WO2018201017A1 - Dendritic cell targeted adenovirus for vaccination - Google Patents
Dendritic cell targeted adenovirus for vaccination Download PDFInfo
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- WO2018201017A1 WO2018201017A1 PCT/US2018/029900 US2018029900W WO2018201017A1 WO 2018201017 A1 WO2018201017 A1 WO 2018201017A1 US 2018029900 W US2018029900 W US 2018029900W WO 2018201017 A1 WO2018201017 A1 WO 2018201017A1
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- dendritic cell
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- virus
- adenoviral vector
- structural gene
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Definitions
- Sequence Listing which is a part of the present disclosure, includes a computer readable form and a written sequence listing comprising nucleotide and/or amino acid sequences.
- the sequence listing information recorded in computer readable form is identical to the written sequence listing.
- the subject matter of the Sequence Listing is incorporated herein by reference in its entirety.
- the present teachings relate to adenovirus vaccines against Zika virus, Dengue virus, yellow fever, Chikungunya virus, or West Nile virus.
- Dendritic Cells have the unique ability to prime immune responses. Few reports have shown either induction of innate immune responses following vaccination or correlation of such responses with protective responses.
- the innate immune system is the first line of defense against pathogens and is known to shape adaptive immune responses.
- Conventional myeloid DC cells link innate and adaptive immunity and are responsible for the initiation and regulation of immune responses against various antigens via the activation of T cells, natural killer cells and B cells.
- the cDC include phenotypically, ontogenically, and functionally different subsets and form a complex network capable of integrating multiple signals leading to immunity or tolerance.
- cDCl express CD141 (BDCA3), XCR1 (lymphotactin), Clec9A, and CADMl
- cDC2 express CDlc (BDCAl), CDl lc, and SIRPa (Dutertre, C.A., Cell Immunol., 2014, 291, 3-10).
- CD141 was used as a specific marker for cDCl, but recent data support the view that XCR1 is an exclusive identifier of cDCl population across species including non-human primates (NHP) (Dutertre, C.A., et al., Cell Immunol., 2014, 291, 3-10; Dutertre, C.A., et al, J.
- NHS non-human primates
- cDCl Human cDCl are present in blood, lymph nodes, bone marrow, and tonsil, and the latter contain a higher percentage of cDCl than cDC2 among isolated mononuclear cells. cDCl are more efficient than cDC2 in cross-presenting soluble antigens as well as necrotic virus-infected cells to CD8+ T cells (Jongbloed, S.L., et al., J. Exp. Med., 2010, 207, 1247-1260).
- the cDCl also have high expression of TLR3, produce IL-12p70 and IFNb, and have a superior capacity to induce Thl helper T cell responses, when compared with the cDC2 (Dutertre, C.A., et al., Cell Immunol., 2014, 291, 3-10; Jongbloed, S.L., J. Exp. Med., 2010, 207, 1247-1260).
- Adenoviral vector targeting to DC As discussed above, DC are key regulators of T cell responses, orchestrating innate and adaptive immune responses. We have explored the ability of Ad vectors to target DC. We hypothesized that enhanced and/or specific transduction of DC by Ad vectors could improve vaccine efficacy. To establish proof-of- concept, we exploited bi-specific retargeting adapters to retarget Ad vectors to CD40, a costimulatory molecule of the TNF receptor superfamily expressed on DC (Brandao, J.G., et al., Vaccine, 2003, 21, 2268-2272; Korokhov, N., et al., J. Virology, 2003, 77, 12931-12940; Pereboev, A.V.
- CD40-retargeted Ad vectors induced significantly enhanced antitumor immunity compared to conventional serotype Ad5-based vectors (Tillman, B.W., et al., Cancer Research, 2000, 60, 5456-5463, Hangalapura, B.N., et al., J. Gene Medicine, 2012, 14, 416-427). These results have been confirmed in a range of models and with distinct molecular species serving the role of bispecific adapter for Ad vector retargeting (Pereboev, A.V., Molecular Therapy, 2004, 9, 712-720; Williams, B.J., et al., PloS one, 2012, 7, e46981).
- Zika virus is a member of the genus Flavivirus, which also includes Dengue (DENV), yellow fever (YFV) and West Nile (WNV) viruses (Lazear, H.M., et al., J. Virol., 2016, 90, 4864-4875). Although the majority of human ZIKV infections result in
- ZIKV also can be spread by mother-to-child transmission (Martines, R.B., et al., Morbidity and Mortality Weekly Report, 2016, 65, 159- 160; Mlakar, J., et al., N. Engl. J. Med., 2016, 374, 951-958; Quicke, K.M., et al., Cell Host Microbe, 2016, 20, 83-90), sexual contact (Venturi, G., et al., Euro. Surveill., 2016, 21, 30148; Moreira, J., et al., Clin. Infect. Dis., 2016, 63, 141-142; D'Ortenzio, E. et al., N. Engl. J.
- ZIKV is an enveloped positive-sense RNA virus with a 10.7 kb genome.
- the genome encodes a polyprotein that is cleaved post-translationally into three structural proteins—the capsid (C), membrane precursor (prM), and envelope (E)--and seven non-structural (NS) proteins (Kuno, G., et al., Arch. Virol., 2007, 152, 687-696).
- the C protein binds to the viral RNA to form a nucleocapsid, prM prevents premature fusion with host membranes and forms heterodimers with the E protein that is essential for virion assembly.
- the pr region of the prM protein is subsequently cleaved by cellular furin-like proteases leaving the M-E proteins on mature virions (Zhu, Z. et al., Emerging Microbes & Infections, 2016, 5, e22).
- the E protein mediates cellular attachment, entry, and fusion (Mukhopadhyay, S. et al., Nat. Rev.
- the prM protein also is a target of the anti-flavivirus response, however anti-prM antibodies generally are non-neutralizing (de Alwis, R., et al., PLoS Negl. Trop. Dis., 2011, 5, el 188; Dejnirattisai, W., et al, Science, 2010, 328, 745-748).
- CD8 + T cell responses augmented vaccine immunity (Shrestha, B., et al, Vaccine 2008, 26, 2020-2033).
- DENV-specific CD4 + T cells can produce IFN- ⁇ , TNF-a, and IL-2, and this polyfunctionality correlated with an asymptomatic presentation (Hatch, S., et al., J. Infect. Dis., 2011, 203, 1282-1291; Mangada, MM., J. Infect. Dis., 2002, 185, 1697- 1703).
- the possible protective roles of T cell responses need to be considered and optimized in the design of ZIKV vaccines.
- Chikungunya virus is an alphavirus that has caused periodic but sporadic outbreaks in tropical Africa. Asia has recently (2005-2007) had the largest outbreak of this virus in recorded history. Over 260,000 cases ( ⁇ l/3 of the population) were reported in Reunion Island (France) (Pialoux, G., et al., Lancet Infect. Dis., 2007, 7, 319-327) with 1.39 million cases reported in India (Mavalankar, D., et al., Lancet Infect Dis., 2007, 7, 306-307).
- the disease usually involves weeks to months of debilitating arthralgia/arthritis, and can involve myalgia, fever, headache, nausea, vomiting and/or a rash (Brighton, S.W., et al., S. Afr. Med. J., 1983, 63, 313-315) with
- Clec9A targeting induces follicular T helper (TFH) cells that are essential for germinal center formation and crucial for generating long-lived plasma cells (LLPCs) and memory B cells (MBCs), which are required for durable and anamnestic antibody responses (Kato, Y. et al., J Immunol. 2015, 195, 1006-1014).
- TFH follicular T helper
- Adenoviral vector for flavi virus vaccine development can be vaccine platforms to stimulate innate and adaptive immune responses (Hartman, Z.C., et al, Virology, 2007, 358, 357-372; Huang, X., et al., Human Gene Therapy, 2009, 20, 293- 301 ; Lore, K., et al, J. Immunology, 2007, 179, 1721-1729).
- Ad2 and Ad5 the most commonly employed vectors are based on serotypes 2 and 5 (Ad2 and Ad5) (Abbink, P., et al., J.
- Ad5 vectors are utilized with clinical trials ongoing in the cancer vaccine and infectious disease fields (Gene Therapy Clinical Trials Worldwide, wiley.com). Ad vectors have been used for the development of DENV vaccines (Holman, D.H., Clin.
- Vaccine Immunol. 2007, 14, 182- 189) including a tetravalent vaccine expressing domain III of the E protein (E-DIII) from the four different DENV serotypes (Khanam, S., et al., Vaccine 2009, 27, 6011-6021).
- This vaccine candidate was tested using Ad5 vector as a priming immunization and DNA immunization as a boosting and induced neutralizing antibodies and T cell responses against all DENV serotypes.
- Murine models of ZIKV Recently, the Diamond laboratory and other groups have developed mouse models of ZIKV pathogenesis that recapitulate many features of human disease (Aliota, M.T., et al., PLoS Negl. Trop. Dis., 2016, 10, e0004750; Aliota, M.T., PLoS Negl. Trop. Dis., 2016, 10, e0004682; Lazear, H.M., et al., Cell Host Microbe, 2016, 19, 720- 730; Rossi, S.L., et al., Am. J. Trop. Med. Hyg., 2016, 94, 1362-1369).
- mice did not develop overt clinical illness after infection with contemporary clinical strains of ZIKV
- mice lacking the ability to produce or respond to type I interferon (IFN) e.g., Ifnarl '1' mice
- IFN type I interferon
- WT mice treated with a blocking anti-ifnar antibody (MAR1-5A3)
- MAR1-5A3 a less severe model of ZIKV pathogenesis which also resulted in replication of ZIKV in several organs was developed (Lazear, H.M., et al., Cell Host Microbe, 2016, 19, 720-730).
- the Diamond laboratory has generated an adapted ZIKV strain that causes significant morbidity and mortality in adult WT mice treated with a blocking anti-Ifnar antibody; this model allows for induction of vaccine- derived immune responses in WT immunocompetent mice, and then after administration of the anti-Ifnar antibody, a stringent challenge model of protection against ZIKV infection.
- the Diamond laboratory also has generated an in utero transmission model of ZIKV infection and pathogenesis.
- Ads recombinant adenoviral vectors
- Ad vectors poorly infect DCs because of a lack in expression of the Coxsackie and adenovirus receptor mediating infectious uptake (Stockwin, L.H., et al., J. Immunological Methods, 2002, 259, 205-215).
- This limitation could be overcome by using a bispecific adapter molecule that encompasses a fusion of an
- the present inventors have developed adenoviral vectors that can be targeted to dendritic cells (DCs) and can exhibit enhanced potency.
- the inventors have generated single domain camelid antibodies (sdAb) with specificity for DC cell surface markers such as Clec9A and CD40 and have also modified the adenoviral capsid to allow incorporation of camelid antibody (sdAb) targeting moieties, including sdAb moieties with specificities for DC cell surface markers such as Clec9A and CD40.
- an adenoviral vector of the present teachings can exhibit enhanced and selective transduction of dendritic cells with genes for foreign antigens or tumor antigens, which can provide enhanced potency compared to current adenoviral vaccines against viruses and other infectious agents (e.g., alpha viruses and flavi viruses such as Zika, Chikungunya, and Dengue) as well as cancer.
- viruses and other infectious agents e.g., alpha viruses and flavi viruses such as Zika, Chikungunya, and Dengue
- the present teachings provide for a camelid sdAb against a dendritic cell surface antigen, which can be a camelid sdAb against a dendritic cell surface antigen other than carcinoembryonic antigen or EGFR.
- the dendritic cell surface antigen can be Clec9A and a camelid sdAb can be against Clec9A.
- the dendritic cell surface antigen can be CD40 and a camelid sdAb can be against CD40.
- the present teachings provide for a dendritic cell- targeted adenovirus which can comprise a camelid sdAb against a dendritic cell surface antigen other than carcinoembryonic antigen and EGFR.
- the dendritic cell surface antigen can be Clec9A.
- the dendritic cell surface antigen can be CD40.
- the dendritic cell targeted adenovirus can be deleted for an E1A/B genetic region.
