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US20150191704A1 - Poxvirus-plasmodium recombinants, compositions containing such recombinants, uses thereof, and methods of making and using same - Google Patents

Poxvirus-plasmodium recombinants, compositions containing such recombinants, uses thereof, and methods of making and using same Download PDF

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US20150191704A1
US20150191704A1 US14/576,578 US201414576578A US2015191704A1 US 20150191704 A1 US20150191704 A1 US 20150191704A1 US 201414576578 A US201414576578 A US 201414576578A US 2015191704 A1 US2015191704 A1 US 2015191704A1
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poxvirus
vaccine
nyvac
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Enzo Paoletti
Randall L. Weinberg
Scott J. Goebel
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V-CORE TECHNOLOGIES Inc
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Definitions

  • the present invention relates to modified poxvirus and to the methods of making and using the same.
  • the invention relates to recombinant poxvirus, which virus expresses exogenous or heterologous gene product(s), e.g., from Plasmodium , a specific poxvirus replication regulator and an adjuvant for immune-response enhancement, and immunogenic compositions or vaccines containing such poxvirus, and methods for providing immunity, e.g., protective immunity, against Plasmodium infections.
  • poxviruses such as Chordopoxvirinae subfamily poxviruses (poxviruses of vertebrates), for instance, orthopoxviruses and avipoxviruses, e.g., vaccinia virus (e.g., Wyeth Strain, WR Strain (e.g., ATCC® VR-1354), Copenhagen Strain, NYVAC, NYVAC.1, NYVAC.2, MVA, MVA-BN), canarypox virus (e.g., Wheatley C93 Strain, ALVAC), fowlpox virus (e.g., FP9 Strain, Webster Strain, TROVAC), dovepox, pigeonpox, quailpox, and raccoon pox, inter alia, synthetic or non-naturally occurring recombinants thereof, uses thereof, and methods for making and using such recombinants may be found in scientific and patent literature, such as:
  • NYVAC- Plasmodium recombinant known as VP1209 or NYVAC-Pf7
  • Tine et al “NYVAC-Pf7: a poxvirus-vectored, multiantigen, falciparum malaria multistage vaccine candidate for Plasmodium ,” Infect. Immun. 1996, 64(9):3833, and Ockenhouse et al, “Phase InIa Safety, Immunogenicity, and Efficacy Trial of NYVAC-Pf7, a Pox-Vectored, Multiantigen, Multistage Vaccine Candidate for Plasmodium falciparum Malaria,” 1998; 177:1664-73, each of which is incorporated herein by reference.
  • malaria is considered the most important parasitic disease in the world. It is estimated that malaria caused over 200 million clinical episodes worldwide resulting in 655,000 deaths, mostly African children; see WHO Global Malaria Program 2011. Furthermore the economic losses are magnified as most of the endemic countries are impoverished, costing some 3 billion dollars in Africa alone; see Teklehaimanot A. J. Trop. Med. Hyg. 2007; 77(6): 138-44. There have been substantial efforts and resources directed to methods and approaches for control-intervention such as indoor spraying, insecticidal nets, rapid diagnostics for testing, especially pregnant woman and children; see Aponte J J. Lancet 2009; 374(9700): 1533-44., Menendez C. Lancet Infect. Dis. 2007; 7(2): 126-35.
  • the present invention recognizes and endeavors to address poxvirus (e.g., recombinant poxvirus) immunological or immunogenic composition or vaccine induction of only weak or suboptimal immune correlatives; see, e.g., Smith, J M. AIDS Res. Hum. Retroviruses 2004; 20: 1335-1347, Hanke, T. J. Gen. Virol. 2007; 88: 1-12, Sandstrom, E. J. Inf. Dis. 2008; 198: 1482-90, Walker, BD. Science 2008; 320: 760-4, Sekaly, RP. J. Exp. Med. 2008; 205: 7-12. Rerks-Ngarm S. 2009; N Engl J Med 361: 2209-2220.
  • poxvirus e.g., recombinant poxvirus
  • poxvirus includes members of the Chordopoxvirinae subfamily, such as orthopoxviruses and avipoxviruses, e.g., vaccinia virus (e.g., Wyeth Strain, WR Strain (e.g., ATCC® VR-1354), Copenhagen Strain, NYVAC, MVA, MVA-BN), canarypox virus (e.g., Wheatley C93 Strain, ALVAC), fowlpox virus (e.g., FP9 Strain, Webster Strain, TROVAC), dovepox, pigeonpox, quailpox, and raccoon pox, inter alia; and it especially includes poxviruses of documents cited herein, including poxviruses that also express transcription and/or translation factor(s) of U.S. Pat. Nos. 5,990,091, 6,130,066 and 6,004,777.
  • vaccinia virus e.g.
  • the invention provides a poxvirus that is a synthetic or non-naturally occurring, i.e., an engineered, synthetic or a non-naturally-occurring poxvirus, e.g., through recombination, advantageously an attenuated poxvirus as to a mammal, such as NYVAC, NYVAC.1, NYVAC.2, avipox, canarypox, fowlpox, ALVAC, TROVAC, MVA, MVA-BN, that through such engineering contains DNA encoding Flagellin (or an operable binding portion thereof) and/or vaccinia host range gene K1L, and expresses such DNA.
  • the poxvirus contains and expresses DNA encoding Flagellin (or an operable binding portion thereof) and vaccinia host range gene K1L.
  • the invention comprehends such a poxvirus that is synthetic or non-naturally occurring, i.e., that has been engineered or manipulated, e.g., through recombination, to contain, advantageously in a non-essential region, DNA encoding Flagellin (or an operable binding portion thereof) and/or vaccinia host range gene K1L, and express such DNA.
  • the synthetic or non-naturally occurring or engineered or recombinant poxvirus that contains and expresses DNA encoding Flagellin (or an operable binding portion thereof) and/or vaccinia host range gene K1L can also be manipulated, engineered to contain and express DNA coding for one or more antigen(s), immunogen(s) or protein(s) that is/are foreign or exogenous or heterologous to the poxvirus.
  • the invention also comprehends compositions containing such an engineered or synthetic or non-naturally-occurring or recombinant poxvirus, e.g., immunogenic or immunological or vaccine compositions, uses of such a poxvirus or composition, e.g., to stimulate an immune response, such as a protective immune response, for example for generation of antibodies for use either in vivo, in vitro or ex vivo, and methods of making such poxviruses and compositions, and methods of using such poxviruses and compositions.
