTTAATCCACATGAGG CTATAGATTG GATTAACGGC CAACCCTGGG TAGGCTCCTT TGTGTTGTCTCTACTAACTA CAGACTTTGA GTCCCCAGGT AAAGAATTTA TGGACCAGAT CGCAGATGAC TACGTACACT AGTACCTCGC AGAATGCATGGATGCTACCC CGTAGTTGCA TATGAGATCC GTGACTTTTT AGGAGGATCA TGCTGACCTG TGGGAGCCAT TAGACATCCCGACGCTATCA ACGAAGCTTT CCCAATCTGG CCTCCGCAGCGTTTTACTGGAGTAAGAAGG AAAACCCCAC AATGGCAGGCCCACCATCCA GCCGGGCGCAAGTGTCAAGG TGCCCGGTAT AGGCGCCGCG AGATATCTCG CGGGGAGGACGGGGCAGAGC TCTCAGGTGA GATTTCTGCC TGATAGGTGT GACTGGTCTAAACTAAAAAA CAATGAACAA ACCAATAAAA AACCAAATGC GGCAACCCCT CCGCAACCTGTGATGAGTTC CGACCTCCGG CTGACACTGC CAGGAGGCTC AATGGCAACGCGACCATCGA GTCTGGTCGA CTCCCTGGAG GACGAAGAAG ATCCCCAGAC GGTCACCAAG ACCACGGAAG ATGCATTGAC CCAACCAGTCGACCAGCTCC CAGGAAGAAC CCCTCCATGA TCTCAGACCC AGCAGCTGTC GAATGATGAG CTAATCAAGA AGTTAGTGAC GGAGCTGGCCGAGAATAGCA TGAGGAGGTG CGGGGCACTC TTGGAGACAT CTCGGCTCGCATCGAGGCAG GGTTTGAGTC CCTGTCCGCC CTCCAAGTGG AAACCATCCA GACAGCTCAGCGGTGCGACC CATCAGGATC CTCGGCGAGA ACATCAAGAT CAATGATGGA GACAATGAAG CTCATGATGG AGAAGGTGGA CCTCCTCTACGCATCAACCG CCGTTGGGAC CTCTGCACCC ATGTTGCCCT CCCATCCTGC AGCTCCCAAG TGCCCCGACA GAAAGAGAACAAATTGTTTTATGATAAATATATTTATAAAATAAA GTATAAAAAACTTTTACAATATATTTTTTTATTTTGAACGTATATAA ATTGTGTCTAAAAGAAATGAAAACGGATTCTAGCATATAAACAAATATAGAAGAGCAATTATTTTATTACATGTTCGAACAGATTTAAAAG ATAGACCAGTTTCATTTTGTGATTTTAGAAAAGGTGAATTATATAA TTATTTAAATGCTTATAGTGAAGGAGATCTATGTATAATAATTTCC AAATCAAATACAAGTTTTGGTTTTAGATGCCCCGAAAATACAAAA AAAATGCCAGAAAAATGTTTTACTCAAGTATATGAAAAAGGCTAT CTACATGATTCCTATAAAATTAATACTAAAAATATTATTAACTATT CATTTGAAAATCCAGAATATGCATTAGCTGGTTTTAATTATACATT AACAAAATCATATCAATTTGAATGTCATTGTGTAGATAAAGAAAC AGAACAAATTGTAAAAACTGTTTTAGTAAAATATGTAAATGAAGA TGAAATATATGATTATAATGATTTACCATTGGTTAATCATAAATCT ATTGTTGCACATCCAAATAAAACACATCTATGTGACTTTATGACAT CTGATAATATGTTATCACCTAAAAAAGAAGATTCAGTAAATTATGT TTGTAATGTATTTCCAAAACCATTAGAATATGTAGCATTACATTGT CCAACCAATATAGTAGATGTAGAAAATGAAGATGATATTTCAAGA CTATCAAAGGAAATGCATAAAGAAGAAATAAAAGCGGAATTAAGAATCAAACTTCREFERENCES1. 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Claims

CLAIMSThe embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:

1. A Plasmodium-species sporozoite that is attenuated to arrest the Plasmodium-species sporozoite’s developmental progression during liver stage and prevent subsequent red blood cell infection, said sporozoite comprising a heterologous transgene encoding an immunomodulator, a functional fragment thereof, or an engineered analogue thereof, thereby providing a liver-specific vector for introduction of the expressible heterologous transgene into hepatocytes.

2. The Plasmodium-species sporozoite of claim 1, wherein the immunomodulator is an antagonist of at least one of a human interferon regulatory factor (IRF) or an interferon signaling protein.