- the dendritic cell- targeted adenovirus can further comprise a nucleic acid sequence encoding a polypeptide heterologous to the adenovirus, wherein the sequence can be an insertion in the deleted EIA/B genetic region.
- a dendritic cell-targeted adenovirus can further comprise in the deleted El A/B genetic region a promoter heterologous to the adenovirus, wherein the promoter can be operably linked to the nucleic acid sequence encoding a polypeptide heterologous to the adenovirus.
- the heterologous promoter can be a cytomegalovirus promoter.
- the polypeptide heterologous to the adenovirus can be a structural polypeptide of a heterologous virus.
- the heterologous virus can be a flavivirus or an alphavirus.
- the flavivirus can be Zika virus, Dengue virus, yellow fever virus or West Nile Virus.
- the alphavirus can be Chikungunya virus.
- the heterologous virus can be Zika virus, Chikungunya virus or Dengue virus.
- the heterologous virus can be a Zika virus.
- the polypeptide can comprise, consist of or consist essentially of a full length prM gene and an ectodomain of an E gene of a Zika virus. In various configurations, the polypeptide can comprise, consist of or consist essentially of the E gene of a Zika virus. In various configurations, the polypeptide heterologous to the adenovirus can be a tumor antigen. In various configurations of the present teachings, a vaccine can comprise, consist of or consist essentially of a dendritic cell-targeted adenoviral vector in accordance with the present teachings.
- a dendritic cell targeted adenoviral vector can comprise a nucleic acid sequence encoding a fiber-fibritin chimeric shaft, a deletion of the adenoviral El A/B genetic region, a sequence encoding a camelid sdAb against a cell surface protein of a dendritic cell and a sequence encoding an antigen inserted in the deleted El A/B genetic region, wherein the antigen can be a heterologous virus structural gene or a tumor antigen.
- the heterologous virus structural gene can be Zika virus, Chikungunya virus, Dengue virus or West Nile virus structural gene.
- the heterologous virus structural gene can be at least one Zika structural gene.
- the at least one Zika virus structural gene can comprise, consist of or consist essentially of a full length prM gene and the ectodomain of an E gene of Zika.
- the at least one structural gene of Zika can be an E gene of Zika.
- the heterologous virus gene can be at least one Chikungunya virus structural gene.
- the at least one Chikungunya virus structural gene can be isolated from virus obtained in Reunion Island.
- the at least one Chikungunya virus structural gene can be inserted in the right hand of the genome.
- the at least one Chikungunya virus structural gene can be an El, E2, capsid gene of Chikungunya virus or a combination thereof. In various configurations, the at least one Chikungunya virus structural gene can be at least two Chikungunya virus structural genes. In various configurations, the at least one Chikungunya virus structural gene can be at least three Chikungunya virus structural genes. In various configurations, the at least one Chikungunya virus structural gene can be an El , E2 and capsid gene of Chikungunya virus.
- the camelid sdAb can be against Clec9A. In various configurations, the camelid sdAb can be against CD40. In various configurations, the dendritic cell targeted adenovirus can further comprise a sequence encoding a polypeptide targeting a cancer cell antigen, such as, without limitation, prostate specific membrane antigen (PSMA); Genbank
- a polypeptide targeting a cancer cell antigen can encode a PSMA.
- the PSMA can be a human PSMA.
- a vaccine can comprise, consist of or consist essentially of a dendritic cell-targeted adenoviral vector in accordance with the present teachings.
- a method of vaccinating a subject against a virus such as, without limitation, Zika, Chikungunya, or Dengue can comprise administering to a subject an adenovirus vector comprising a fiber-fibritin chimeric shaft, a deleted E1A/B genetic region, a camelid sdAb against a cell surface protein of a dendritic cell; and at least one heterologous viral structural gene encoded in the deleted EIA/B genetic region.
- the camelid sdAb against a cell surface protein of a dendritic cell can be against Clec9A.
- the camelid sdAb against a cell surface protein of a dendritic cell can be against CD40.
- an adenoviral vector of the present teachings can comprise a sequence encoding a fiber-fibritin chimeric shaft, a deleted EIA/B genetic region, a sequence encoding a camelid sdAb against a cell surface protein of a dendritic cell, and a sequence encoding an antigen inserted in the deleted EIA/B genetic region, wherein the antigen can be a structural gene of a heterologous virus.
- the heterologous virus structural gene can be an El gene, an E2 gene, a capsid gene, a prM gene or a combination therof.
- an adenoviral vector of the present teachings can comprise a sequence encoding a fiber-fibritin chimeric shaft, a deleted EIA/B genetic region, a sequence encoding a camelid sdAb against a cell surface protein of a dendritic cell, and a sequence encoding an antigen inserted in the deleted El A/B genetic region, wherein the antigen can be a tumor antigen.
- the tumor antigen can be PSMA, CEA, mesothelin or MUC.1.
- an adenoviral vector of the present teachings can comprise an N-terminal segment of Ad5 fiber tail sequence, at least 2 pseudorepeats of an Ad5 fiber shaft domain sequence, a portion of a third Ad5 fiber shaft domain sequence, a carboxy-terminal segment of a T4 fibritin bacteriophage trimerization domain sequence, a linker sequence and a camelid single chain antibody sequence against a dendritic cell surface antigen other than carcinoembryonic antigen and EGFR.
- the dendritic cell surface antigen can be C1ec9A.
- the dendritic cell surface antigen can be CD40.
- FIG. 1 illustrates an adenoviral vector targeted to dendritic cells.
- FIG. 2 illustrates sequences of selected sdAb that bind to murine DC markers CD40 and Clec9A.
- the top panel shows amino acid sequences and alignments of unique sdAb clones selected against mClec9A, and the bottom panel shows amino acid sequences and alignments of unique sdAb clones selected against mCD40 antigens.
- FIG. 3 illustrates the evaluation of sdAb binding to murine DC markers CD40 and Clec9A by tracking ELISA binding of mClec9A.
- sdAb's JPQ-B4, JPQ-B9, JPQ-C3, JPQ-C5, JPQ-D10, JPQ-E5 and JPQ-G9 are against mClec9A; JPP-F8 is against CD40.
- FIG. 4 illustrates the evaluation of sdAb binding to murine DC markers CD40 and Clec9A by tracking ELISA binding of mCD40.
- sdAb's JPP-F8, JPP-G1 , and JPP-H7 are against Mcd40;
- JPQ-B4 is against mClec9A.
- FIG. 5 illustrates sdAbs generated against murine CD40 and Clec9A that exhibit strong binding and are able to cross-react with simian mDC.
- FIG. 6 illustrates ZIKV protein E expression using Ad5ZprM-E-ecto vector.
- FIG. 7 illustrates a structural comparison between wild type Ad5 fiber and a fiber- fibritin-ligand chimera, which comprises a phage T4 fibritin trimerization foldon to replace the Ad5 fiber knob.
- the sdAb Nbl.8 targeting ligand is fused to the foldon C-terminus.
- FIG. 8 illustrates Ad5FF1.8 vector targeting to DC via the fiber knob replacement with sdAb Nbl.8.
- Upper panels iBMDC monolayers infected with the indicated Ad vectors were imaged 40 h post infection using epifluorescence microscopy and representative images are shown at a magnification of 40*.
- Lower panels gene transfer levels in iBMDC transduced with Ad5FFl .8 or control Ad5 vector as measured by flow cytometry analysis of the percentage GFP+CD1 l c+ cells.
- FIG. 9 illustrates viremia measured by RT-PCR in a lethal challenge model of WT mice with ZIKV and protection with antibodies.
- FIG. 10 illustrates daily mouse weights in a lethal challenge model of WT mice with ZIKV and protection with antibodies.
- FIG. 11 illustrates survival analysis in a lethal challenge model of WT mice with ZIKV and protection with antibodies.
- FIG. 12 illustrates viremia in mice after challenge with the Reunion Island isolate of CHIK virus.
- FIG. 13 illustrates foot swelling after challenge with the Reunion Island isolate of CHIK virus.
- FIG. 14 illustrates viremia after challenge with the Asian isolate of CHIK virus.
- FIG. 15 illustrates foot swelling after challenge with the Asian isolate of CHIK virus.
- FIG. 16 illustrates an antitumor effect of a CD40-targeted Ad5-huPSMA vaccine determined using the RM-1-PSMA mouse model.
- the present inventors have developed adenoviral vectors that can be targeted to dendritic cells (DCs) and can exhibit enhanced potency.
- the inventors have generated single domain camelid antibodies (sdAb) with specificity for DC cell surface markers such as Clec9A and CD40 and have also modified the adenoviral capsid to allow incorporation of sdAb targeting moieties, including sdAb moieties with specificities for DC cell surface markers such as Clec9A and CD40. As illustrated in FIG.
- the adenoviral knob domain was deleted to ablate its broad tropism to CAR-expressing cells, and a 95 amino acid (aa) trimerization domain of the T4 phage fibritin protein was inserted to maintain fiber stability while allowing display of a targeting ligand binding such as an sdAb to an alternative receptor on the cell surface.
- This example illustrates generation of sdAb ligands binding Clec9A and CD40.
- PBMCs Peripheral blood mononuclear cells
- ELISA plates were coated with 1 ⁇ g/ml of either mClec9A protein (FIG. 3) or mCD40 protein (FIG. 4), blocked, and then exposed to a dilution series of each sdAb (VHH) at the indicated concentrations (nM). Binding was detected using secondary HRP-conjugated Ab against E-tag, which is present in the C-terminus of each sdAb protein. The optical absorbance values were detected at 450 nm (A450) and plotted versus the concentration of sdAb.
- the sdAb recognizing the other receptor target JPQ-B4 for mCD40 and JPP-F8 for mClec9A
- Positive sdAbs were also analyzed by flow cytometry for their DC-binding ability.
- the recognition of murine dendritic cells by anti-CD40 or anti-Clec9A sdAbs was tested, in both bone marrow-derived and spleen isolated dendritic cells.
- Commercial anti-Clec9A (Miltenyi Biotech Clone: 7H1 .1) and anti-CD40 (Biolegend Clone: 3/23) were used as controls. Cells were stained with individual sdAb (1 ⁇ /100 ⁇ 1) followed by a secondary PE- conjugated Ab against E-tag.
- FIG. 5 the left panel is a representative flow cytometry profile of Anti-CD40 sdAb (Unfilled Areas) in comparison to the staining by a commercially available a-CD40 monoclonal antibody (Filled Area) using bone marrow derived DCs.
- the center panel is a flow cytometry profile of anti- Clec9A sdAb.
- the dot matrix area shows a more effective binding cognition of the DCs subsets than the commercial monoclonal antibody tested (white).
- the right panel is a flow cytometric analysis of anti-Clec9A sdAb binding to rhesus macaque mDC.
- the anti-CD40 and anti-Clec9A sdAbs had a more efficient binding to the cells than the commercially available antibodies.
- the panel of generated sdAb against Clec9A showed a high degree of cross-reactivity with simian mDC.
- This example illustrates expression of ZIKV soluble E protein by recombinant Ad vector.
- Ad5 vector To validate the expression of ZIKV proteins by Ad5 vector we incorporated the DNA sequence encoding the full prM gene and the ectodomain of E of ZIKV (strain H/PF/2013 from French Polynesia) containing a heterologous N-terminal IL-2 signal peptide and C- terminal hexahistidine tag (6-His) under transcriptional control of cytomegalovirus (CMV) immediate early promoter in place of the early El A/B genes deleted in Ad5 genome.
- CMV cytomegalovirus
- This construct produces high levels of soluble E protein only, as prM/M is cleaved completely by the host signalase, and in the absence of the transmembrane domain of E, prM and E do not stably associate as heterodimers (Cockburn J. J., et al., EMBO J., 2012, 31, 767-779).
- the generated Ad5ZprM-E-ecto vector was used to infect A549 cells to validate the expression of secreted E protein. As shown in FIG. 6, the 6-His-tagged E protein band with molecular mass of approximately 48 kDa was detected 48 and 72 hours post-infection in both cell lysates and culture medium by Western blotting.