  • Such compositions can contain an amount of poxvirus akin to the amount of recombinant poxvirus found in prior art recombinant poxvirus immunogenic or immunological or vaccine compositions.
  • the amount of composition and/or poxvirus to be administered can be akin to the amount administered in prior art methods for inducing an immune or protective immune response by recombinant poxvirus compositions or recombinant poxviruses.
  • NYVAC expressing Flagellin (FliC) can be a novel vaccine directed to poxvirus infections, including smallpox.
  • the invention provides a poxvirus that is a synthetic or non-naturally occurring, i.e., an engineered, synthetic or a non-naturally-occurring poxvirus, e.g., through recombination, advantageously an attenuated poxvirus as to a mammal, such as NYVAC, NYVAC.1, NYVAC.2, avipox, canarypox, fowlpox, ALVAC, TROVAC, MVA, MVA-BN, that through such engineering contains, advantageously in a non-essential region, DNA encoding Flagellin (or an operable binding portion thereof) and/or vaccinia host range gene K1L, and expresses such DNA, and DNA encoding gene product(s) of Plasmodium and expresses such DNA encoding gene product(s) of Plasmodium .
  • a poxvirus that is a synthetic or non-naturally occurring, i.e., an engineered, synthetic or a non-
  • the invention comprehends such a poxvirus that is synthetic or non-naturally occurring, i.e., that has been engineered or manipulated, e.g., through recombination, to contain DNA encoding Flagellin (or an operable binding portion thereof) and/or vaccinia host range gene K1L and express such DNA, and DNA encoding gene product(s) of Plasmodium and express such DNA encoding gene product(s) of Plasmodium .
  • the engineered, synthetic, non-naturally occurring and/or recombinant poxvirus of the invention thus co-expresses gene product(s) of Plasmodium , and Flagellin (or an operable binding portion thereof) (and optionally also K1L).
  • the invention also comprehends compositions containing such an engineered or synthetic or non-naturally-occurring or recombinant poxvirus, e.g., immunogenic or immunological or vaccine compositions, uses of such a poxvirus or composition, e.g., to stimulate an immune response, such as a protective immune response, for example for generation of antibodies for use either in vivo, in vitro or ex vivo, and methods of making such poxviruses and compositions, and methods of using such poxviruses and compositions.
  • Such compositions can contain an amount of poxvirus akin to the amount of recombinant poxvirus found in prior art recombinant poxvirus immunogenic or immunological or vaccine compositions.
  • the amount of composition and/or poxvirus to be administered can be akin the amount administered in prior art methods for inducing an immune or protective immune response by recombinant poxvirus compositions or recombinant poxviruses.
  • Immunogenic or immunological compositions stimulate an immune response that may, but need not be, protective.
  • a vaccine stimulates a protective immune response.
  • a vaccine against Plasmodium or malaria provides at least 80% protective efficacy against P. falciparum (protection in at least 80% of subjects receiving the vaccine).
  • the skilled person may employ a complementing host cell or helper virus, see, e.g., U.S. Pat. No. 5,766,882.
  • the DNA encoding gene product(s) of Plasmodium advantageously codes for Plasmodium antigen(s) or immunogen(s), e.g., SERA, ABRA, Pfhsp70, AMA-1, Pfs25, Pfs16, CSP, PfSSP2, LSA-1 repeatless, MSA-1, AMA-1 or combination(s) thereof.
  • the DNA encoding gene product(s) of Plasmodium advantageously codes for sequences for CSP, PfSSP2, LSA-1-repeatless, MSA-1, SERA, AMA-1 and Pfs25, akin to NYVAC-Pf7.
  • the vector is advantageously NYVAC.
  • the vector can also express a translation and/or transcription factor, such as in U.S.
  • the Flagellin (or an operable binding portion thereof) when expressed in an attenuated vector, such as a NYVAC vector may have an adjuvant or immunostimulatory effect.
  • a NYVAC vector this is advantageously an “enhanced” NYVAC vector (i.e., it also contains and expresses vaccinia K1 L).
  • an “enhanced” replication competent NYVAC vector that contains and expresses Flagellin (or an operable binding portion thereof) also contains and expresses Plasmodium falciparum CSP, PfSSP2, LSA-1-repeatless, MSA-1, SERA, AMA-1 and Pfs25.
  • Such a vector has the capacity for a level of limited replication in humans while retaining the established vector safety profile of NYVAC with open reading frames for virulence factors deleted or disrupted, and can obtain an immunological or immunogenic response that is desired for a malaria vaccine.
  • compositions of the invention can contain an amount of engineered, synthetic, non-naturally occurring or recombinant Flagellin- Plasmodium -poxvirus (that advantageously also contains and expresses vaccinia K1 L) as in NYVAC-Pf7 compositions; and, in methods for inducing an immune or protective immune response of the invention, the amount of composition and/or poxvirus to be administered can be akin the amount administered in prior art methods involving NYVAC-Pf7.
  • the invention provides self-adjuvanting immunogenic, immunological or vaccine compositions (by expression of Flagellin or an operable binding portion thereof by the poxvirus, especially with expression of vaccinia K1L).
  • These vectors are capable of triggering innate immunity and important pro-inflammatory cascade(s) critical for the development of robust adaptive immune responses that can provide protective immunity, e.g. against Plasmodium infection.
  • the invention thus provides a replication competent, engineered, synthetic, non-naturally occurring or recombinant poxvirus useful for the production of Plasmodium immunogen(s) or antigen(s), in vivo or in vitro; and, the resulting immunogen(s) or antigen(s).
  • the invention relates to a recombinant poxvirus containing therein DNA encoding at least one Plasmodium antigen or immunogen and at least one DNA sequence encoding Flagellin or an operable binding portion thereof and/or the vaccinia host range gene K1L—and advantageously both the DNA sequence encoding Flagellin or an operable binding portion thereof and the vaccinia host range gene K1L—advantageously in a nonessential region of the poxvirus genome.
  • the poxvirus is advantageously NYVAC.
  • the recombinant poxvirus expresses Plasmodium SERA, ABRA, Pfhsp70, AMA-1, Pfs25, Pfs16, PfSSP2, LSA-1, LSA-1-repeatless, MSA-1, CSP, MSA-1 N-terminal p83 or MSA-1 C-terminal gp42 gene.