3. The Plasmodium-species sporozoite of claim 1 or claim 2. wherein the immunomodulator is an antagonist of at least one of IRF3, IRF1, and IRF7.

4. The Plasmodium-species sporozoite of claim 1 or claim 2, wherein the immunomodulator is an antagonist of the interferon signaling protein type 1 interferon (IFN-1).

5. The Plasmodium-species sporozoite of claim 3, wherein the immunomodulator is an antagonist of IRF3 and the antagonist is selected from the group consisting of Flavivirus Nonstructural Protein 1 (NS1), Human Immunodeficiency Virus Viral Protein R (HIV VPR), West Nile Virus (WNV) NS1, Severe Acute Respiratory Syndrome Coronavirus 2 Nonstructural Protein 3 (SARS Cov-2 NSP3), SARS Cov-2 NSP13, SARS Cov-2 NSP15, SARS Cov-2 Open Reading Fram 6 (ORF 6), SARS Cov-2 ORF3b, SARS Cov-2 NSP1, Ebolavirus VP35, and Ebola virus Nucleoprotein (NP).

6. The Plasmodium-species sporozoite of any one of claims 1-5, wherein the heterologous transgene encoding the immunomodulator is inserted into a P230p locus within the Plasmodium genome.

7. The Plasmodium-species sporozoite of any one of claims 1-6, wherein the heterologous transgene encoding the immunomodulator is operably linked to a promoter sequence of the Plasmodium liver stage protein UIS4.

8. The Plasmodium-species sporozoite of any one of claims 1-6, wherein the heterologous transgene encoding the immunomodulator is operably linked to a promoter sequence of the Plasmodium circumsporozoite protein (CSP) PEXEL.

9. The Plasmodium-species sporozoite of claim 5, comprising a nucleotide sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 40 or 41.

10. The Plasmodium-species sporozoite of claim 1, wherein the immunomodulator is human interleukin 2 (hIL2).

11. The Plasmodium-species sporozoite of claim 1, wherein the heterologous transgene encodes the engineered hIL-2 analogue cIL-2eRa (SEQ ID NO: 38).

12. The Plasmodium-species sporozoite of any one of claims 1-11, wherein the Plasmodium- species sporozoite is of human host range.

13. The Plasmodium-species sporozoite of claim 12, wherein the Plasmodium-species sporozoite is P. falciparum.

14. The Plasmodium-species sporozoite of any one of claims 1-13, wherein the Plasmodium- species sporozoite arrests at late liver stage.

15. The Plasmodium-species sporozoite of claim 14, wherein the Plasmodium-species sporozoite lacks: (i) a functional LINUP gene: (ii) a functional Mei2 gene; or (iii) both, a functional LINUP gene and a functional Mei2 gene.

16. The Plasmodium-species sporozoite of claim 15, wherein the Plasmodium-species sporozoite lacks a functional Mei2 gene.

17. The Plasmodium-species sporozoite of claim 15, wherein the Plasmodium-species sporozoite lacks both, a functional LINUP gene and a functional Mei2 gene.

18. A Plasmodium-species sporozoite which is attenuated to arrest the Plasmodium-species sporozoite developmental progression during liver stage and prevent subsequent red blood cell infection, said sporozoite comprising one or more heterologous expressible transgenes encoding one or more antigens of an infectious hepatotropic pathogen or antigenic fragments thereof, thereby providing a liver-specific vector for introduction of the heterologous expressible trans gene into hepatocytes.

19. The Plasmodium-species sporozoite of claim 18. wherein the Plasmodium-species sporozoite arrests at late liver stage.

20. The Plasmodium-species sporozoite of claim 19, wherein the Plasmodium-species sporozoite is of human host range.

21. The Plasmodium-species sporozoite of any one of claims 18-21, wherein the Plasmodium- species sporozoite is P. falciparum.

22. The Plasmodium-species sporozoite of any one of claims 18-21, wherein the infectious hepatotropic pathogen is hepatitis B virus (HBV), and the one or more antigens of HBV comprise HBxAg, HBpolAg, HBsAg, HBcAg, an antigenic fragment thereof, or any combination thereof.

23. The Plasmodium-species sporozoite of claim 22, further comprising a Plasmodium falciparum (Pf) PfCSP (SEQ ID NO: 25), PfEXPl(SEQ ID NO: 31), PfLSAl (SEQ ID NO: 32), or PfHSP70 (SEQ ID NO: 29) promoter, wherein said promoter is configured to drive expression of the heterologous transgene.