- A549 cells were infected with Ad5ZprM-E-ecto vector at a multiplicity of infection (MOI) of 900.
- the cells and culture medium samples mixed with Laemmli loading buffer, boiled, and run on 4-20% gradient SDS-PAGE as follows.
- Electrophoretically separated proteins were transferred to PVDF membrane and probed with anti-His tag and secondary anti-mouse AP-conjugated antibodies.
- the ZIKV protein E purified from culture medium of HEK293 cells transiently expressing the same CMV-driven prM-E-ecto plasmid was used as a positive control.
- This example illustrates Ad targeting to DCs via functional replacement of fiber knob with an sdAb ligand.
- Ad5 cellular entry is mediated by distinct binding and internalization events; the knob domain of Ad5 fiber initiates attachment through interactions with coxsackie virus and adenovirus receptor (CAR) expressed on epithelial cells (Bergelson, J.M., et al., Science
- Ad5FF1.8 showed enhanced gene transfer into DCs compared to the control Ad5 vector, as demonstrated by the markedly increased number of GFP-positive DCs (FIG. 8).
- sdAb Nbl.8 we transduced DCs with Ad5FF1.8 in the presence or absence of soluble Nbl.8. Increasing the concentration of soluble Nbl.8 resulted in a dose-dependent decrease of gene transfer by Ad5FF1.8 (data not shown). Without being limited by theory, these data demonstrate the feasibility of Ad vector targeting to DCs via genetic sdAb incorporation into viral capsid.
- This example illustrates the generation of sdAb ligands that bind Clec9A.
- This example illustrates mouse models of ZIKV pathogenesis.
- C57BL/6 mice were passively transferred 2 mg of anti-Ifnar 1 mAb and 250 ⁇ gs of the indicated mAbs (CHK-166, ZV-54, or ZV-57) via an intraperitoneal injection one day before subcutaneous inoculation with 10 5 FFU of adapted ZIKV Dakar 41519.
- FIG. 9 on day 3 after infection, serum was collected for analysis of viremia by qRT-PCR, and survival curves were constructed.
- the present methods thus allow for immunization in WT mice with a stringent requirement for protection against challenge.
- an African ZIKV strain is used, although concern is mitigated by immunization with an Ad encoding structural genes from a contemporary Asian (or American) isolate).
- This example illustrates CHIK challenge of CAdVax-CHIK immunized C57BL/6 mice.
- CAdVax-CHIK vaccine Wang, D., et al., J. Virol. 2006, 80, 2738-2746
- a single insert encoding the structural polyprotein (comprising the envelope glycoproteins El, E2 and capsid) of CHIKV was inserted in the right hand of the genome.
- An advantage of this configuration of the CAdVax is that it prevents the generation of replication-competent adenovirus through homologous recombination in the packaging cell line, HEK293, a common problem of first generation Ad5 vectors.
- the antigen sequences are from a CHIKV isolate from the recent epidemic on Reunion Island, or from an Asian isolate.
- the complete structural polyprotein gene was expressed in order to retain the native processing sequences.
- a single dose of the vaccine completely protected mice against viraemia and disease in recently developed adult wild-type mouse models of CHIKV-induced arthritis (Tsetsarkin, K.A., et al., PloS One, 2009, 4, e6835).
- FIG. 12 illustrates viremia after challenge with the Reunion Island isolate. Viremia was significantly different between CAdVax-CHIK and CAdVax-control vaccinated groups on days 1-3 (all p ⁇ 0.037, Mann Whitney U test).
- FIG. 13 illustrates foot swelling after challenge with the Reunion island isolate.
- FIG. 14 illustrates viremia after challenge with the Asian isolate. Viremia was significantly different between CAdVax-CHIK and CAdVax-control vaccinated groups on days 1-4 (all p ⁇ 0.014, Mann Whitney U test).
- FIG. 14 illustrates foot swelling after challenge with the Asian isolate. Swelling was significantly different between CAdVax-CHIK and CAdVax-control vaccinated groups on days 3-10 (all p ⁇ 0.04, Mann Whitney U test). Our DC targeting can improve these vaccine outcomes.
- This example illustrates the assessment of tumor growth after vaccine treatment with
- DC -targeted adenovirus vaccine DC -targeted adenovirus vaccine.
- RM-1-PSMA mouse model C57BL/6 mice were immunized by intraperitoneal injection of lxl 0 8 ifu of untargeted Ad5-huPSMA or CD40- targeted Ad5-huPSMA. The animals received a boost immunization at 10 days after the initial immunization. At the 14th day after the second immunization, the mice were challenged subcutaneously with 46105 RM-1 parental cells or RM-l-PSMA clone 1 cells. Three days later, the treatment groups were injected at the site of tumor cell injection with IxlO 8 ifu of Ad5-lFNy or with normal saline. At day 24 after initiation of the experiment, each mouse received 4xl0 6 RM-1 parental cells (which do not express the human PSMA antigen) or 4xl0 6 RM-l-PSMA clone 1 cells injected
- tumor volume 1 ⁇ 2 x (length x width 2 ) where length is the longest distance of the tumor.
- Each data point represents the mean volume of 15 tumors ⁇ standard error.
- immunization with CO40-targeted Ad5-huPSMA alone or with non-targeted Ad5-huPSMA + Ad5-IFNy similarly diminish tumor growth in animals challenged with RM- 1-PSMA cells.
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Abstract
Disclosed are camelid single domain antibody (sdAb) against dendritic cell surface antigens such as Clec9A and CD40. Also disclosed are chimeric adenoviruses which can be used as vaccines against a pathogen or cancer. A chimeric adenovirus targeted to dendritic cells incorporates a sequence encoding an sdAb against a dendritic cell surface antigen. A chimeric adenovirus includes a phage T4 fibritin trimerization foldon to replace the Ad5 fiber knob. Furthermore, the E1A/B genetic region of a chimeric adenovirus can be replaced with a heterologous sequence such as a structural gene from a heterologous virus or a sequence encoding a tumor antigen. Examples of heterologous viruses include flaviviruses such as Zika, Chikungunya, Dengue, yellow fever, and West Nile virus. Exemplary Zika sequences introduced into a chimeric adenovirus include full length prM, and the E ectodomain.
Description
Dendritic Cell Targeted Adenovirus for Vaccination
Reference to Prior Application
This application claims benefit of and priority to US Provisional Application
62/490,764 filed April 27, 2017. US Provisional Application 62/490,764 is hereby incorporated by reference in its entirety.
Incorporation by Reference of Sequence Listing
The Sequence Listing, which is a part of the present disclosure, includes a computer readable form and a written sequence listing comprising nucleotide and/or amino acid sequences. The sequence listing information recorded in computer readable form is identical to the written sequence listing. The subject matter of the Sequence Listing is incorporated herein by reference in its entirety.
Statement of Government Support
This invention was made with government support under grant All 31254 awarded by the National Institutes of Health. The government has certain rights in the invention.
Technical Field
The present teachings relate to adenovirus vaccines against Zika virus, Dengue virus, yellow fever, Chikungunya virus, or West Nile virus.
Introduction
Dendritic Cells (DC) have the unique ability to prime immune responses. Few reports have shown either induction of innate immune responses following vaccination or correlation of such responses with protective responses. The innate immune system is the first line of defense against pathogens and is known to shape adaptive immune responses. Conventional myeloid DC cells (cDC) link innate and adaptive immunity and are responsible for the initiation and regulation of immune responses against various antigens via the activation of T cells, natural killer cells and B cells. The cDC include phenotypically, ontogenically, and functionally different subsets and form a complex network capable of integrating multiple signals leading to immunity or tolerance. While much of cDC research has been carried out in mice, progress has been made in defining cDC subsets across species, including humans and monkeys (Khanam S., et al., Vaccine 2009, 27, 6011-6021). Similar to the mouse, humans and monkeys have two major subsets of cDC: cDCl and cDC2. Human cDCl express
CD141 (BDCA3), XCR1 (lymphotactin), Clec9A, and CADMl while cDC2 express CDlc (BDCAl), CDl lc, and SIRPa (Dutertre, C.A., Cell Immunol., 2014, 291, 3-10). initially, CD141 was used as a specific marker for cDCl, but recent data support the view that XCR1 is an exclusive identifier of cDCl population across species including non-human primates (NHP) (Dutertre, C.A., et al., Cell Immunol., 2014, 291, 3-10; Dutertre, C.A., et al, J.
Immunol, 2014, 192, 4697-4708). Human cDCl are present in blood, lymph nodes, bone marrow, and tonsil, and the latter contain a higher percentage of cDCl than cDC2 among isolated mononuclear cells. cDCl are more efficient than cDC2 in cross-presenting soluble antigens as well as necrotic virus-infected cells to CD8+ T cells (Jongbloed, S.L., et al., J. Exp. Med., 2010, 207, 1247-1260). The cDCl also have high expression of TLR3, produce IL-12p70 and IFNb, and have a superior capacity to induce Thl helper T cell responses, when compared with the cDC2 (Dutertre, C.A., et al., Cell Immunol., 2014, 291, 3-10; Jongbloed, S.L., J. Exp. Med., 2010, 207, 1247-1260).
Adenoviral vector targeting to DC. As discussed above, DC are key regulators of T cell responses, orchestrating innate and adaptive immune responses. We have explored the ability of Ad vectors to target DC. We hypothesized that enhanced and/or specific transduction of DC by Ad vectors could improve vaccine efficacy. To establish proof-of- concept, we exploited bi-specific retargeting adapters to retarget Ad vectors to CD40, a costimulatory molecule of the TNF receptor superfamily expressed on DC (Brandao, J.G., et al., Vaccine, 2003, 21, 2268-2272; Korokhov, N., et al., J. Virology, 2003, 77, 12931-12940; Pereboev, A.V. et al., Gene therapy, 2002, 9, 1189-1193; Pereboev, A.V., Molecular Therapy, 2004, 9, 712-720; Tillman, B.W. et al., J. Immunology, 1999, 162, 6378-6383; Tillman, B.W., et al., Cancer Research, 2000, 60, 5456-5463). Specifically, we retargeted Ad vectors to CD40 via retargeting "adapter" molecules. Utilizing a fusion between the ectodomain of the native Ad receptor (sCAR) and an anti-CD40 single chain antibody (scFv) we were able to show in vitro enhanced transduction and DC activation. In addition to enhancing DC transduction, targeting Ad vectors to CD40 appears to activate DC, resulting in enhanced antigen presentation and immune responses in vitro (Pereboev, A.V., Molecular Therapy, 2004, 9, 712-720; Tillman, B.W. et al., J. Immunology, 1999, 162, 6378-6383; Tillman, B.W., et al, Cancer Research, 2000, 60, 5456-5463). We then tested these adapter- retargeted Ad vectors in vivo, using m urine models of cancer vaccination (melanoma and prostate). Studies in several distinct murine models demonstrated that CD40-retargeted Ad
vectors induced significantly enhanced antitumor immunity compared to conventional serotype Ad5-based vectors (Tillman, B.W., et al., Cancer Research, 2000, 60, 5456-5463, Hangalapura, B.N., et al., J. Gene Medicine, 2012, 14, 416-427). These results have been confirmed in a range of models and with distinct molecular species serving the role of bispecific adapter for Ad vector retargeting (Pereboev, A.V., Molecular Therapy, 2004, 9, 712-720; Williams, B.J., et al., PloS one, 2012, 7, e46981). Previously we had defined genetic capsid modifications which allowed Ad tropism modification for retargeting applications. Using this strategy, we demonstrated that we could genetically modify the Ad capsid to allow incorporation of CD40L, the native ligand for CD40 (Belousova, N., et al., J. Virology, 2003, 77, 11367-11377; Korokhov, N., et al., Molecular Pharmaceutics, 2005, 2, 218-223). These genetically retargeted Ad vectors were able to transduce and activate DC similar to the adapter-retargeted Ad vectors described above. In addition, we demonstrated DC-specific targeting in vivo of these Ad vectors in a preclinical model based on explanted human skin plugs (Korokhov, N., et al., Molecular pharmaceutics, 2005, 2, 218-223). These studies have demonstrated the potential efficacy of targeting DC while revealing the lack of molecular flexibility and ease of manufacturing required for clinical translation. Previous studies using camelid antibodies against bone marrow dendritic cells, human
carcinoembryonic antigen and EGFR have demonstrated proof of concept for adenoviral cell targeting; however other dendritic cell surface markers have not been tested
(WO/2015/161314 by Curiel, D.T. et al.).