  • Plasmodium genes are co-expressed in the host by the recombinant inventive poxvirus, NYVAC e.g., CSP, PfSSP2, LSA-1-repeatless, MSA-1, SERA, AMA-1 and Pfs25; in combination with at least one or both of the vaccinia host range gene K1L and DNA encoding Flagellin, or at least an operable binding portion of Flagellin.
  • inventive poxvirus e.g., CSP, PfSSP2, LSA-1-repeatless, MSA-1, SERA, AMA-1 and Pfs25
  • the recombinant poxvirus NYVAC contains the K1L gene providing the capacity for limited replication in humans, yet retaining attenuated virulence; and, this NYVAC contains DNA coding for and expresses the CSP, PfSSP2, LSA1-repeatless, MSA-1, SERA, AMA-1, Pfs25, ABRA, Pfhsp70, or Pfs16, P. falciparum antigens, and advantageously this NYVAC that contains K1L and the foregoing DNA encoding P. falciparum antigens also contains DNA encoding Flagellin, or an operable binding portion of Flagellin.
  • the invention comprehends recombinant poxviruses, e.g., NYVAC, expressing one or more or only some of these P. falciparum antigens, as well as Flagellin or an operable binding portion thereof and/or K1 L.
  • poxviruses e.g., NYVAC
  • the foregoing P. falciparum antigens individually or in combinations can be expressed by single poxvirus vectors (e.g., NYVACs) that also contain and express Flagellin, or an operable binding portion of Flagellin and also advantageously K1L, and these single poxvirus vectors can be used in combination with each other in an immunogenic, immunological or vaccine composition.
  • the invention also comprehends poxvirus, e.g., NYVAC single recombinants expressing the CSP, PfSSP2, LSA1-repeatless, SERA, or MSA-1 N-terminal p83 and C-terminal gp42 processing fragments in combination with at least one of the genes K1L and flagellin or an operable binding portion of Flagellin.
  • poxvirus e.g., NYVAC single recombinants expressing the CSP, PfSSP2, LSA1-repeatless, SERA, or MSA-1 N-terminal p83 and C-terminal gp42 processing fragments in combination with at least one of the genes K1L and flagellin or an operable binding portion of Flagellin.
  • the invention is also directed to the methods of making and using the replication competent poxvirus expressing malaria or Plasmodium genes for the production of Plasmodium gene products, either in vivo or in vitro as well as to the recombinant gene products.
  • the invention relates to a composition for inducing an immunological response in a host animal inoculated with the composition.
  • the composition can include an adjuvant for the induction of innate immunity.
  • the composition can contain a synthetic or engineered or non-naturally occurring or recombinant poxvirus, e.g.
  • NYVAC that contains, advantageously in a nonessential region thereof, DNA encoding one or more antigens or immunogens, e.g., one or more Plasmodium antigens or immunogens, and Flagellin or an operable binding portion thereof, and optionally also K1L, as well as to methods for inducing such an immunological response in an animal by inoculating or administering to the animal the composition or a poxvirus of the composition.
  • the immunological response can be a protective immunological response and hence the composition can be a vaccine; but, it need not elicit a protective immune response and can be an immunogenic or immunological composition.
  • DNA in the poxvirus codes for and the poxvirus expresses one or more and advantageously all of SERA, ABRA, Pfhsp70, AMA-1, Pfs25, Pfs16, PfSSP2, LSA-1, LSA-1-repeatless, MSA-1, CSP, MSA-1 N-terminal p83 and MSA-1 C-terminal gp42 of Plasmodium , in combination with the Flagellin or at least an operable binding portion of Flagellin, and K1L.
  • a portion of Flagellin that is essential to trigger the TLR5 PAMP is an operable binding portion of Flagellin.
  • a poxvirus a plurality of Plasmodium genes is advantageously co-expressed in the host or animal, e.g., CSP, PfSSP2, LSA-1-repeatless, MSA-1, SERA, AMA-1, and Pfs25; and preferably the poxvirus contains the host range gene K1L and also expresses Flagellin or an operable binding portion thereof; and, preferably the poxvirus is a NYVAC poxvirus.
  • a poxvirus has the capacity for limited replication in mammals, e.g., humans while retaining the attenuated virulence profile. Accordingly, animals or hosts in this description are advantageously mammals, such as humans.
  • FIG. 1 shows primer locations with regard to Example 1.
  • FIG. 1 discloses “IKSRR” as SEQ ID NO: 6.
  • FIG. 2A shows the K1L expression cassette nucleotide sequence (SEQ ID NO: 20).
  • FIG. 2B is a diagram of the K1L expression cassette for insertion between the XhoI and SpeI sites in FIG. 1 for generation of Pf7.2
  • FIGS. 3A , 3 B, 3 C, 3 D contain the results of expression by inventive recombinants.
  • vaccinia provides an exciting new avenue for the generation of recombinant vaccines, perhaps with the potential to be the “universal immunization vehicle”.
  • Recombinant vaccinia vectors were rapidly embraced by the veterinary industry for the development of new vaccine technologies. (Yilma T D. Vaccine 1989; 7: 484-485, Brochier B. Nature 1991; 354: 520-22, Wiktor, TJ. Proc. Natl Acd. Sci. 1984; 81: 7194-8, Rupprecht, C E. Proc.
  • MVA Modified Vaccinia Ankara
  • NYVAC Two highly attenuated strains of vaccinia, Modified Vaccinia Ankara (MVA) and NYVAC have emerged as two of the most predominately studied, non-replicating vectors in human tissues. Recombinants of both MVA and NYVAC have been extensively studied pre-clinically and many have made their way through late phase II/III clinical trials. Both viruses have been extensively studied and characterized at the genomic level.
  • MVA was developed during the 1970s, by high serial passage of vaccinia Ankara on primary chicken embryo fibroblasts (CEF).
  • CEF primary chicken embryo fibroblasts
  • the NYVAC strain was derived from a plaque isolate of the Copenhagen strain of vaccinia by the precise deletion of 18 open reading frames (ORFs) that were implicated in pathogenesis, virulence and host range regulatory functions.
  • ORFs open reading frames
  • One method of enhancing expression of target antigens from NYVAC is to re-engineer NYVAC to allow the virus to proceed later into the infectious cycle, potentially providing some limited level of avirulent replication.
  • replication competence does not have to exclude attenuation.
  • this level of replication would be enhanced compared to NYVAC but less than that obtained from the parent Copenhagen strain.
  • More robust replication and expression may provide more antigen load for processing and importantly, better mimicking of the naturally occurring viral infectious cycle, potentially triggering stronger innate immune responses.
