Patent Application
20230256084
The new Severe Acute Respiratory Syndrome beta-coronavirus 2 (SARS-CoV-2), emerged in late 2019 in Wuhan, China, is extraordinarily contagious and fast-spreading across the world (Guo et al., 2020). Compared to the previously emerged SARS or Middle East Respiratory Syndrome (MERS) coronaviruses, SARS-CoV-2 causes unprecedented threat on global health and tremendous socio-economic consequences. Therefore, the development of effective prophylactic vaccines against SARS-CoV-2 is of absolute imperative to contain the spread of the epidemic and to attenuate the onset of CoronaVirus Disease 2019 (COVID-19), such as deleterious inflammation and progressive respiratory failure (Amanat and Krammer, 2020). Although lung is the organ of predilection for SARS-CoV-2, its neurotropism, like that of SARS-CoV and Middle East Respiratory Syndrome (MERS)-CoV, (Glass et al., 2004; Li et al., 2016; Netland et al., 2008) has been reported (Aghagoli et al., 2020; Fotuhi et al., 2020; Hu et al., 2020; Liu et al., 2020; Politi et al., 2020; Roman et al., 2020; von Weyhern et al., 2020; Whittaker et al., 2020). Moreover, expression of Angiotensin Converting Enzyme 2 (ACE2) in neuronal and glial cells has been described (Chen et al., 2020; Xu and Lazartigues, 2020). Accordingly, COVID-19 human patients can present symptoms like headache, myalgia, anosmia, dysgeusia, impaired consciousness and acute cerebrovascular disease (Bourgonje et al., 2020; Hu et al., 2020; Mao et al., 2020). Viruses can gain access to the brain through neural dissemination or hematogenous route (Desforges et al., 2014). Analysis of autopsies of COVID-19 deceased patients demonstrated presence of SARS-CoV-2 in nasopharynx and brain and virus entry into central nervous system (CNS) via neural-mucosal interface of olfactory mucosa (Meinhardt et al., 2020). Therefore, it is critical to focus hereinafter on the protective properties of COVID-19 vaccine candidates, not only in the respiratory tracts, but also in the brain.
Coronaviruses are enveloped, non-segmented positive-stranded RNA viruses, characterized by their envelop-anchored Spike (S) glycoprotein (Walls et al., 2020). The SARS-CoV-2 S (SCoV-2) is a (180 kDa)3 homotrimeric class I viral fusion protein, which engages the carboxypeptidase Angiotensin-Converting Enzyme 2 (ACE2), expressed on host cells. The monomer of SCoV-2 protein possesses an ecto-domain, a transmembrane anchor domain, and a short internal tail. SCoV-2 is activated by a two-step sequential proteolytic cleavage to initiate fusion with the host cell membrane. Subsequent to SCoV-2-ACE2 interaction, which leads to a conformational reorganization, the extracellular domain of SCoV-2 is first cleaved at the highly specific furin 682RRAR685 (SEQ ID NO: 99) site (Guo et al., 2020; Walls et al., 2020), a key factor determining the pathological features of the virus, linked to the ubiquitous furin expression (Wang et al., 2020). The resulted subunits are constituted of: (i) S1, which harbors the ACE2 Receptor Binding Domain (RBD), with the atomic contacts restricted to the ACE2 protease domain and also harbors main B-cell epitopes, targeted of NAbs (Walls et al., 2020), and (ii) S2, which bears the membrane-fusion elements. Like for SCoV-1, the shedding of S1 renders accessible on S2 the second proteolytic cleavage site 7978, namely S2′ (Belouzard et al., 2009). According to the cell or tissue types, one or several host proteases, including furin, trypsin, cathepsins or TransMembrane Protease Serine Protease (TMPRSS)-2 or -4, can be involved in this second cleavage step (Coutard et al., 2020). The consequent “fusogenic” conformational changes of S result in a highly stable postfusion form of SCoV-2 that initiates the fusion reaction with the host cell membrane (Sternberg and Naujokat, 2020) and lead to the exposure of a Fusion Peptide (FP), adjacent to S2′. Insertion of FP to the host cell/vesicle membrane primes the fusion reaction, whereby the viral RNA release into the host cytosol (Lai et al., 2017). The facts that the SCoV-2-ACE2 interaction is the only mechanism, thus far identified for the host cell infection by SARS-CoV-2, and that the RBD contains numerous conformational B-cell epitopes (Walls et al., 2020), designate this viral envelop glycoprotein as the main target for neutralization antibodies (nAbs). Like envelop glycoproteins of several other viruses including respiratory syncytial virus, HIV, Ebola virus, human metapneumovirus, and Lassa virus (Bos et al., 2020), it is possible to engineer SCoV-2 to avoid its conformational dynamics and its stabilization under its prefusion conformation that will possibly better maintain exposure of the S1 B-cell epitopes and possibly improve immunogen availability (McCallum et al., 2020).