Zika virus (ZIKV) is a member of the genus Flavivirus, which also includes Dengue (DENV), yellow fever (YFV) and West Nile (WNV) viruses (Lazear, H.M., et al., J. Virol., 2016, 90, 4864-4875). Although the majority of human ZIKV infections result in
asymptomatic or mild illness, there is strong epidemiological evidence associating ZIKV infection during pregnancy with severe congenital defects including microcephaly and spontaneous miscarriage (Zika Virus Microcephaly and Guillain-Barre Syndrome Situation Report, apps.who.int; Cauchemez, S., et al., Lancet, 2016, 387, 2125-2132). The
unprecedented geographic expansion of the virus, with up to 1.3 million estimated human infections and 35 countries in the Americas reporting active ZIKV transmission (Zika Virus Microcephaly and Guillain-Barre Syndrome Situation Report, apps.who.int) has prompted the World Health Organization to declare that the ZIKV outbreak constituted a "Public Health Emergency of International Concern" (Gulland, A., BM J 2016, 352, i657). Besides the main
mode of ZIKV transmission through mosquitoes of the Aedes genus (Fernandez-Salas, I., et al., Antiviral Res., 2015, 124, 30-42). ZIKV also can be spread by mother-to-child transmission (Martines, R.B., et al., Morbidity and Mortality Weekly Report, 2016, 65, 159- 160; Mlakar, J., et al., N. Engl. J. Med., 2016, 374, 951-958; Quicke, K.M., et al., Cell Host Microbe, 2016, 20, 83-90), sexual contact (Venturi, G., et al., Euro. Surveill., 2016, 21, 30148; Moreira, J., et al., Clin. Infect. Dis., 2016, 63, 141-142; D'Ortenzio, E. et al., N. Engl. J. Med., 2016, 374, 2195-2198; Deckard, D.T., et al., M.M.W.R., 2016, 65, 372-374; Musso, D., et al., Emerging infectious diseases, 2015, 21, 359-361, and blood transfusion (Marano, G., Blood Transfusion, 2016, 14, 95-100; Musso, D. et al., Euro Surveill., 2014, 19, pii=20761). There is an urgent need to expedite preclinical development and clinical translation of vaccines that can control current and future outbreaks of ZIKV infection in Latin America and prevent its further geographic spread.
ZIKV is an enveloped positive-sense RNA virus with a 10.7 kb genome. The genome encodes a polyprotein that is cleaved post-translationally into three structural proteins—the capsid (C), membrane precursor (prM), and envelope (E)--and seven non-structural (NS) proteins (Kuno, G., et al., Arch. Virol., 2007, 152, 687-696). The C protein binds to the viral RNA to form a nucleocapsid, prM prevents premature fusion with host membranes and forms heterodimers with the E protein that is essential for virion assembly. The pr region of the prM protein is subsequently cleaved by cellular furin-like proteases leaving the M-E proteins on mature virions (Zhu, Z. et al., Emerging Microbes & Infections, 2016, 5, e22). The E protein, mediates cellular attachment, entry, and fusion (Mukhopadhyay, S. et al., Nat. Rev.
Microbiol. 2005, 3, 13-22). Based on the biology of other flaviviruses, the E protein represents an attractive target for vaccine development as neutralizing antibodies against the E protein mediate protection from disease (Pierson, T.C., et al., Cell Host Microbe, 2008, 4, 229-238). However, many of the most potently inhibitory anti-E antibodies against other flaviviruses (e.g., WNV and DENV) recognize quaternary epitopes that are present on E only when it is arranged on the icosahedral virion (Barba-Spaeth, G., et al., Nature, 2016, 536, 48- 53; Rouvinski, A. et al., Nature, 2015, 520, 109-113; Dejnirattisai, W. et al., Nat. Immunol., 2015, 16, 170-177; Vogt, M.R., et al., J. Virology, 2009, 83, 6494-6507; Kaufmann, B., et al., Proc. Natl. Acad. Sci. U.S.A., 2010, 107, 18950-18955; Fibriansah, G., et al., Science, 2015, 349, 88-91 ; Fibriansah, G., et al, Nat. Commun., 2015, 6, 6341 ; Fibriansah, G., et al., EMBO Mol. Med., 2014, 6, 358-371). The prM protein also is a target of the anti-flavivirus response,
however anti-prM antibodies generally are non-neutralizing (de Alwis, R., et al., PLoS Negl. Trop. Dis., 2011, 5, el 188; Dejnirattisai, W., et al, Science, 2010, 328, 745-748). Cryo- electron microscopy studies indicate that the ZIKV virion is highly mature, which would result in most of the pr antigen being cleaved in the late Golgi by furin-like proteases (Kostyuchenko, V.A., et al., Nature 2016, 533, 425-428; Sirohi, D., et al, Science, 2016, 352, 467-470). Along with neutralizing antibodies, T cell responses contribute to protection against flaviviruses. In DENV infections, CD84' T cells are associated with protection in patients living in hyperendemic areas (Weiskopf, D., et al., Proc. Natl. Acad. Sci. U.S.A., 2013, 110, E2046-2053), a role also observed in murine models (Yauch, L.E., et al., J.
Immunol, 2009, 182, 4865-4873). Analogously, in the context of an inactivated WNV virion- based vaccine, CD8+ T cell responses augmented vaccine immunity (Shrestha, B., et al, Vaccine 2008, 26, 2020-2033). DENV-specific CD4+ T cells can produce IFN-γ, TNF-a, and IL-2, and this polyfunctionality correlated with an asymptomatic presentation (Hatch, S., et al., J. Infect. Dis., 2011, 203, 1282-1291; Mangada, MM., J. Infect. Dis., 2002, 185, 1697- 1703). Thus, the possible protective roles of T cell responses need to be considered and optimized in the design of ZIKV vaccines.
Chikungunya virus (CHIKV) is an alphavirus that has caused periodic but sporadic outbreaks in tropical Africa. Asia has recently (2005-2007) had the largest outbreak of this virus in recorded history. Over 260,000 cases (~l/3 of the population) were reported in Reunion Island (France) (Pialoux, G., et al., Lancet Infect. Dis., 2007, 7, 319-327) with 1.39 million cases reported in India (Mavalankar, D., et al., Lancet Infect Dis., 2007, 7, 306-307). Numerous imported cases have also been reported in Europe (Fernandez-Salas, I., et al., Antiviral Res 2015, 124, 30-42; Hochedez, P., et al., Eurosurveillance, 2007, 12, pii=679), Asia (Lim, P.L., et al., J. Travel Medicine, 2009, 16, 289-291) and the United States (Centers for Disease C, M.M.W.R., 2007, 56, 276-277). No vaccine or effective drug for CHIKV disease is currently commercially available. The disease usually involves weeks to months of debilitating arthralgia/arthritis, and can involve myalgia, fever, headache, nausea, vomiting and/or a rash (Brighton, S.W., et al., S. Afr. Med. J., 1983, 63, 313-315) with
arthritis/arthralgia affecting 73-80% of patients (Pialoux, G., et al., Lancet Infect. Dis., 2007, 7, 319-327). Disease can persist for two years or more in some patients (de Andrade, D.C., et al., BMC Infectious Diseases, 2010, 10, 31; Larrieu, S., et al., J. Clin. Virol., 2010, 47, 85-88; Borgherini, G., et al., Clin. Infect. Dis., 2008, 47, 469-475). The recent epidemic involved the
emergence of a new clade of CHIKV within the large East-, Central-, and South-African phylogroup (Schuffenecker, I., et al., PLoS Med, 2006, 3, e263). Viruses within the new clade of CHIKV are efficiently transmitted by Aedes albopictus mosquito, whereas the usual vector for CHIKV is Aedes aegypti (Tsetsarkin, K.A., et al, PloS One, 2009, 4, e6835;
Tsetsarkin, K.A., et al., PLoS Pathog., 2007, 3, e201). The new viruses also appear to be associated with more severe disease in humans (Charrel, R.N., et al., The New England J. Medicine, 2007, 356, 769-771; Ng, L.F., et al., PloS One, 2009, 4, e4261). Viruses within this new clade have been associated for the first time with human mortality (Suryawanshi, S.D. et al., The Indian J. Medical Research, 2009, 129, 438-441; Economopoulou, A., et al., Epidemiology and Infection, 2009, 137, 534-541; Mavalankar, D., et al., Emerg. Infect. Dis., 2008, 14, 412-415).
Targeting of antigen to different molecules on APCs can polarize the immune response (Khanam, S., et al., Vaccine, 2009, 27, 6011-6021; Raviprakash, K., et al., J.
Virology, 2008, 82, 6927-6934). While targeting of vaccine antigens to MHC II molecules increases TH2 and IgGl antibody responses, targeting to chemokine receptor XCR1 enhances THI and IgG2 responses, in addition to CD8+ T cell responses (Grodeland, G., et al., PLoS One, 2013, 8, e80008; Grodeland, G., et al., Frontiers in Immunology, 2015, 6, 367;
Grodeland, G., et al., J. Immunol, 2013, 191, 3221-3231). The targeting to Clec9A+ DCs has been shown to enhance presentation of both MHC class I and Il-restricted antigens
(Caminschi, I., et al., Blood, 2008, 112, 3264-3273). Targeting of DCs via Clec9A also induced robust, long-lasting humoral responses even without adjuvants (Rossi, S.L., et al., Am. J. Trop. Med. Hyg., 2016, 94, 1362-1369) in mice and non-human primates (Li, J., et al., European Journal of Immunology, 2015, 45, 854-864). Clec9A targeting induces follicular T helper (TFH) cells that are essential for germinal center formation and crucial for generating long-lived plasma cells (LLPCs) and memory B cells (MBCs), which are required for durable and anamnestic antibody responses (Kato, Y. et al., J Immunol. 2015, 195, 1006-1014).
Adenoviral vector for flavi virus vaccine development: Adenovirus (Ad)-based vectors can be vaccine platforms to stimulate innate and adaptive immune responses (Hartman, Z.C., et al, Virology, 2007, 358, 357-372; Huang, X., et al., Human Gene Therapy, 2009, 20, 293- 301 ; Lore, K., et al, J. Immunology, 2007, 179, 1721-1729). Of the 51 known human Ad serotypes, the most commonly employed vectors are based on serotypes 2 and 5 (Ad2 and Ad5) (Abbink, P., et al., J. Virology, 2007, 81 , 4654-4663; Barouch, D.H., et al., Vaccine,
201 1 , 29, 5203-5209; Bassett, J.D., et al., Expert Rev. Vaccines, 2011, 10, 1307-1319). Ad5 vectors are utilized with clinical trials ongoing in the cancer vaccine and infectious disease fields (Gene Therapy Clinical Trials Worldwide, wiley.com). Ad vectors have been used for the development of DENV vaccines (Holman, D.H., Clin. Vaccine Immunol., 2007, 14, 182- 189) including a tetravalent vaccine expressing domain III of the E protein (E-DIII) from the four different DENV serotypes (Khanam, S., et al., Vaccine 2009, 27, 6011-6021). This vaccine candidate was tested using Ad5 vector as a priming immunization and DNA immunization as a boosting and induced neutralizing antibodies and T cell responses against all DENV serotypes. In rhesus macaques, tetravalent vaccination using a mixture of two bivalent Ad vectors encoding both the prM and the E proteins of DENV induced neutralizing antibodies and T cells responses against the 4 vaccination serotypes and protected monkeys against a live DENV challenge occurring at 4 or 24 weeks after two immunizations
(Raviprakash, K., et al., J. Virology, 2008, 82, 6927-6934).