  • Virally encoded genes that were specifically deleted from Copenhagen to generate NYVAC or lost upon serial passage in primary chick cells in generating MVA, were predominately viral gene functions that had evolved particularly for the modulation and or inhibition of antiviral host immune responses. Such factors are referred to as pathogenicity factors. These factors can determine viral host range, pathology and virulence in a given host. (McFadden G. Nat. Rev. 2005; 3: 201-13.) The focus of a large body of research has been devoted to study these virulence factors and their importance in determining host range. The understanding of how these host range genes interact with specific host targets has elucidated functionality with respect to viral pathogenesis and the abrogation of specific host immune responses.
  • C7L and K1L previously identified have been the obvious targets of choice to reinsert back into the attenuated genome of NYVAC to enable the virus to proceed further into its replicative cycle.
  • C7L is known to inhibit host antiviral action induced by type I interferons. (Meng, X J. Virol. 2009; 83:10627-636, Backes, S. J. Gen. Virol.
  • C7L and K1L inhibits the phosphorylation of eIF2 alpha and the induction of apoptosis, through the inhibition of PKR activity in infected cells.
  • PKR activity infected cells.
  • the NYVAC vector containing both C7L and K1L was found to be replication competent in a variety of different cultured human cells. Importantly, (NYVAC+C7L, K1L) was found to still retain the highly attenuated phenotype in comparison to wild type replication competent strains, such as Copenhagen and NYCBH. Bio-distribution analysis indicated that other genomic modifications such as the deletion of B19R allowed for further attenuation compared to (NYVAC+C7L, K1L), potentially through the activation of PKR through INF I activation, resulting in the induction of the pathogen-associated molecular pattern (PAMP) sensors. (Kibler, KV. 2011)
  • a specific inventive embodiment of the invention is NYVAC or another attenuated (as to mammals) poxvirus containing the Host Range gene K1L, e.g., NYVAC vectors modified to contain the host range gene K1L (NYVAC+K1L) so that these vectors are further developed to specifically replicate in human tissues to a level intermediate of that of the more virulent parental replication competent strain Copenhagen and the replication deficient stain NYVAC or MVA or MVA-BN or canarypox or fowlpox or ALVAC or TROVAC, and to co-express at least one vaccine target antigen(s).
  • a vector also contains DNA coding for and expresses Flagellin or an operable binding portion thereof.
  • Prime boost is routinely used in vaccination protocols to increase the immune response.
  • Classical immune studies have shown that the immune system once activated and allowed to rest then reactivated will result in a significant boost to both B-cell and T-cell responses.
  • strong anti-vector immune response induced by the priming vaccine
  • the boost vaccine is pondered before viral expression from host cells can express foreign protein, along with immune signals and therefore provides little advantage to the original vaccination.
  • it is critical to use a different vector for the boost.
  • Prime boost protocols are well known in the art. (Hu, S L. Science 1992; 255: 456-459, Richmond, J F L. Virology 1997; 230: 265-274, Brown, S. Viruses 2010; 2: 435-467) Prime boost regimens expressing the antigens of interest from another vector system, such as DNA, then boosting with the recombinant virus vector have enhanced vaccine efficacy (Ramsay, A J. Immunol. Cell Biol. 1997; 75: 382-388, Ramshaw, I A. Immunol. Today 2000; 21: 163-165, Estcourt, M J. Int. Immunol. 2002; 14: 31-37, Hodge, J W. Can. Res.
  • T-cell co-stimulation Another approach to optimizing immunization is through T-cell co-stimulation.
  • T-cell activation depends on the interaction of MHC peptide complexes with T-cell receptors, along with the interaction of co-stimulatory molecules with antigen presenting cells (APC) and the corresponding receptors on T-cells.
  • APC antigen presenting cells
  • Co-stimulation is particularly important when expressed antigens are only poorly immunogenic such as TAAs.
  • co-stimulatory molecules such as B7.1, the ligand for T-cell surface antigens CD28 and CTLA-4 and a triad of human co-stimulatory molecules (TRICOM) have been studied extensively. (Damle, NK. J. Immunol, 1992; 148: 1985-92, Hodge, J W. Can. Res.
  • the rational for using an immune adjuvant is to enhance the immune response to a vaccine by interaction with Antigen Presenting Cells (APCs) and T-cells.
  • APCs Antigen Presenting Cells
  • T-cells T-cells.
  • cytokines such as GM-CSF, IL-2, and FLT-3 ligand
  • DCs dendritic cells
  • the co-expression of cytokines has been highly utilized in oncology based vaccines, to boost responses, again to poorly immunogenic TAAs.
  • Immune responses have been classically categorized into innate and adaptive immunity. Adaptive responses are further subdivided into cellular and humoral. In comparative analysis of innate and adaptive responses, adaptive immunity is driven by the specificity of the T-cell and B-cell antigen specific receptors resulting in further induction of immune cell, cytokine and antibody trafficking to converge on the invading pathogen. Additionally, memory T and B-cell responses are generated so that any subsequent adaptive response to the same pathogen can be more rapidly regenerated (Janeway 2002). Innate immunity is found in all vertebrates. Originally, innate responses were viewed as a vestige of ancient host defenses and were simply used as an immediate host defense, a temporary and highly non-specific reaction until more important adaptive responses could take over.
  • TLRs Toll-Like Receptors
  • TLRs are type one integral membrane glycoproteins, with an excellular domain having a leucine rich repeat region (LRR) and a cytoplasmic signaling domain.
  • LRR domain is important for ligand binding.
  • TLR-4 specific TLRs (TLR-4) were involved with the recognition of lipopolysaccharide (LPS), the cell wall component of gram-negative bacteria.
  • LPS lipopolysaccharide
  • PAMP Pathogen-Associated Molecular Pattern
  • TLRs can recognize a variety of components derived from bacteria and viral pathogens.
  • LPS a cell wall component
  • bacterial and viral DNA are recognized through (CpG) by TLR-9 (Hemmi, H. Nature 2000; 408: 740-5), ssRNA by TLRs 7 and 8 (Hemmi, H. Nat. Immunol. 2002; 3: 196-200, Diebold, S.
  • TLR-3 Alexopoulou, L. Nature 2001; 413: 732-38
  • bacterial proteins such as Flagellin, a component of bacterial Flagella.
  • Flagella are responsible for bacterial motility, and are detected by TLR-5 (Hayashi, F. Nature 2001; 410: 1099-1103, Uematsu, S. Nat. Immunol. 2006; 7: 868-874.)