Several vaccine alternatives have significant drawbacks. Specifically: (i) attenuated or inactivated viral vaccine candidates which require extensive safety testing, (ii) the nucleic acids encoding for S do not have proven efficacy on long term protection, (iii) protein vaccines require the use of adjuvants and boosting, and (iv) pre-existing immunity exists for viral vectors, such as adenoviral vectors, can generate strong anti-vector immune response, which largely reduces their immunogenicity (Rosenberg et al., 1998; Schirmbeck et al., 2008).
Among viral vectors, lentiviral vectors exist under integrative (ILV) and non-integrative (NILV) forms which are permissive to insertion of up to 8 kb-length transgenes of vaccinal interest and possess outstanding potential of gene transfer to the nuclei of host cells (Di Nunzio et al., 2012; Hu et al., 2011; Ku et al., 2020; Zennou et al., 2000). Lentivectors display in vivo tropism for immune cells, notably dendritic cells, are non-replicative, non-cytopathic and scarcely inflammatory, and induce long-lasting B- and T-cell immunity (Di Nunzio et al., 2012; Hu et al., 2011; Ku et al., 2020; Zennou et al., 2000). Pseudo-typed at their envelop with the surface glycoprotein of Vesicular Stomatitis Virus, to which the human population has been barely exposed, LV are not target of specific preexisting immunity in humans, in net contrast to adenoviral vectors (Rosenberg et al., 1998; Schirmbeck et al., 2008). In addition, the safety of LV has been established in human in a phase I/II Human Immunodeficiency Virus (HIV)-1 vaccine trial (2011-006260-52 EN).
A need exists for compositions and methods of inducing a protective immune response against SARS-CoV-2. This disclosure meets these and other needs.
To develop a vaccine candidate capable of preventing COVID-19 or decreasing its severity, LV coding for: (i) full-length, membrane anchored form of S (LV::SFL/LV::SFL), (ii) S1-S2 ecto-domain, without the transmembrane and internal tail domains (LV::S1-52), (iii) S1 alone (LV::S1), (iv) mutated S deleted of a sequence encompassing the furin site and substituted at residues K986P and V987P to introduce consecutive proline residues in S2 (2P mutation) (LV::SΔF2P) thereby providing a stabilized (2P) and prefusion (ΔF) form of the protein were generated. Additional vaccine candidates were generated, including LV coding for: (i) the spike protein of variant B1.351 (so called South African or β variant), (ii) the spike protein of variant B1.1.7 (so called UK or alpha variant), (iii) the spike protein of variant B1.351 substituted at residues K986P and V987P, (iv) the full-length, membrane anchored form of S combined with a D614G substitution (LV::SFL-D614G), and (v) the spike protein of variant P.1 (so called Manaus or gamma variant). The data presented in the examples establish in particular that LV::SFL and LV::SΔF2P either in the integrative or non integrative version of the vector(i) induced neutralizing antibodies specific to the Spike glycoprotein (S) of SARS-CoV-2, the etiologic agent of COVID-19, with neutralizing activity comparable to those found in a cohort of SARS-CoV-2 patients, and (ii) induced Spike-specific CD8+ T cells. Moreover, using golden hamsters highly susceptible to SARS-CoV-2 replication, a strong prophylactic effect of LV::SFL or LV::SΔF2P immunization against the replication of a SARS-CoV-2 clinical isolate was demonstrated. Similar results were obtained in a mouse model in which the expression of human ACE2 (hACE2) was induced in the respiratory tracts by an adenoviral vector serotype 5 (Ad5). Besides, in transgenic mice generated as a preclinical model showing unprecedent permissibility to SARS-CoV-2 replication including in brain, the inventors were able to demonstrate that a LV encoding a prefusion form of spike glycoprotein of SARS-CoV-2 such as LV::SΔF2P induces substantial protection of respiratory tracts and CNS against SARS-CoV-2. Unexpectedly the generated transgenic mice enabled addressing the capability of protection of the CNS by the developed LV encoding the Spike protein or a derivative or a fragment thereof according to the definition provided below and illustrated in the experimental examples. In addition, the inventors have demonstrated that a single intranasal administration of a LV encoding a prefusion form of Spike glycoprotein of SARS-CoV-2 induces substantial protection of respiratory tracts and totally avoids pulmonary inflammation in the susceptible hamster model. Importantly also, the upper respiratory tract mucosal boost/target immunization with LV::SFL or with LV::SΔF2P was instrumental in the protection efficacy in stringent preclinical model constituted by the generated transgenic mice. The presented virological, immunological and histopathological data demonstrates: (i) marked prophylactic effects of a LV-based vaccination strategy against SARS-CoV-2, (ii) the fact that LV-based immunization represents a promising strategy to develop vaccine candidates against coronaviruses, and (iii) mucosal immunization enables vigorous protective lung immunity and protective CNS immunity. In the particular context of SARS-CoV-2 exhibiting tropism for multiple organs in the infected host, lentiviral vector in any of its forms harboring the lentiviral sequences essential for targeting host cells and enabling expression of a transgene, for instance encoding the Spike protein of SARS-CoV-2 or a derivative or fragment thereof bearing B epitopes and T epitopes, has shown capability to induce and/or activate immune response against the transgene antigen. The inventors have in particular proven the capability of the lentiviral vector to retain or support a conformation of the S antigen (whether wild type or mutated as disclosed herein) that enables effective presentation of the epitopes, especially of the B-epitopes, to the immune system of the host. In addition, the experimental data disclosed herein show that an administration route encompassing a step of administration to upper respiratory tract of the host may improve the immune response in some tissues or organs targeted by the virus. These results are surprising and unexpected.