Murine models of ZIKV. Recently, the Diamond laboratory and other groups have developed mouse models of ZIKV pathogenesis that recapitulate many features of human disease (Aliota, M.T., et al., PLoS Negl. Trop. Dis., 2016, 10, e0004750; Aliota, M.T., PLoS Negl. Trop. Dis., 2016, 10, e0004682; Lazear, H.M., et al., Cell Host Microbe, 2016, 19, 720- 730; Rossi, S.L., et al., Am. J. Trop. Med. Hyg., 2016, 94, 1362-1369). Whereas 4- to 6- week-old wild-type (WT) mice did not develop overt clinical illness after infection with contemporary clinical strains of ZIKV, mice lacking the ability to produce or respond to type I interferon (IFN) (e.g., Ifnarl'1' mice) developed severe neurological disease that was associated with high viral loads in the brain and spinal cord, chronic infection in the testes, and substantial lethality. In a complementary approach using WT mice treated with a blocking anti-ifnar antibody (MAR1-5A3), a less severe model of ZIKV pathogenesis which also resulted in replication of ZIKV in several organs was developed (Lazear, H.M., et al., Cell Host Microbe, 2016, 19, 720-730). These animals, however, survived infection and did not develop neurological disease. In more recent studies, the Diamond laboratory has generated an adapted ZIKV strain that causes significant morbidity and mortality in adult WT mice treated with a blocking anti-Ifnar antibody; this model allows for induction of vaccine- derived immune responses in WT immunocompetent mice, and then after administration of the anti-Ifnar antibody, a stringent challenge model of protection against ZIKV infection. In addition to these models, the Diamond laboratory also has generated an in utero transmission
model of ZIKV infection and pathogenesis. To evaluate ZIKV infection during pregnancy, Miner, J.J., et al., Cell, 2016, 165, 1081-1091 used Ifnarl 1' females crossed to WT males and used pregnant WT females treated with an anti-ifnar-blocking antibody. These studies revealed that ZIKV infects pregnant dams including the placenta, and can result in damage to the placental barrier, infection of the developing fetus, placental insufficiency and intrauterine growth restriction. In severe cases, ZIKV infection can lead to fetal demise. These findings establish models for studying mechanisms of in utero transmission and testing of vaccines that could prevent or mitigate intrauterine infection with ZIKV.
Previous reports have shown that ex vivo DC-based vaccines can induce specific antitumor T-cell responses in patients (Fishman, M., Expert Opinion on Biological Therapy, 2009, 9, 1565-1575; Waeckerle-Men, Y., et al., Cancer Immunology, Immunotherapy, 2006, 55, 1524-1533; Fuessel, S., et al., The Prostate, 2006, 66, 811-821 ; Mlakar, J., N. Engl. J. Med., 2016, 374, 951-958; Quicke, K.M., et al., Cell Host Microbe, 2016, 20, 83-90; Venturi, G., et al., Euro. Surveill., 2016, 21, 30148). Despite these clinical successes, this approach is limited from widespread clinical application because manipulating DCs through ex vivo culture and antigen loading is laborious, expensive, and time consuming. Likewise, ex vivo- prepared DCs show limited migration to the lymph nodes for subsequent activation of T-cells (Verdijk, P., et al., Expert Opinion on Biological Therapy, 2008, 8, 865-874). This problem has been addressed by in situ loading of DCs with tumor associated antigens using viral and non-viral vectors (Bolhassani, A., et al., Molecular Cancer, 2011, 10, 3). Among the viral vectors, recombinant adenoviral vectors (Ads) have received much attention for cancer therapy because of their high capacity and robust gene expression (Rein, D.T., et al., Future Oncology, 2006, 2, 137-143). Nonetheless, Ad vectors poorly infect DCs because of a lack in expression of the Coxsackie and adenovirus receptor mediating infectious uptake (Stockwin, L.H., et al., J. Immunological Methods, 2002, 259, 205-215). This limitation could be overcome by using a bispecific adapter molecule that encompasses a fusion of an
extracellular domain of the native Coxsackie and adenovirus receptor and the mouse CD40 ligand linked by a trimerization motif from the T4 bacteriophage fibritin protein (Pereboev, A.V. et al., Molecular Therapy, 2004, 9, 712-720; Brandao, J.G., et al., Vaccine 2003, 21, 2268-2272). More recently, this adapter was used successfully for DC-based immunotherapy in a mouse model of melanoma (Hangalapura, B.N., et al ., Cancer Research, 2011, 71 , 5827- 5837; Hangalapura, B.N., et al., J. Immunotherapy 2010, 33, 706-715). However, other
tumor/antigen combinations have not been tested.
Summary
The present inventors have developed adenoviral vectors that can be targeted to dendritic cells (DCs) and can exhibit enhanced potency. The inventors have generated single domain camelid antibodies (sdAb) with specificity for DC cell surface markers such as Clec9A and CD40 and have also modified the adenoviral capsid to allow incorporation of camelid antibody (sdAb) targeting moieties, including sdAb moieties with specificities for DC cell surface markers such as Clec9A and CD40. In various embodiments, an adenoviral vector of the present teachings can exhibit enhanced and selective transduction of dendritic cells with genes for foreign antigens or tumor antigens, which can provide enhanced potency compared to current adenoviral vaccines against viruses and other infectious agents (e.g., alpha viruses and flavi viruses such as Zika, Chikungunya, and Dengue) as well as cancer.
In some embodiments, the present teachings provide for a camelid sdAb against a dendritic cell surface antigen, which can be a camelid sdAb against a dendritic cell surface antigen other than carcinoembryonic antigen or EGFR. In some configurations, the dendritic cell surface antigen can be Clec9A and a camelid sdAb can be against Clec9A. In various configurations, the dendritic cell surface antigen can be CD40 and a camelid sdAb can be against CD40. In various configurations, the present teachings provide for a dendritic cell- targeted adenovirus which can comprise a camelid sdAb against a dendritic cell surface antigen other than carcinoembryonic antigen and EGFR. In some configurations, the dendritic cell surface antigen can be Clec9A. In various configurations, the dendritic cell surface antigen can be CD40. In various configurations, the dendritic cell targeted adenovirus can be deleted for an E1A/B genetic region. In various configurations, the dendritic cell- targeted adenovirus can further comprise a nucleic acid sequence encoding a polypeptide heterologous to the adenovirus, wherein the sequence can be an insertion in the deleted EIA/B genetic region. In various configurations, a dendritic cell-targeted adenovirus can further comprise in the deleted El A/B genetic region a promoter heterologous to the adenovirus, wherein the promoter can be operably linked to the nucleic acid sequence encoding a polypeptide heterologous to the adenovirus. In various configurations, the heterologous promoter can be a cytomegalovirus promoter. In various configurations, the polypeptide heterologous to the adenovirus can be a structural polypeptide of a heterologous virus. In various configurations, the heterologous virus can be a flavivirus or an alphavirus. In
various configurations, the flavivirus can be Zika virus, Dengue virus, yellow fever virus or West Nile Virus. In various configurations, the alphavirus can be Chikungunya virus. In various configurations, the heterologous virus can be Zika virus, Chikungunya virus or Dengue virus. In various configurations, the heterologous virus can be a Zika virus. In various configurations, the polypeptide can comprise, consist of or consist essentially of a full length prM gene and an ectodomain of an E gene of a Zika virus. In various configurations, the polypeptide can comprise, consist of or consist essentially of the E gene of a Zika virus. In various configurations, the polypeptide heterologous to the adenovirus can be a tumor antigen. In various configurations of the present teachings, a vaccine can comprise, consist of or consist essentially of a dendritic cell-targeted adenoviral vector in accordance with the present teachings.
In various embodiments, a dendritic cell targeted adenoviral vector can comprise a nucleic acid sequence encoding a fiber-fibritin chimeric shaft, a deletion of the adenoviral El A/B genetic region, a sequence encoding a camelid sdAb against a cell surface protein of a dendritic cell and a sequence encoding an antigen inserted in the deleted El A/B genetic region, wherein the antigen can be a heterologous virus structural gene or a tumor antigen. In various configurations, the heterologous virus structural gene can be Zika virus, Chikungunya virus, Dengue virus or West Nile virus structural gene. In various configurations, the heterologous virus structural gene can be at least one Zika structural gene. In various configurations, the at least one Zika virus structural gene can comprise, consist of or consist essentially of a full length prM gene and the ectodomain of an E gene of Zika. In various configurations, the at least one structural gene of Zika can be an E gene of Zika. In various configurations, the heterologous virus gene can be at least one Chikungunya virus structural gene. In various configurations, the at least one Chikungunya virus structural gene can be isolated from virus obtained in Reunion Island. In various configurations, the at least one Chikungunya virus structural gene can be inserted in the right hand of the genome. In various configurations, the at least one Chikungunya virus structural gene can be an El, E2, capsid gene of Chikungunya virus or a combination thereof. In various configurations, the at least one Chikungunya virus structural gene can be at least two Chikungunya virus structural genes. In various configurations, the at least one Chikungunya virus structural gene can be at least three Chikungunya virus structural genes. In various configurations, the at least one Chikungunya virus structural gene can be an El , E2 and capsid gene of Chikungunya virus.
In various configurations, the camelid sdAb can be against Clec9A. In various configurations, the camelid sdAb can be against CD40. In various configurations, the dendritic cell targeted adenovirus can further comprise a sequence encoding a polypeptide targeting a cancer cell antigen, such as, without limitation, prostate specific membrane antigen (PSMA); Genbank
Accession No. AAA60209.1,
encoding a polypeptide targeting a cancer cell antigen can encode a PSMA. In some configurations, the PSMA can be a human PSMA. In some configurations, a vaccine can comprise, consist of or consist essentially of a dendritic cell-targeted adenoviral vector in accordance with the present teachings.
In various embodiments, a method of vaccinating a subject against a virus such as, without limitation, Zika, Chikungunya, or Dengue can comprise administering to a subject an adenovirus vector comprising a fiber-fibritin chimeric shaft, a deleted E1A/B genetic region, a camelid sdAb against a cell surface protein of a dendritic cell; and at least one heterologous viral structural gene encoded in the deleted EIA/B genetic region. In some configurations, the camelid sdAb against a cell surface protein of a dendritic cell can be against Clec9A. In various configurations, the camelid sdAb against a cell surface protein of a dendritic cell can be against CD40.
In various embodiments, an adenoviral vector of the present teachings can comprise a sequence encoding a fiber-fibritin chimeric shaft, a deleted EIA/B genetic region, a sequence encoding a camelid sdAb against a cell surface protein of a dendritic cell, and a sequence encoding an antigen inserted in the deleted EIA/B genetic region, wherein the antigen can be a structural gene of a heterologous virus. In various configurations, the heterologous virus structural gene can be an El gene, an E2 gene, a capsid gene, a prM gene or a combination therof.
In various embodiments, an adenoviral vector of the present teachings can comprise a sequence encoding a fiber-fibritin chimeric shaft, a deleted EIA/B genetic region, a sequence encoding a camelid sdAb against a cell surface protein of a dendritic cell, and a sequence encoding an antigen inserted in the deleted El A/B genetic region, wherein the antigen can be a tumor antigen. In various configurations, the tumor antigen can be PSMA, CEA, mesothelin or MUC.1. In various embodiments, an adenoviral vector of the present teachings can comprise an N-terminal segment of Ad5 fiber tail sequence, at least 2 pseudorepeats of an
Ad5 fiber shaft domain sequence, a portion of a third Ad5 fiber shaft domain sequence, a carboxy-terminal segment of a T4 fibritin bacteriophage trimerization domain sequence, a linker sequence and a camelid single chain antibody sequence against a dendritic cell surface antigen other than carcinoembryonic antigen and EGFR. In various configurations, the dendritic cell surface antigen can be C1ec9A. In various configurations, the dendritic cell surface antigen can be CD40.