  • TLRs can be divided as to cellular localization, TLR 1,2,4-6 are on the cell surface, TLR 3,7-9 are within endosomes. (Kumar H. Biochem. Biophy. Res. Commun. 2009; 388: 621-5).
  • TIR-Domaincontaining Inducing interferon-B TIR-Domaincontaining Inducing interferon-B
  • MyD88 Myeloid Differentiation Primary Response Gene
  • TLRs were involved in controlling viral infection came from the finding that some viruses expressed genes specifically targeting and blocking TLR signaling responses. TLRs have been shown to be involved in antiviral responses to a wide variety of virus families, in context with many different viral macromolecules; the list is long and reviewed extensively (Carty, M. Clinical and Exp. Immunol. 2010; 161: 397-406). Plasmacytoid dendritic cells (pDC) are specialized immune cells that produce type I IFN and are critical for antiviral responses. (Gilliet M. Nat. Rev. Immunolo. 2008; 8: 594-606, Theofilopoulos A N. Ann. Rev. Immunol. 2005; 23: 307-36).
  • TLRs 7 and 9 signaling by viral nucleic acids in the endosome promotes activation of pDCs.
  • TLR9 detects CpG in DNA
  • TLRs 7 and 8 detect G/U rich ssRNA.
  • TLR 7-9 signaling is mediated through adaptor MyD88.
  • Vaccinia has been shown to activate pDCs upon infection.
  • human cells such as monocytes, macrophages and keratinocytes
  • activation of NF-KB is mediated through TLR 2, 3 and 4.
  • TLR 2 3 and 4.
  • A/T rich viral DNA was detected by TLR 8, resulting in INF responses from activated pDCs.
  • this response in mice was shown to be independent of TLR-9.
  • human pDCs do not express TLR8, only TLR-7 and 9.
  • human conventional DCs do express TLR8 and these may play a role in IFN responses.
  • vaccinia encodes several genes targeting modes of TLR signaling. A46R has been shown to inhibit the activity of MyD88, while A52R and C4L inhibits TLR mediated NF-KB activation. (Stack, J. J. Exp. Med. 2005; 201: 1007-18, Maloney, G. J. Biol.
  • TLRs lie at the forefront of the host defense system, and provide a system wide network for the detection of pathogens. In humans, the network of 10 different expressed TLRs have been determined for a variety of different cell types. Importantly, TLRs are found not only on cells of the immune system but are also expressed on epithelial cells of the intestine, urogenital and respiratory tracts, areas potentially important to the site of invading pathogens. (Guillot, L. J. Bio. Chem. 2004; 280: 5571-80, Vora, P. J. Immunol.
  • TLR expression profile by cell type has been well established; mDCs express TLRs (1-6, 8), pDCs express TLR (7, 9), neutrophils express TLR (1, 2, 4-10), NK cells express TLR1, monocytes express all except TLR3, B-lymphocytes express TLR (9,10), activated T-cells express TLR 2, regulatory T-cells express TLR (8, 10).
  • mDCs express TLRs (1-6, 8)
  • pDCs express TLR (7, 9)
  • neutrophils express TLR (1, 2, 4-10)
  • NK cells express TLR1, monocytes express all except TLR3, B-lymphocytes express TLR (9,10), activated T-cells express TLR 2, regulatory T-cells express TLR (8, 10).
  • TLR activating components for vaccine optimization; as to site or route of vaccine inoculation (dermal, mucosol intranasal), type of desired immune response (cellular, humoral or both, TH 1 or 2), type of vaccine (subunit, viral or bacterial, live, killed).
  • Innate immune activation is optimized for each class of infection that directs appropriate acquired immune responses to similar infections.
  • Most immune responses to antigens expressed by a viral vector, such as NYVAC would be expected to be anti-viral.
  • novel inclusion of a bacterial PAMP, such as flagellin, to a viral vector would induce the expected antiviral responses plus an additional array of anti bacterial responses—this vaccine induction of two classes of innate responses should enhance the vigor and breadth of immune responses when encountering Plasmodium infection with induction of both antiviral and antibacterial responses (an unnatural response dictated by the nature of the novel vaccine).
  • innate Plasmodium immune activators may be used to further enhance vaccine efficacy.
  • Flagellin is the integral component of Flagella, structures that certain bacteria have that are responsible for motility. In isolates of Salmonella there are two genes that encode the flagellar antigens. FliC encodes phase I flagellin and FljB encodes phase II flagellin. (Zieg J. Science 1977; 196: 170-2.) Both the FliC and FljB encode N and C domains that form part of the flagellar structure. (McQuiston J R. J. Clin. Microbio. 2004; 42: 1923-32.) Importantly, both contain motifs that are recognized by TLR5.
  • Flagellin the ligand for TLR5
  • Flagellin has been shown to be an effective adjuvant in physical association (formulation mixtures) within vaccine antigen preparations, or expressed as a fusion with targeted antigens or lastly, co-incorporated into viral particles with target antigens such as in virus-like particles, (VLPs).
  • the invention accordingly include: Coexpression by a recombinant or synthetic or engineered or non-naturally occurring poxvirus of one or more exogenous or heterologous antigens or immunogens and one or more PAMP modulators as an adjuvant.
  • the invention comprehends a poxvirus vector developed to specifically express the Flagellin PAMP responsible for activation of TLR5 for enhanced adaptive immune responses to co-expressed antigen(s) or immunogen(s).
  • the poxvirus is an attenuated (as to mammals) poxvirus, such as NYVAC, MVA, MVA-BN, canarypox, fowlpox, ALVAC, TROVAC.
  • the poxvirus contains DNA coding for and expresses the entire or operable binding portion of the bacterial protein Flagellin.
  • the operable binding portion of the Flagellin is the portion responsible for binding to and activating the TLR5 receptor, resulting in a cascade of immune stimulatory pro-inflammatory responses to the targeted vaccine antigen.
  • the Flagellin or operable binding portion is expressed either as peptide or fusion with antigen(s) or immunigen(s) provides for a multiplicity of options; the key to Flagellin or operable binding portion thereof expression is that the Flagellin operably and specifically agonize TLR5 to further stimulate “adjuvant” adaptive immune responses to expressed antigen(s) or immunogen(s).