24. The Plasmodium-species sporozoite of claim 19, wherein the Plasmodium-species sporozoite lacks: (i) a functional LINUP gene: (ii) a functional Mei2 gene; or (iii) both, a functional LINUP gene and a functional Mei2 gene.

25. The Plasmodium-species sporozoite of claim 18, wherein the Plasmodium-species sporozoite comprises both (i) a heterologous transgene encoding a human immunomodulator, a functional fragment thereof, or an engineered analogue thereof: and (ii) one or more heterologous transgenes encoding one or more antigens of an infectious hepatotropic pathogen or antigenic fragments thereof.

26. A vaccine composition comprising the Plasmodium-species sporozoite of any one of claims 14, 19, or 25 and a carrier.

27. The vaccine composition of claim 26, wherein the carrier is selected from the group consisting of phosphate buffered saline (PBS), saline, human serum albumin, or any combination thereof.

28. The vaccine composition of claim 26 further comprising an adjuvant.

29. The vaccine composition of claim 28, wherein the adjuvant is 7DW8-5.

30. A method of expressing one or more heterologous transgenes intra-hepatically in a human subject, the method comprising administering to the subject an effective amount of the Plasmodium-species sporozoite vaccine composition of claim 26.

31. A method of generating an immune reaction to a human hepatic pathogen intra-hepatically in a human subject, the method compnsing administering to the subject an effective amount of the Plasmodium-species sporozoite vaccine compositions of claim 26.

32. A method of preventing or reducing the likelihood of an infection of a human pathogen in a human subject comprising administering to the subject an effective amount of the Plasmodium-species sporozoite vaccine compositions of claim 26.

33. A method of treating a hepatic infection in a human subject in need thereof, the method comprising administering to the subject an effective amount of the Plasmodium-species sporozoite vaccine compositions of claim 26.

34. The methods of any one of claims 30-33, wherein the administration is by parenteral injection.

35. A method of treating a hepatic infection in a human subject in need thereof comprising administration to the human subject, a Plasmodium-species sporozoite of human host range which is attenuated to arrest the Plasmodium-species sporozoite developmental progression in liver stage and prior to red blood cell infection.

36. A hepatotropic vector for introduction / delivery of an expressible heterologous transgene into hepatocytes, the vector comprising:a genetically attenuated Plasmodium-species sporozoite comprising at least one heterologous expressible transgene, wherein the Plasmodium-species sporozoite is attenuated to arrest the Plasmodium-species sporozoite’s developmental progression during liver stage and prevent subsequent red blood cell infection.

37. The hepatotropic vector of claim 36, wherein the Plasmodium-species sporozoite arrests at late liver stage.

38. The hepatotropic vector of claim 37. wherein the genetically attenuated Plasmodium- species sporozoite lacks a functional LINUP gene (SEQ ID NO: 19), a functional LINUP protein (SEQ ID NO: 37), or both.

39. The hepatotropic vector of claim 37. wherein the genetically attenuated Plasmodium- species sporozoite lacks a functional Mei2 gene (SEQ ID NO: 18) or a functional Mei2 protein (SEQ ID NO: 20).

40. The hepatotropic vector of claim 37. wherein the genetically attenuated Plasmodium- species sporozoite lacks a functional LINUP gene (SEQ ID NO: 19) and a functional Mei2 gene (e.g., SEQ ID NO: 18).

41. The hepatotropic vector of any one of claims 36-40, wherein the Plasmodium-species sporozoite is of human host range.

42. The hepatotropic vector of claim 41, wherein the Plasmodium-species sporozoite is Plasmodium falciparum.

43. The hepatotropic vector of claim 40 or claim 42, wherein the at least one heterologous expressible transgene encodes a human immunomodulator, a functional fragment thereof, or an engineered analogue thereof.

44. The hepatotropic vector of claim 43, wherein the immunomodulator is an antagonist of at least one of an interferon regulatory factor (IRF) or an interferon signaling protein.

45. The hepatotropic vector of any one of claims 36-44, further comprising a second heterologous transgene encoding one or more antigens or fragments thereof derived from a hepatotropic pathogen.

46. The hepatotropic vector of claim 45, wherein the hepatotropic pathogen is hepatitis B virus (HBV), and the one or more antigens of HBV comprise HBxAg, HBpolAg, HBsAg, HBcAg, an antigenic fragment thereof, or any combination thereof.

47. The hepatotropic vector of any one of claims 36-46, wherein the hepatotropic vector further comprises a promoter, and optionally an export signal.

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