Brief Description of the Drawings
FIG. 1 illustrates an adenoviral vector targeted to dendritic cells.
FIG. 2 illustrates sequences of selected sdAb that bind to murine DC markers CD40 and Clec9A. The top panel shows amino acid sequences and alignments of unique sdAb clones selected against mClec9A, and the bottom panel shows amino acid sequences and alignments of unique sdAb clones selected against mCD40 antigens.
FIG. 3 illustrates the evaluation of sdAb binding to murine DC markers CD40 and Clec9A by tracking ELISA binding of mClec9A. sdAb's JPQ-B4, JPQ-B9, JPQ-C3, JPQ-C5, JPQ-D10, JPQ-E5 and JPQ-G9 are against mClec9A; JPP-F8 is against CD40.
FIG. 4 illustrates the evaluation of sdAb binding to murine DC markers CD40 and Clec9A by tracking ELISA binding of mCD40. sdAb's JPP-F8, JPP-G1 , and JPP-H7 are against Mcd40; JPQ-B4 is against mClec9A.
FIG. 5 illustrates sdAbs generated against murine CD40 and Clec9A that exhibit strong binding and are able to cross-react with simian mDC.
FIG. 6 illustrates ZIKV protein E expression using Ad5ZprM-E-ecto vector.
FIG. 7 illustrates a structural comparison between wild type Ad5 fiber and a fiber- fibritin-ligand chimera, which comprises a phage T4 fibritin trimerization foldon to replace the Ad5 fiber knob. The sdAb Nbl.8 targeting ligand is fused to the foldon C-terminus.
FIG. 8 illustrates Ad5FF1.8 vector targeting to DC via the fiber knob replacement with sdAb Nbl.8. Upper panels, iBMDC monolayers infected with the indicated Ad vectors were imaged 40 h post infection using epifluorescence microscopy and representative images are shown at a magnification of 40*. Lower panels, gene transfer levels in iBMDC transduced with Ad5FFl .8 or control Ad5 vector as measured by flow cytometry analysis of the percentage GFP+CD1 l c+ cells.
FIG. 9 illustrates viremia measured by RT-PCR in a lethal challenge model of WT mice with ZIKV and protection with antibodies.
FIG. 10 illustrates daily mouse weights in a lethal challenge model of WT mice with ZIKV and protection with antibodies.
FIG. 11 illustrates survival analysis in a lethal challenge model of WT mice with ZIKV and protection with antibodies.
FIG. 12 illustrates viremia in mice after challenge with the Reunion Island isolate of CHIK virus.
FIG. 13 illustrates foot swelling after challenge with the Reunion Island isolate of CHIK virus.
FIG. 14 illustrates viremia after challenge with the Asian isolate of CHIK virus. FIG. 15 illustrates foot swelling after challenge with the Asian isolate of CHIK virus. FIG. 16 illustrates an antitumor effect of a CD40-targeted Ad5-huPSMA vaccine determined using the RM-1-PSMA mouse model.
Detailed Description
The present inventors have developed adenoviral vectors that can be targeted to dendritic cells (DCs) and can exhibit enhanced potency. The inventors have generated single domain camelid antibodies (sdAb) with specificity for DC cell surface markers such as Clec9A and CD40 and have also modified the adenoviral capsid to allow incorporation of sdAb targeting moieties, including sdAb moieties with specificities for DC cell surface markers such as Clec9A and CD40. As illustrated in FIG. 1, the adenoviral knob domain was deleted to ablate its broad tropism to CAR-expressing cells, and a 95 amino acid (aa) trimerization domain of the T4 phage fibritin protein was inserted to maintain fiber stability while allowing display of a targeting ligand binding such as an sdAb to an alternative receptor on the cell surface.
Methods
The methods and compositions described herein utilize laboratory techniques well known to skilled artisans, and can be found in laboratory manuals such as Sambrook, J., et al., Molecular Cloning: A Laboratory Manual, 3rd ed. Cold Spring Harbor Laboratory Press,
Cold Spring Harbor, NY, 2001; Spector, D. L. et ah, Cells: A Laboratory Manual, Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1998; Nagy, A., Manipulating the
Mouse Embryo: A Laboratory Manual (Third Edition), Cold Spring Harbor, NY, 2003 and
Harlow, E., Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press,
Cold Spring Harbor, NY, 1999. Methods of administration of pharmaceuticals and dosage
regimes, can be determined according to standard principles of pharmacology well known skilled artisans, using methods provided by standard reference texts such as Remington: the Science and Practice of Pharmacy (Alfonso R. Gennaro ed. 19th ed. 1995); Hardman, J.G., et al., Goodman & Gilman's The Pharmacological Basis of Therapeutics, Ninth Edition, McGraw-Hill, 1996; and Rowe, R.C., et al., Handbook of Pharmaceutical Excipients, Fourth Edition, Pharmaceutical Press, 2003. As used in the present description and the appended claims, the singular forms "a", "an" and "the" are intended to include the plural forms as well, unless the context indicates otherwise.
Examples
The present teachings include descriptions provided in the examples that are not intended to limit the scope of any aspect or claim. Unless specifically presented in the past tense, an example can be a prophetic or an actual example. The following non-limiting examples are provided to further illustrate the present teachings. Those of skill in the art, in light of the present disclosure, will appreciate that many changes can be made in the specific embodiments that are disclosed and still obtain a like or similar result without departing from the spirit and scope of the present teachings.
Example 1
This example illustrates generation of sdAb ligands binding Clec9A and CD40.
To develop high fidelity ligands allowing Ad vector targeting to dendritic cells (DC) we generated single domain antibodies (sdAb) based on the camelid heavy chain-only antibody (Krah, S., et al., Immunopharmacol. Immunotoxicol., 2016, 38, 21-28). To this end, we employed recombinant murine Clec9A (DNGR-1) and CD40 proteins (MyBioSource, Inc., San Diego, CA) to immunize alpacas (Maass, D.R., et al., J. Immunological Methods, 2007, 324, 13-25; Mukherjee, J., et al., PloS One, 2012, 7, e29941; Tremblay, J.M., et al., Toxicon, 2010, 56, 990-998; Kaliberov, S.A., et al, Lab Invest., 2014, 94, 893-905; Yang, Z., J. Infect. Dis., 2014, 210, 964-972). Peripheral blood mononuclear cells (PBMCs) were used to isolate total RNA for cDNA synthesis, and a phage-display sdAb library was constructed as previously described (Maass, D.R., et al., J. Immunological Methods, 2007, 324, 13-25; Mukherjee, J., et al, PloS One, 2012, 7, e29941; Tremblay, J.M., et al., Toxicon, 2010, 56, 990-998). To detect phages displaying sdAbs which were developed against Clec9A, the library was first incubated with murine Clec9A protein immobilized on ELISA plates
allowing sdAb binding. The plates were washed extensively while bound phages were recovered directly by adding bacteria to the wells. The eluted phages were then amplified and used for a second panning cycle performed similarly to the first. Panning, phage recovery, and clone fingerprinting was performed essentially as previously described (Maass, D.R., et al., International Journal for Parasitology, 2007, 37, 953-962). The development of sdAb against mCD40 was carried out essentially as described for Clec9A above. As illustrated in FIG. 2, seven unique sdAb clones were selected against mClec9A (SEQ ID NO: 1-7):
These putative positive sdAbs were engineered for soluble expression, purified, and evaluated by ELISA for mClec9A and mCD40 recognition. As shown in FIG. 3 and FIG. 4, most of the selected sdAbs showed high Clec9A or CD40 binding efficiency in the range of 1
nM or less (EC50 is similar to Kd). Only one sdAb from each group appeared to be a weak binder in EL1SA (JPP-H7 for mCD40 and JPQ-B9 for mClec9A) indicating that sdAb targeting either mCD40 or mClec9A could be selected from each group (FIG. 3 and FIG. 4). ELISA plates were coated with 1 μg/ml of either mClec9A protein (FIG. 3) or mCD40 protein (FIG. 4), blocked, and then exposed to a dilution series of each sdAb (VHH) at the indicated concentrations (nM). Binding was detected using secondary HRP-conjugated Ab against E-tag, which is present in the C-terminus of each sdAb protein. The optical absorbance values were detected at 450 nm (A450) and plotted versus the concentration of sdAb. The sdAb recognizing the other receptor target (JPQ-B4 for mCD40 and JPP-F8 for mClec9A) were included in each ELISA to serve as negative controls and these showed no binding as expected.
Positive sdAbs were also analyzed by flow cytometry for their DC-binding ability. The recognition of murine dendritic cells by anti-CD40 or anti-Clec9A sdAbs was tested, in both bone marrow-derived and spleen isolated dendritic cells. Commercial anti-Clec9A (Miltenyi Biotech Clone: 7H1 .1) and anti-CD40 (Biolegend Clone: 3/23) were used as controls. Cells were stained with individual sdAb (1μ§/100μ1) followed by a secondary PE- conjugated Ab against E-tag. Cells were then additionally stained for mDC (CD3-, CD20-, DR+, CD14-, CD16-, CD1 lc+, CD123-) and analyzed by flow cytometry. In FIG. 5, the left panel is a representative flow cytometry profile of Anti-CD40 sdAb (Unfilled Areas) in comparison to the staining by a commercially available a-CD40 monoclonal antibody (Filled Area) using bone marrow derived DCs. The center panel is a flow cytometry profile of anti- Clec9A sdAb. The dot matrix area shows a more effective binding cognition of the DCs subsets than the commercial monoclonal antibody tested (white). The right panel is a flow cytometric analysis of anti-Clec9A sdAb binding to rhesus macaque mDC. In both cultured and splenocyte-derived dendritic cell the anti-CD40 and anti-Clec9A sdAbs had a more efficient binding to the cells than the commercially available antibodies. Furthermore, the panel of generated sdAb against Clec9A showed a high degree of cross-reactivity with simian mDC. These data demonstrate our ability to produce high-fidelity sdAb ligands specific for DC markers.
Example 2
This example illustrates expression of ZIKV soluble E protein by recombinant Ad vector.
To validate the expression of ZIKV proteins by Ad5 vector we incorporated the DNA sequence encoding the full prM gene and the ectodomain of E of ZIKV (strain H/PF/2013 from French Polynesia) containing a heterologous N-terminal IL-2 signal peptide and C- terminal hexahistidine tag (6-His) under transcriptional control of cytomegalovirus (CMV) immediate early promoter in place of the early El A/B genes deleted in Ad5 genome. This construct produces high levels of soluble E protein only, as prM/M is cleaved completely by the host signalase, and in the absence of the transmembrane domain of E, prM and E do not stably associate as heterodimers (Cockburn J. J., et al., EMBO J., 2012, 31, 767-779). The generated Ad5ZprM-E-ecto vector was used to infect A549 cells to validate the expression of secreted E protein. As shown in FIG. 6, the 6-His-tagged E protein band with molecular mass of approximately 48 kDa was detected 48 and 72 hours post-infection in both cell lysates and culture medium by Western blotting. A549 cells were infected with Ad5ZprM-E-ecto vector at a multiplicity of infection (MOI) of 900. The cells and culture medium samples mixed with Laemmli loading buffer, boiled, and run on 4-20% gradient SDS-PAGE as follows. (1) Pre- stained protein standards, molecular masses are indicated in kDa on the left.; (2) Culture medium 48-h post-infection (pi); (3) Culture medium 72-h pi; (4) Uninfected cells culture medium; (5) Cells 48-h pi; (6) Cells 72-h pi; (7) Mock-infected cells; (8) Purified ZIKV protein E (25 ng/lane). Electrophoretically separated proteins were transferred to PVDF membrane and probed with anti-His tag and secondary anti-mouse AP-conjugated antibodies. The ZIKV protein E purified from culture medium of HEK293 cells transiently expressing the same CMV-driven prM-E-ecto plasmid was used as a positive control. These data demonstrate the feasibility of soluble ZIKV E expression using recombinant Ad5ZprM-E- ecto vector.
Example 3
This example illustrates Ad targeting to DCs via functional replacement of fiber knob with an sdAb ligand.