  • the invention thus comprehends a synthetic, engineered, recombinant or non-naturally occurring poxvirus, e.g., vaccinia, vector developed to specifically replicate in human tissues to a level intermediate of that of the parental replication competent strain, e.g., Copenhagen, and the replication deficient stain e.g., NYVAC, MVA, MVA-BN (e.g., via K1L being present in the vector) and further developed to co-express Flagellin or an operable binding portion thereof (e.g., to deliver the Flagellin PAMP responsible for activation of TLR5) and at least one antigen or immunogen for which an adaptive immune response is desired whereby the poxvirus provides agonist(s) for one or several TLRs, e.g., TLR5 and a resulting cascade of immune stimulatory pro-inflammatory responses to the antigen(s) or immunogen(s).
  • NYVAC vectors are preferred.
  • Malaria is considered one of the most important parasitic diseases in the world. It is estimated that malaria caused over 200 million clinical episodes worldwide resulting in 655,000 deaths, mostly African children (WHO Global Malaria Program 2011). Furthermore the economic losses are magnified as most of the endemic countries are impoverished, costing some 3 billion dollars in Africa alone. (Teklehaimanot A. J. Trop. Med. Hyg. 2007; 77(6): 138-44. There have been substantial efforts and resources directed to methods and approaches for control-intervention such as indoor spraying, insecticidal nets, rapid diagnostics for testing, especially pregnant woman and children (Aponte J J. Lancet 2009; 374(9700): 1533-44., Menendez C. Lancet Infect. Dis. 2007; 7(2): 126-35).
  • Intensive malaria vaccine research has encompassed several decades and has yet to overcome substantial hurdles associated with complexities of the parasite life cycle, specifically, antigen expression during different parasite life stages and variability of antigens or important epitopes from different parasite isolates. Although many develop anti-parasitic immunity by repeated natural exposure, reproducing this by vaccination has been difficult. (Langhorne, J. Nat. Immunity 2008; 9: 725-32., Goodman A L. Ann. Trop. Med. Parasitol. 2010; 104: 189-211). Vaccine candidates have targeted the pre-erythrocytic liver stage, blood stage or transmission blocking stage. (Dubovsky F. In: Plotkin S A Vaccines 2004; p1283-9., Plos Med 2011; 8(1): e1000400., Aide P. Arch Dis. Child. 2007; 92(6): 476-9.)
  • pre-erythrocytic vaccines the immune response would direct antibodies to invading Plasmodium sporozoites delivered by mosquitoes and target infected liver cells with humoral and cellular immunity with the hope to prevent or limit parasites from entering red blood cells, thus avoiding clinical symptomology and any risk of further infection and transmission.
  • Pre-erythrocytic vaccines were the first attempted modern vaccines against malaria, and currently the basis of the GlaxoSmithKline (GSK) RTS,S vaccine, the furthest along the clinical pipeline currently in phase III trials. (Nussenzweig R S. Nature 1967; 216(5111): 160-2., Rieckmann K H. Bull. WHO 1979; 57(Suppl.
  • the GSK vaccine uses the central repeat region of the circumsporozoite protein (CSP) and hepatitis B surface antigen (HBsAg) as an immunogenic carrier. Efficacy results of the RTS,S in adults and children are reviewed. (Bojang K A. Vaccine 2005; 23(32): 4148-57., Macete E. Trop. Int. Med. Health 2007; 12(1): 37-46., Alonso P L. Lancet 2004; 364(9443): 1411-20., Alonso P L.
  • NYVAC-Pf7 directs immune responses to sporozoites, and all other life cycle stages, including induction of antibodies that have been shown to block Plasmodium transmission by mosquitoes-NYVAC-Pf7.1 and NYVAC-Pf7.2 are expected to enhance immunogenicity.
  • the asexual blood stage vaccines attempt to block parasite infection of red blood cells. It is this stage of rapid parasite replication that leads to the onset of clinical symptoms of the disease. Vaccines directed to this stage would only hope to limit or reduce the level of infection and therefore the severity of the symptoms, therefore blood stage vaccines should only be considered as part of a multi-component malaria vaccine. (Thera M A N. Jour. Med. 2011; 365(11): 1004-13).
  • Targeted blood stage antigens that have been evaluated are the apical membrane protein (AMA1), merozoite surface protein (MSP)1, 2 and 3, (SERA5) erythrocyte binding antigen 175(EBA 175), glutamine-rich protein long synthetic peptide (GRURP) and ring-infected erythrocyte surface antigen (RESA).
  • AMA1 apical membrane protein
  • MSP merozoite surface protein
  • SERA5 erythrocyte binding antigen 175(EBA 175)
  • GURP glutamine-rich protein long synthetic peptide
  • RESA ring-infected erythrocyte surface antigen
  • Antigenic variation of the blood stage antigens represents one of the biggest hurdles for vaccines directed to these antigens.
  • Ellis R D. Human Vaccines 2010; 6(8): 627-34 Naturally acquired immunity (or the bites of one thousand irradiated mosquitoes) induces resistance to Plasmodium infection—this encourages development of novel vaccines such as NYVAC-Pf7.1.
  • Sexual Stage vaccines or transmission blocking vaccines are vaccines that target the sexual stage of Plasmodia by blocking the fertilization of gametes in the mosquito midgut, thus preventing further development in the vector and subsequent rounds of new infections.
  • ingested sexual stage antibodies, complement and cytokines inhibit oocyst development in the vector.
  • P25 is the only sexual stage antigen to reach later stage vaccine clinical trials.
  • Additional transmission blocking vaccine targets would include antigens of the ookinete.
  • antibodies generated against the mosquito mid-gut antigen aminopeptidase-N(AgAPN 1) are effective in blocking ookinete invsion.
  • Specific embodiments of the invention include: Coexpression of Flagellin or an operable binding portion thereof and Plasmodium antigen(s) or immunogen(s), advantageously by a poxvirus vector, and more advantageously by a poxvirus vector that has reproductive capability via K1L.
  • poxvirus e.g., vaccinia
  • the invention comprehends poxvirus, e.g., vaccinia, vectors developed to specifically deliver the Flagellin PAMP responsible for activation of TLR5 for enhanced adaptive immune responses to co-expressed Malaria antigen(s).
  • the invention thus comprehends a non-naturally occurring or synthetic or engineered or recombinant poxvirus, e.g., vaccinia vector that contains DNA for and expression of multiple P.
  • the binding portion of Flagellin is the portion responsible for binding to and activating the TLR5 receptor, resulting in a cascade of immune stimulating pro-inflammatory responses to the co-expressed P. falciparum antigen(s).