The data in the examples also demonstrate: (i) strong CD8+ T-cell responses induced by NILV::SCoV-2 Wuhan at the systemic level, (ii) notable proportions of IFN-γ-producing lung CD8+ T cells, specific to several SCoV-2 epitopes, (iii) high proportions of lung CD8+ T cells with effector memory (Tem) and resident memory (Trm) phenotye, (iv) recruitment of CD8+ T cells in the olfactory bulbs, detectable in mice vaccinated and challenged with SARS-CoV-2 Wuhan or SARS-CoV-2 P.1 variant. Remarkably, all murine and human CD8+ T-cell epitopes identified on SCoV-2 Wuhan sequence are preserved in the mutated SCoV-2 Manaus P.1. These observations indicate the strong potential of NILV at inducing full protection of lungs and brain against ancestral and emerging SARS-CoV-2 variants by eliciting marked B and T cell-responses. In contrast to the B-cell epitopes which are targets of NAbs, the so far identified T-cell epitopes have not been impacted by mutations accumulated in the SCoV-2 of the emerging variants. These results are surprising and unexpected.
The data in the examples further demonstrate: (i) sera from mice immunized with LV::SCoV-2 B1.1.7 neutralized at high EC50 pseudo-viruses harboring SCoV-2 Wuhan and LV::S SCoV-2 B1.1.7, but poorly pseudo-viruses harboring SCoV-2 B1.351 and LV::S SCoV-2 P.1.
(ii) sera from mice immunized with LV::S SCoV-2 P.1 neutralized at high EC50 pseudo-viruses harboring SCoV-2 P.1 and LV::SCoV-2 B1.351, but poorly pseudo-viruses harboring SCoV-2 Wuhan and LV::SCoV-2 B1.1.7.
(iii) sera from mice immunized with LV::SCoV-2 B1.351 not only neutralized at high EC50 pseudo-viruses carrying SCoV-2 P.1 and LV::SCoV-2 B1.351 but also pseudo-viruses harboring SCoV-2 Wuhan and LV::SCoV-2 B1.1.7.
These results designate the Spike sequence from the B1.351 (South African or β) variant as the most cross-reactive immunogen in terms of neutralizing antibodies.
Furthermore, the data showed that in the context of LV, Spike stabilization by K986P-V987P substitutions (2P) considerably improves the (cross) neutralizing antibody activity.
Taken together the data surprisingly and unexpectedly show that one particularly effective antigen is the full-length Spike from the B1.351 (South African or β) variant with 2P.