Ad5 cellular entry is mediated by distinct binding and internalization events; the knob domain of Ad5 fiber initiates attachment through interactions with coxsackie virus and adenovirus receptor (CAR) expressed on epithelial cells (Bergelson, J.M., et al., Science
1997, 275, 1320-1323), whereas internalization is mediated by distinct interactions between integrins and the RGD motif within the Ad5 penton (Wickham, T.J., et al., Cell, 1993, 73,
309-319). To give Ad5 vectors a CAR-independent tropism we previously developed a
genetic targeting platform, based on key fiber modifications. Specifically, the knob domain was deleted to ablate its broad tropism to CAR-expressing cells, and a 95 amino acid (aa) trimerization domain of the T4 phage fibritin protein was inserted to maintain fiber stability while allowing display of a targeting ligand binding to alternative receptor on the cell surface (Noureddini, S.C., et al., Virus Res., 2006, 1 16, 185-195; Korokhov, N., et al., J. Virology, 2003, 77, 12931-12940; Alberti, M.O., et al., PloS One, 2012, 7, e37812) as illustrated in FIG. 7. To generate a DC-targeted Ad, we employed the nanobody Nbl.8, a camelid sdAb raised against murine bone marrow-derived DCs (BMDC), which recognizes immature BMDC (iBMDC) in vitro and in vivo (De Groeve, K., et al., J. Nuclear Medicine, 2010, 51, 782-789). The sdAb Nbl.8-coding sequence was incorporated in-frame following fiber- fibritin fusion within Ad5 genome by homologous recombination in E. coli and the resultant viral genome was rescued in 293F28 cells (Belousova, N., et al., J. Virology, 2003, 77, 11367-11377) to generate replication incompetent Ad5FF1.8 vector essentially as described (Kaliberov, S.A., et al., Laboratory Investigation; a J. Technical Methods and Pathology, 2014, 94, 893-905). Both fiber and Nbl.8 incorporation in the context of assembled virions were verified by Western blotting on purified Ad5FFl .8 particles (data not shown).
Example 4
This example illustrates that Ad5FF1.8 vector enhances DC transduction.
To establish the efficacy of the Ad5FF1.8 vector gene transfer in a relevant animal DC substrate, we used murine iBMDC to assess their transduction using GFP reporter gene expression. Ad5FF1.8 showed enhanced gene transfer into DCs compared to the control Ad5 vector, as demonstrated by the markedly increased number of GFP-positive DCs (FIG. 8). To confirm that the specificity of Ad5FF1.8 transduction is mediated by sdAb Nbl.8, we transduced DCs with Ad5FF1.8 in the presence or absence of soluble Nbl.8. Increasing the concentration of soluble Nbl.8 resulted in a dose-dependent decrease of gene transfer by Ad5FF1.8 (data not shown). Without being limited by theory, these data demonstrate the feasibility of Ad vector targeting to DCs via genetic sdAb incorporation into viral capsid.
Example 5
This example illustrates the generation of sdAb ligands that bind Clec9A.
To target Ad vector to specific DC subsets we identified several surface markers that are conserved across species and present on CD8a+/CD141+ DCs (Khanam, S., et al.,
Vaccine, 2009, 27, 6011-6021; Borgherini, G. et al., Clin. Infect. Dis., 2008, 47, 469-475; Charrel, R.N., et al., The New England Journal of Medicine, 2007, 356, 769-771) including the C-type lectin receptor Clec9A (or DNGR-1), a damage-associated molecular pattern (DAMP) receptor (Borgherini, G. et al., Clin. Infect. Dis., 2008, 47, 469-475; Schuffenecker, I., et al, PLoS Med., 2006, 3, e263). The generation of sdAb binding Clec9A was carried out (Tillman, B.W., et al., Cancer Research, 2000, 60, 5456-5463; Hangalapura, B.N., et al., J. Gene Medicine, 2012, 14, 416-427; Hangalapura, B.N., et al., Cancer Res., 2011, 71, 5827- 5837; Thacker, E.E., et al., Vaccine, 2009, 27, 7116-7124; Williams, B.J., et al., PloS One, 2012, 7, e46981) using the recombinant murine Clec9A protein (MyBioSource, Inc., San Diego, CA) to immunize alpacas. Seven unique sdAb clones were selected using the constructed phage-display sdAb library and validated by ELISA for Clec9A recognition. Most of the selected sdAbs showed high binding efficiency in the range of 1 nM or less (data not shown) thus providing us with several sdAb-coding sequences to be exploited for genetic 5 bh4capsid incorporation to achieve GAd vector targeting to CD8a+/Clec9A+ DC subset.
Example 6
This example illustrates mouse models of ZIKV pathogenesis.
Several mouse models of ZIKV pathogenesis in mice deficient in type I interferon
(1FN) signaling have been developed. A loss of Ifhar expression or blockade of Ifnar function is necessary because ZIKV does not replicate efficiently in wild-type (WT) mice due in part to a species-specific lack of antagonism of mouse Stat2 (Tsetsarkin, K.A., et al., PloS One
2009, 4, e6835) a key signaling intermediate downstream of type I IFN signaling. The different adult mouse models have utility for vaccine testing, each with its own limitations.
Lethal infection of contemporary ZIKV strains in adult Ifnar1' mice (De Groeve, K., et al., J.
Nuclear Medicine, 2010, 51, 782-789) provides the advantage of a stringent challenge of protection, however, immunization occurs in an immunocompromised Ifhar'' mouse, which could skew or dampen responses. Inoculation of WT adult C57BL/6 mice treated with an anti-Ifnar blocking antibody and contemporary American/Asian ZIKV strains (De Groeve,
K., et al., J. Nuclear Medicine, 2010, 51, 782-789) allows immunization in WT mice, however, it provides only a virological read-out because infection causes mild morbidity and no mortality. The present teachings allow for lethal infection of an adapted African ZIKV strain in adult WT mice (FIG. 9-11). For preliminary studies using this model, adult WT
C57BL/6 mice were passively transferred 2 mg of anti-Ifnar 1 mAb and 250 μgs of the
indicated mAbs (CHK-166, ZV-54, or ZV-57) via an intraperitoneal injection one day before subcutaneous inoculation with 105 FFU of adapted ZIKV Dakar 41519. FIG. 9: on day 3 after infection, serum was collected for analysis of viremia by qRT-PCR, and survival curves were constructed. FIG. 10: daily weights were measured. For FIG. 9 and FIG. 10, statistical significance was analyzed by a one-way ANOVA (**, P < 0.01 ; ***, P < 0.001). The results are pooled from independent experiments; n = 8-9 mice for each treatment condition. The data indicate that anti-ZIKV mAbs provided statistically significant protection in the percentage of surviving animals compared to the control CHK-166 mAb (***, P < 0.001, log rank test for ZV-54 and ZV-67). The present methods thus allow for immunization in WT mice with a stringent requirement for protection against challenge. However an African ZIKV strain is used, although concern is mitigated by immunization with an Ad encoding structural genes from a contemporary Asian (or American) isolate). Given that this last model is high-throughput (WT mice can be purchased in large cohorts from commercial vendors), uses immune-competent mice for induction of vaccine responses, and challenges with a heterologous ZIKV strain (which can account for breadth/diversity of response) it is preferred for vaccination and lethal challenge.
In addition to these infection models in adult mice, parallel models (Ifnar '~ pregnant females infected with contemporary ZIKV strains or WT pregnant females treated with an Ifnar blocking mAb and infected with contemporary or adapted ZIKV strains) of in utero transmission of ZIKV are available (Kaliberov, S.A., Laboratory Investigation, 2014, 94, 893-905). Depending on the specific model, we observed placental infection, injury and insufficiency, fetal resorption, and fetal brain injury and neuronal cell death. Moreover, we identified ZIKV within trophoblasts and fetal endothelial cells in the placenta, consistent with a tropism for cells lining the maternal-fetal interface and a trans-placental route of infection. The different in utero models have possible utility for vaccine testing, again each with limitations. Based on the rationale described above, we can immunize WT females with GAd encoding ZIKV genes, allow for immune responses to develop, breed them with WT males, administer a blocking anti-Ifnar antibody, challenge with the adapted African ZIKV strain, and monitor clinical and virological parameters in the mother and developing fetus according to published methods (Kaliberov, S.A., et al., Laboratory Investigation, 2014, 94, 893-905).
Example 7
This example illustrates CHIK challenge of CAdVax-CHIK immunized C57BL/6
mice.
Herein we describe a CH1KV vaccine. In this CAdVax-CHIK vaccine (Wang, D., et al., J. Virol. 2006, 80, 2738-2746), a single insert encoding the structural polyprotein (comprising the envelope glycoproteins El, E2 and capsid) of CHIKV was inserted in the right hand of the genome. An advantage of this configuration of the CAdVax is that it prevents the generation of replication-competent adenovirus through homologous recombination in the packaging cell line, HEK293, a common problem of first generation Ad5 vectors. The antigen sequences are from a CHIKV isolate from the recent epidemic on Reunion Island, or from an Asian isolate. The complete structural polyprotein gene was expressed in order to retain the native processing sequences. A single dose of the vaccine completely protected mice against viraemia and disease in recently developed adult wild-type mouse models of CHIKV-induced arthritis (Tsetsarkin, K.A., et al., PloS One, 2009, 4, e6835).
Mice (n=3-6 per group) were vaccinated with CAdVax-CHIK, a control CAdVax vaccine or PBS. At 6.5 weeks post-immunization, mice were challenged with CHIKV. FIG. 12 illustrates viremia after challenge with the Reunion Island isolate. Viremia was significantly different between CAdVax-CHIK and CAdVax-control vaccinated groups on days 1-3 (all p<0.037, Mann Whitney U test). FIG. 13 illustrates foot swelling after challenge with the Reunion island isolate. Swelling (represented as a cross sectional area in mm2) was significantly different between CAdVaxCHIK and CAdVax-control vaccinated groups on days 3-8 (all p<0.02, Mann Whitney U test). FIG. 14 illustrates viremia after challenge with the Asian isolate. Viremia was significantly different between CAdVax-CHIK and CAdVax-control vaccinated groups on days 1-4 (all p<0.014, Mann Whitney U test). FIG. 14 illustrates foot swelling after challenge with the Asian isolate. Swelling was significantly different between CAdVax-CHIK and CAdVax-control vaccinated groups on days 3-10 (all p<0.04, Mann Whitney U test). Our DC targeting can improve these vaccine outcomes.
Example 8
This example illustrates the assessment of tumor growth after vaccine treatment with
DC -targeted adenovirus vaccine.
Antitumor effect of a CD40-targeted Ad5-huPSMA vaccine was determined using the
RM-1-PSMA mouse model. C57BL/6 mice were immunized by intraperitoneal injection of
lxl 08 ifu of untargeted Ad5-huPSMA or CD40- targeted Ad5-huPSMA. The animals received a boost immunization at 10 days after the initial immunization. At the 14th day after the second immunization, the mice were challenged subcutaneously with 46105 RM-1 parental cells or RM-l-PSMA clone 1 cells. Three days later, the treatment groups were injected at the site of tumor cell injection with IxlO8 ifu of Ad5-lFNy or with normal saline. At day 24 after initiation of the experiment, each mouse received 4xl06 RM-1 parental cells (which do not express the human PSMA antigen) or 4xl06 RM-l-PSMA clone 1 cells injected
subcutaneously. Three days later, the treatment groups were injected at the site of tumor cell injection with IxlO8 ifu of Ad5-IFNy or with normal saline. Beginning at the time of tumor cell challenge (day 0), the tumors were measured and volumes calculated by the formula: tumor volume = ½ x (length x width2) where length is the longest distance of the tumor. Each data point represents the mean volume of 15 tumors ± standard error. As shown in FIG. 16, immunization with CO40-targeted Ad5-huPSMA alone or with non-targeted Ad5-huPSMA + Ad5-IFNy similarly diminish tumor growth in animals challenged with RM- 1-PSMA cells. This tumor growth was significantly delayed compared with the animals immunized with the CD40-targeted Ad5-huPSMA but challenged with parental RM-1 cells. Mice immunized with the CD40-targeted Ad5-huPSMA and AdS-IFNy demonstrated the greatest inhibition of tumor growth when challenged with RM- 1-PSMA cells compared with the other groups.