  • falciparum antigen(s) provides for a multiplicity of options; the key is that the expressed Flagellin or portion thereof is operable to specifically agonize TLR5 to further stimulate adjuvant adaptive immune responses to co-expressed Malaria antigens.
  • a particularly preferred embodiment is a non-naturally occurring or recombinant or synthetic or engineered poxvirus, e.g., vaccinia, that co-expresses K1 L, Flagellin or an operable binding portion thereof and one or more Malaria antigen(s) or immunogen(s).
  • An enhanced NYVAC or MVA or MVA-BN vector replicates in human tissues to a level intermediate of that of the more virulent parental replication competent strain Copenhagen and the replication deficient stain NYVAC or MVA or MVA-BN.
  • an enhanced vector further co-express at least one P. falciparum antigen(s) or immunogen(s) for which adaptive immune responses are desired and the entire or a binding portion of the bacterial protein Flagellin (wherein the binding portion of the Flagellin is the portion responsible for binding to and activating the TLR5 receptor), a cascade of immune stimulating pro-inflammatory responses to the co-expressed P. falciparum antigen(s) or immunogen(s) results.
  • the Flagellin sequence and species and mode in which Flagellin is expressed is selected to specifically agonize TLR5 to further stimulate adaptive immune responses to P. falciparum.
  • the invention also comprehends P. falciparum antigen(s) or immunogen(s) co-expressed with Flagellin or an operable binding portion thereof in vitro. After infecting cells in vitro with an inventive recombinant, the expression products are collected and the collected malarial expression products can then be employed in a vaccine, antigenic or immunological composition which also contains a suitable carrier.
  • the viral vector system, especially the preferred poxvirus vector system, of the invention can itself be employed in a vaccine, immunological or immunogenic composition which also contains a suitable carrier.
  • the recombinant poxvirus in the composition expresses the malarial products and Flagellin or a binding operable portion thereof in vivo after administration or inoculation.
  • the poxvirus has some reproductive capacity, e.g., from K1L being present in an attenuated (as to mammals) poxvirus such as a NYVAC, ALVAC, TROVAC, MVA, MVA-BN, avipox, canarypox, or fowlpox.
  • the antigenic, immunological or vaccine composition of the invention either containing products expressed or containing a recombinant poxvirus is administered in the same fashion as typical malarial antigenic immunological or vaccine compositions (e.g., NYVAC-Pf7).
  • typical malarial antigenic immunological or vaccine compositions e.g., NYVAC-Pf7.
  • One skilled in the medical arts can determine dosage from this disclosure without undue experimentation, taking into consideration such factors as the age, weight, and general health of the particular individual.
  • the inventive recombinant poxvirus and the expression products therefrom stimulate an immune or antibody response in animals.
  • monoclonal antibodies can be prepared and, those monoclonal antibodies, can be employed in well known antibody binding assays, diagnostic kits or tests to determine the presence or absence of particular malarial antigen(s) and therefrom the presence or absence of malaria or, to determine whether an immune response to malaria or malarial antigen(s) has simply been stimulated.
  • Monoclonal antibodies are immunoglobulins produced by hybridoma cells. A monoclonal antibody reacts with a single antigenic determinant and provides greater specificity than a conventional, serum-derived antibody.
  • Embodiments of this invention include: NYVAC-PF7.1 (AMA1 repair+FliC) and NYVAC-PF7.2 (AMA1 repair+FliC+K1L).
  • NYVAC-PF7.1 AMA1 repair+FliC
  • NYVAC-PF7.2 AMA1 repair+FliC+K1L
  • Dry pellets of Salmonella enterica are readily available and were obtained from the University of New Hampshire (e.g., Robert Mooney).
  • the S. enterica coding sequence and flanking sequences were amplified using primers RW3 and RW4 then digested with BamHI and EcoRI generating a 1.5 kb fragment.
  • RW3 (SEQ ID NO: 1) TATTCAAGCTTGAATTCGTGTCGGTGAATCAATCG
  • RW4 (SEQ ID NO: 2) AACTCTAGAGGATCCAATAACATCAAGTTGTAATTG
  • the 1.5 kb BamHI-EcoRI fragment containing the FliC coding sequence was inserted into the 2.7 kbp BamHI-EcoRI fragment of plasmid pSV- ⁇ Gal (Promega, Madison, Wis.), yielding plasmid pRW2.
  • Pi promoted fragment was synthesized by IDT (Coralville, Iowa).
  • the Pi promoted synthetic fragment contained the 5′ and 3′ FIiC coding sequences. This fragment was inserted between the HindIII-XbaI of pZErO-2 (Invitrogen, Carlsbad, Calif.) yielding plasmid pRW8.
  • AMA coding sequences from in the original NYVAC-PF7 had several regions that needed to be modified for complete authentic AMAI expression. Firstly, the constructed repairs removed a 5-amino acid (RRIKS (SEQ ID NO: 5) also called IKSRR (SEQ ID NO: 6), both the same insert with reading from different ends) accidental insertion between amino acids 377 and 378 of AMA1, secondly, it was necessary to modify sequences encoding an early transcription termination signal (T5NT) found between nucleotide positions (1436-1442) in the AMA1 coding sequences and lastly to remove unnecessary DNA sequences 3′ of the original NYVAC-Pf7, AMA1 coding sequences. Preliminary experiments repairing IKSRR (SEQ ID NO: 6) demonstrated a change of small Pf7 plaques on CEF cells to an increase of plaque size approaching the size of NYVAC plaques.
  • RRIKS 5-amino acid
  • IKSRR early transcription termination signal
  • Plasmid pRW55 containing AMA1 repairs and Pi promoted FliC, was constructed in the following manner.
  • Full length Pi promoted FliC was constructed by insertion of a 1.3 kb pRW2 BbsI-KpnI fragment, containing the central coding portion of FIiC, between the BbsI and KpnI sites of pRW8 followed by PCR with the primers VC106/VC107.
  • the product of PCR from NYVAC with the primers VC68NC105 was combined with the VC106/107 fragment for PCR with the primers VC98NC106.
  • PCR fragments derived from Pf7 with the primer pairs VC110NC91, VC103/VC109 and VC108/104 were combined for PCR with the primers VC110NC104. Fragments derived with the primers VC98NC106 and VC110NC104 were combined for PCR with the primers VC97NC98, followed by digestion with Sail for insertion into the Sall site of pUC19 (Yanisch-Perron, C. Gene 1985; 33(1):103-19), yielding plasmid pRW55.