All publications cited are herein incorporated by reference, each in its entirety.
Claims
1. A camelid sdAb against a dendritic cell surface antigen other than carcinoembryonic antigen and EGFR.
2. A camelid sdAb against a dendritic cell surface antigen in accordance with claim 1, wherein the dendritic cell surface antigen is Clec9A.
3. A camelid sdAb against a dendritic cell surface antigen in accordance with claim 1, wherein the dendritic cell surface antigen is CD40.
4. A dendritic cell-targeted adenovirus comprising the camelid sdAb of any one of claims 1-3
5. A dendritic cell-targeted adenovirus in accordance with claim 4, deleted for an E1A/B genetic region.
6. A dendritic cell-targeted adenoviral vector comprising the adenovirus of claim 5, further comprising a nucleic acid sequence encoding a polypeptide heterologous to the adenovirus, wherein the sequence is an insertion in the deleted El A/B genetic region.
7. A dendritic cell-targeted adenoviral vector comprising the adenovirus of claim 6, further comprising a promoter heterologous to the adenovirus in the deleted El A/B genetic region.
8. A dendritic cell-targeted adenoviral vector comprising the adenovirus of claim 7, wherein the heterologous promoter is a cytomegalovirus promoter.
9. A dendritic cell-targeted adenoviral vector in accordance with claim 6, wherein the polypeptide heterologous to the adenovirus is a structural polypeptide of a heterologous virus.
10. A dendritic cell targeted adenoviral vector in accordance with claim 9, wherein the heterologous virus is a flavivirus.
11. A dendritic cell targeted adenoviral vector in accordance with claim 10, wherein the flavivirus is selected from the group consisting of Zika virus. Dengue virus, yellow fever virus and West Nile Virus.
12. A dendritic cell targeted adenoviral vector in accordance with claim 9, wherein the heterologous virus is selected from the group consisting Zika virus, Chikungunya virus and Dengue virus.
13. A dendritic cell targeted adenoviral vector in accordance with claim 9, wherein the heterologous virus is a Zika virus.
14. A dendritic cell targeted adenoviral vector in accordance with claim 13, wherein the polypeptide comprises a full length prM gene and an ectodomain of an E gene of a Zika virus.
15. A dendritic cell targeted adenoviral vector in accordance with claim 13, wherein the nucleic acid sequence encoding a polypeptide heterologous to the adenovirus comprises a sequence encoded by the E gene of a Zika virus.
16. A dendritic cell targeted adenoviral vector in accordance with claim 6, wherein the polypeptide heterologous to the adenovirus is a tumor antigen.
17. A dendritic cell targeted adenoviral vector in accordance with claim 16, wherein the tumor antigen is PSMA.
18. A vaccine comprising a dendritic cell adenoviral vector in accordance with any one of claims 6-17.
19. A dendritic cell targeted adenoviral vector comprising:
a sequence encoding a fiber-fibritin chimeric shaft;
a sequence encoding a camelid sdAb against a cell surface protein of a dendritic cell; and
a sequence encoding an antigen selected from the group consisting of a heterologous virus structural gene and a tumor antigen, wherein the vector is deleted for a sequence encoding a E1A/B genetic region.
20. A dendritic cell targeted adenoviral vector in accordance with claim 19, wherein the heterologous virus structural gene is selected from the group consisting of a Zika virus structural gene, a Chikungunya virus structural gene and a Dengue virus structural gene.
21. A dendritic cell targeted adenoviral vector in accordance with claim 19, wherein the heterologous virus structural gene is a Zika virus structural gene.
22. A dendritic cell targeted adenoviral vector in accordance with claim 21, wherein the Zika virus structural gene comprises a full length prM gene and an ectodomain of a Zika virus E gene.
23. A dendritic cell targeted adenoviral vector in accordance with claim 21 , wherein the Zika virus structural gene is a Zika virus E gene.
24. A dendritic cell targeted adenoviral vector in accordance with claim 19, wherein the heterologous virus structural gene is a CHIK virus structural gene.
25. A dendritic cell targeted adenoviral vector in accordance with claim 24, wherein the CHIK virus structural gene encodes a CHIK virus polyprotein comprising CHIK El, CHIK E2 and CHIK capsid.
26. A dendritic cell targeted adenoviral vector in accordance with claim 24, wherein the CHIK virus structural gene encodes a CHIK virus E gene polypeptide.
27. A dendritic cell targeted adenoviral vector in accordance with claim 19, wherein the camelid sdAb is against Clec9A.
28. A dendritic cell targeted adenoviral vector in accordance with claim 19, wherein the camelid sdAb is against CD40.
29. A vaccine comprising a dendritic cell-targeted adenoviral vector in accordance with any one of claims 19-28.
30. A method of vaccinating a subject against a flavivirus comprising administering to the subject an adenovirus vector comprising:
a fiber-fibritin chimeric shaft;
a deleted E1A/B genetic region;
a camelid sdAb against a cell surface protein of a dendritic cell; and
at least one flavivirus structural gene inserted in the deleted E1A/B genetic region.
31. A method of vaccinating a subject against a flavivirus in accordance with claim 30, wherein the camelid sdAb is against Clec9A.
32. A method of vaccinating a subject against a flavivirus in accordance with claim 30, wherein the camelid sdAb is against CD40.
33. A method of vaccinating a subject against a flavivirus in accordance with claim 30, wherein the flavivirus is a Zika virus.
34. A method of vaccinating a subject against a flavivirus in accordance with claim 30, wherein the flavivirus is a CH1K virus.
35. An adenoviral vector deleted for the E1A/B region, comprising:
a sequence encoding a fiber-fibritin chimeric shaft;
a sequence encoding a camelid sdAb against a cell surface protein of a dendritic cell; and
a sequence encoding an antigen, wherein the antigen is selected from the group consisting of a heterologous virus structural gene and a tumor antigen.
36. An adenoviral vector deleted for the E1A/B region in accordance with claim 35, wherein the sequence encoding an antigen is an insertion in the deleted E1A/B region.
37. An adenoviral vector deleted for the El A/B region in accordance with claim 35, wherein the heterologous virus structural gene is a flavivirus structural gene.
38. An adenoviral vector deleted for the E1A/B region in accordance with claim 37, wherein the flavivirus structural gene is selected from the group consisting of a Zika virus structural gene, a CHIK virus structural gene and a Dengue virus structural gene.
39. A method of vaccinating a subject against CHIK virus, comprising administering to the subject an adenovirus vector comprising:
a fiber-fibritin chimeric shaft;
a deleted El A/B genetic region;
a camelid sdAb against a cell surface protein of a dendritic cell; and
at least one CHIK structural gene inserted in the deleted EIA/B genetic region.
40. A method of vaccinating a subject against Zika in accordance with claim 39, wherein the camelid sdAb is against Clec9A.
41. A method of vaccinating a subject against Zika in accordance with claim 39, wherein the camelid sdAb is against CD40.
42. An adenoviral vector comprising:
a sequence encoding a fiber-fibritin chimeric shaft;
a deleted El A/B genetic region;
a sequence encoding a camelid sdAb against a cell surface protein of a dendritic cell;
and
a sequence encoding an antigen comprising the deleted El A/B genetic region, wherein the antigen is selected from the group consisting of a heterologous virus structural gene and a tumor antigen.
43. An adenoviral vector comprising:
an N-terminal segment of Ad5 fiber tail sequence;
at least 2 pseudorepeats of an Ad5 fiber shaft domain sequence;
a portion of a third Ad5 fiber shaft domain sequence;
a carboxy-tenninal segment of a T4 fibritin bacteriophage trimerization domain sequence;
a linker sequence; and
a sequence encoding a camelid single chain antibody against a dendritic cell surface antigen other than carcinoembryonic antigen and EGFR.
44. An adenoviral vector in accordance with claim 43, wherein the dendritic cell surface antigen is Clec9A.
45. An adenoviral vector in accordance with claim 43, wherein the dendritic cell surface antigen is CD40.
46. An adenoviral vector in accordance with claim 43, wherein the E1A/B genetic region is deleted.
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Cited By (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US11077156B2 (en) | 2013-03-14 | 2021-08-03 | Salk Institute For Biological Studies | Oncolytic adenovirus compositions |
CN113429481A (en) * | 2021-07-07 | 2021-09-24 | 江苏农牧科技职业学院 | Nano antibody molecule of targeted dendritic cell and application |
US11130968B2 (en) | 2016-02-23 | 2021-09-28 | Salk Institute For Biological Studies | High throughput assay for measuring adenovirus replication kinetics |
WO2022013277A1 (en) * | 2020-07-14 | 2022-01-20 | Evaxion Biotech A/S | Apc targeting units for immunotherapy |
US11401529B2 (en) | 2016-02-23 | 2022-08-02 | Salk Institute For Biological Studies | Exogenous gene expression in recombinant adenovirus for minimal impact on viral kinetics |
US11813337B2 (en) | 2016-12-12 | 2023-11-14 | Salk Institute For Biological Studies | Tumor-targeting synthetic adenoviruses and uses thereof |
WO2024051096A1 (en) * | 2022-09-05 | 2024-03-14 | 上海百英生物科技股份有限公司 | Anti-cd40 nanobody, and preparation method therefor and use thereof |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2013053008A2 (en) * | 2011-10-14 | 2013-04-18 | The Walter And Eliza Hall Institute Of Medical Research | Molecules which bind clec9a |
WO2015161314A1 (en) * | 2014-04-18 | 2015-10-22 | Washington University | Adenoviral targeting, compositions and methods therefor |
-
2018
- 2018-04-27 WO PCT/US2018/029900 patent/WO2018201017A1/en active Application Filing
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2013053008A2 (en) * | 2011-10-14 | 2013-04-18 | The Walter And Eliza Hall Institute Of Medical Research | Molecules which bind clec9a |
WO2015161314A1 (en) * | 2014-04-18 | 2015-10-22 | Washington University | Adenoviral targeting, compositions and methods therefor |
Non-Patent Citations (1)
Title |
---|
WILLIAMS, BRIANA JILL ET AL.: "Dendritic cell based PSMA immunotherapy for prostate cancer using a CD 40-targeted adenovirus vector", PLOS ONE, vol. 7, no. 10, 10 August 2012 (2012-08-10), pages e46981, XP055535333, Retrieved from the Internet <URL:http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0046981> [retrieved on 20180715] * |
Cited By (8)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US11077156B2 (en) | 2013-03-14 | 2021-08-03 | Salk Institute For Biological Studies | Oncolytic adenovirus compositions |
US11130968B2 (en) | 2016-02-23 | 2021-09-28 | Salk Institute For Biological Studies | High throughput assay for measuring adenovirus replication kinetics |
US11401529B2 (en) | 2016-02-23 | 2022-08-02 | Salk Institute For Biological Studies | Exogenous gene expression in recombinant adenovirus for minimal impact on viral kinetics |
US11813337B2 (en) | 2016-12-12 | 2023-11-14 | Salk Institute For Biological Studies | Tumor-targeting synthetic adenoviruses and uses thereof |
WO2022013277A1 (en) * | 2020-07-14 | 2022-01-20 | Evaxion Biotech A/S | Apc targeting units for immunotherapy |
CN113429481A (en) * | 2021-07-07 | 2021-09-24 | 江苏农牧科技职业学院 | Nano antibody molecule of targeted dendritic cell and application |
CN113429481B (en) * | 2021-07-07 | 2022-05-31 | 江苏农牧科技职业学院 | Nano antibody molecule of targeted dendritic cell and application |
WO2024051096A1 (en) * | 2022-09-05 | 2024-03-14 | 上海百英生物科技股份有限公司 | Anti-cd40 nanobody, and preparation method therefor and use thereof |
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