  • VC68 AATAGACCTGCTTCGTTGGCCTC (SEQ ID NO: 7)
  • VC91 AGCACTTTTGATCATACTAGCGTTCTTATTTTTG
  • VC97 CCTACAGGTCGACCATTACACCAGGAACATACATACC
  • VC98 CCTACAGGTCGACCATATCCGTTTTTGCCAATATCAC (SEQ ID NO: 10)
  • VC103 GAACGCTAGTATGATCAAAAGTGCTTTTCTTCCCACTGGTGCT (SEQ ID NO: 11)
  • VC104 TAGTCTCCTCGAGCTGACAGATCTATAAAAATTAATAGTATGGTTTTTCCATCAG
  • VC105 GATCTGTCAGCTCGAGGAGACTAGTCGTAGGGCCCGGCCGTGGCAATATTCTGTA
  • VC106 GATGGAAAAACCATACTATTAATTTTTATAGATCTACTGTAAAAATAGA
  • FIG. 1 illustrates primer locations.
  • the vaccinia virus Copenhagen strain K1L promoted K1L coding sequence (Gillard et al., 1986) was synthesized at TOP Gene Technologies (Montreal, Canada) as a fragment similar to the BglII (partial)-HpaI fragment described in Perkus et al., 1989; XhoI was added to the 5′ end and SpeI was added 3′ of HpaI.
  • the synthetic DNA was inserted between the Ascl and Pad sites of an intermediate cloning shuttle pAPG10, yielding plasmid pK1L.
  • Plasmid pRW56 was constructed by insertion of the 1Kb XhoI-SpeI fragment from pK1L, containing the K1L expression cassette, between the XhoI and SpeI sites of pRW55.
  • the synthetic DNA sequence and its position are illustrated in FIGS. 2A , 2 B.
  • IVR In vivo recombination
  • donor plasmid 8 ug
  • Lipofectamine 2000 as per manufacturer specification (Invitrogen, Carlsbad, Calif.) into 1E6 poxvirus infected Vero cells using a multiplicity of infection (MOI) of 0.1.
  • Donor plasmid pRW55 was used in an IVR with NYVAC-PF7 to generate the recombinant NYVAC-PF7.1 containing AMA1 repairs plus FliC.
  • Donor plasmid pRW56 was used in an IVR with NYVAC-PF7.1 to generate NYVAC-PF7.2 containing AMA1 repairs, FliC plus K1 L.
  • PCR polymerase chain reaction
  • virus was serially diluted in 96 well plates. Between 1-10% of each single well was used in PCR analysis. Wells identified as positive by PCR were repeatedly serially diluted for several rounds of infection and further tested by PCR.
  • the second primer set contained sequences present in the original NYVAC-Pf7 that flanked the insertion site; input NYVAC-Pf7 control virus would yield a PCR fragment smaller than a NYVAC-Pf7.1 recombinant containing an insertion.
  • virus was further purified by plaquing under agarose (Perkus M. et al., 1993). Well isolated plaques were picked from agarose, amplified and screened by PCR with both sets of PCR primers. Purification sometimes requires more than one round of plaque purification under agarose.
  • FIGS. 3A , 3 B, 3 C, 3 D demonstrate expression by inventive recombinants.
  • construction of NYVAC-Pf7.1 did not involve modifications of Pfs25 or CSP; it is important to note increased expression levels demonstrated in the Figs that may be due to FliC survival signals expressed by the novel vaccine candidate NYVAC-Pf7.1 or by removal of potentially deleterious effects of RRIKS (SEQ ID NO: 5) from AMA 1.
  • FIG. 3A shows expression of P. falciparum CSP from cell lysates two days post infection: Lysates were separated by 10% SDS-PAGE for western blotting with colorimetric detection. Rabbit antibody (Alpha Diagnostics, San Antonio, Tex.) directed to P. falciparum CSP repeat sequence (NANP) 5 . Lanes: 1 & 4, NYVAC; 2 & 5, NYVAC Pf7.1; 3 & 6, NYVAC Pf7. Compared with lanes 1-3, 20% of lysates were loaded on lanes 4-6.
  • FIG. 3B shows expression of secreted P. falciparum CSP from infected cell media two days post infection: Cell media was separated by 10% SDS-PAGE for western blotting with colorimetric detection. Rabbit antibody (Alpha Diagnostics, San Antonio, Tex.) directed to P. falciparum CSP repeat sequence (NANP) 5 . Lanes: 1, NYVAC Pf7; 2, NYVAC; 3, NYVAC Pf7.1.
  • FIG. 3C shows expression of P. falciparum Pfs25 two days post infection: Lysates were separated by 10% SDS-PAGE for western blotting and colorimetric detection: Rabbit anti-Pfs25 antiserum (ATCC, Manassas, Va.). Lanes: 1, NYVAC Pf7 supernatant; 2, NYVAC Pf7 cell pellet; 3, NYVAC supernatant; 4, NYVAC Pf7.1 supernatant; 5, NYVAC Pf7.1 cell pellet; 6, NYVAC cell pellet; 7, uninfected cell pellet; 8, molecular weight marker.
  • FIG. 3D shows FliC expression two days post infection: 10% SDS-PAGE analyzed by western blotting and colorimetric detection.
  • Mouse anti-FliC BioLegend, San Diego, Calif.
  • Lane 1 NYVAC Pf7.1 cells; 2, NYVAC Pf7.1 supernatant; 3, NYVAC supernatant; 4, NYVAC Pr supernatant; 5 NYVAC P17 cells; 6 NYVAC cells.

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AU2015323736B2 (en) * 2014-09-26 2020-09-10 Bavarian Nordic A/S Methods and compositions for intra-nasal immunization with recombinant MVA encoding flagellin
WO2018237339A1 (fr) * 2017-06-22 2018-12-27 The Government Of The United States Of America As Represented By The Secretary Of The Army Vaccin contre le paludisme utilisant du plasmodium à base de nyvac
US12144853B2 (en) 2020-01-13 2024-11-19 University Of Washington Targeted vaccination in the liver
WO2023157880A1 (fr) * 2022-02-18 2023-08-24 国立大学法人金沢大学 Vaccin contre le paludisme et méthode de prévention/traitement du paludisme
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WO2009079564A2 (fr) * 2007-12-17 2009-06-25 Emory University Compositions immunogènes et leurs procédés d'utilisation
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