Accordingly, in a first aspect this invention provides a method of inducing a protective immune response against Severe Acute Respiratory Syndrome beta-coronavirus 2 (SARS-CoV-2) in a subject, comprising administering to the upper respiratory tract of the subject an effective amount of an agent that induces a protective immune response against SARS-CoV-2. In certain embodiments the agent that induces a protective immune response against SARS-CoV-2 is a pseudotyped LV vector particle encoding a SARS-CoV-2 Spike (S) glycoprotein or a derivative or fragment thereof. In some embodiments the agent is administered by aerosol inhalation. In some embodiments the agent is administered by nasal instillation. In some embodiments the agent is administered by nasal insufflation. In some embodiments the treatment course consists of a single administration to the upper respiratory tract. In some embodiments the treatment course comprises at least one priming administration outside the respiratory tract followed by at least one boosting administration to the upper respiratory tract. In some embodiments the protective immune response comprises production of SARS-CoV-2 neutralizing antibodies in the subject. In some embodiments the neutralizing antibodies comprise IgG antibodies. In some embodiments the neutralizing antibodies comprise IgA antibodies. In some embodiments the protective immune response comprises production of SARS-CoV-2 S-specific T cells in the subject. In some embodiments the SARS-CoV-2 S-specific T cells comprise CD4+ T cells, CD8+ T cells, or both CD4+ and CD8+ T cells. In some embodiments the SARS-CoV-2 S-specific T cells comprise lung CD8+ T cells. In some embodiments the SARS-CoV-2 S-specific T cells comprise IFN-γ-producing T-cells. In some embodiments the CD8+ T cells comprise T cells with an effector memory (Tem) and/or resident memory (Trm) phenotype. In some embodiments the SARS-CoV-2 S-specific T cells are recruited to the olfactory bulb. In some embodiments the protective immune response provides a reduced likelihood of developing SARS-CoV-2 infection-related inflammation in the subject. In some embodiments the SARS-CoV-2 S protein derivative has an amino acid sequence at least 95% identical to SEQ ID NO: 1. In some embodiments the SARS-CoV-2 S protein derivative is expressed from a coding sequence having a nucleotide sequence at least 80% identical to SEQ ID NO: 2. In some embodiments the SARS-CoV-2 S protein fragment comprises a peptide selected from peptide 61-75 (NVTWFHAIHVSGTNG (SEQ ID NO: 15)), peptide 536-550 (NKCVNFNFNGLTGTG (SEQ ID NO: 16)) and peptide 576-590 (VRDPQTLEILDITPC (SEQ ID NO: 17)). In some embodiments the SARS-CoV-2 S derivative or fragment thereof comprises an amino acid modification relative to SEQ ID NO: 1, the modification selected from: (i) 986K→P and 987V→P, (ii) 681PRRARS686 (SEQ ID NO: 22)→681PGSAGS686 (SEQ ID NO: 23), and (iii) 986K→P, 987V→P, and 675QTQTNSPRRAR685 (SEQ ID NO: 24) deletion. In some embodiments, the Severe Acute Respiratory Syndrome beta-coronavirus 2 (SARS-CoV-2) spike (S) protein or a derivative or fragment thereof comprises or consists of an amino acid sequence selected from SEQ ID NOS: 1, 5, 8, 11, 14, 108, 111, 114, 117, and 120.
In some embodiments the SARS-CoV-2 S protein or a derivative or fragment thereof comprises of SEQ ID NO: 1. In some embodiments the SARS-CoV-2 S protein or a derivative or fragment thereof consists of SEQ ID NO: 1.
In some embodiments the SARS-CoV-2 S protein or a derivative or fragment thereof comprises of SEQ ID NO: 5. In some embodiments the SARS-CoV-2 S protein or a derivative or fragment thereof consists of SEQ ID NO: 5.
In some embodiments the SARS-CoV-2 S protein or a derivative or fragment thereof comprises of SEQ ID NO: 8. In some embodiments the SARS-CoV-2 S protein or a derivative or fragment thereof consists of SEQ ID NO: 8.
In some embodiments the SARS-CoV-2 S protein or a derivative or fragment thereof comprises of SEQ ID NO: 11. In some embodiments the SARS-CoV-2 S protein or a derivative or fragment thereof consists of SEQ ID NO: 11.
In some embodiments the SARS-CoV-2 S protein or a derivative or fragment thereof comprises of SEQ ID NO: 14. In some embodiments the SARS-CoV-2 S protein or a derivative or fragment thereof consists of SEQ ID NO: 14.
In some embodiments the SARS-CoV-2 S protein or a derivative or fragment thereof comprises of SEQ ID NO: 108. In some embodiments the SARS-CoV-2 S protein or a derivative or fragment thereof consists of SEQ ID NO: 108.
In some embodiments the SARS-CoV-2 S protein or a derivative or fragment thereof comprises of SEQ ID NO: 111. In some embodiments the SARS-CoV-2 S protein or a derivative or fragment thereof consists of SEQ ID NO: 111.
In some embodiments the SARS-CoV-2 S protein or a derivative or fragment thereof comprises of SEQ ID NO: 114. In some embodiments the SARS-CoV-2 S protein or a derivative or fragment thereof consists of SEQ ID NO: 114.
In some embodiments the SARS-CoV-2 S protein or a derivative or fragment thereof comprises of SEQ ID NO: 117. In some embodiments the SARS-CoV-2 S protein or a derivative or fragment thereof consists of SEQ ID NO: 117.
In some embodiments the SARS-CoV-2 S protein or a derivative or fragment thereof comprises of SEQ ID NO: 120. In some embodiments the SARS-CoV-2 S protein or a derivative or fragment thereof consists of SEQ ID NO: 120.
In some embodiments the administered LV vector particle is integrative (ILV). In some embodiments the administered lentiviral vector particle is nonintegrative with a defective integrase protein (NILV). In some embodiments the administered NILV comprises a D64V mutation in an integrase coding sequence. In some embodiments the administered LV vector particle is pseudotyped with Vesicular Stomatitis Virus envelop Glycoprotein (VSV-G). In some embodiments the LV vector particle is administered as a vaccine formulation comprising the LV vector particle and a pharmaceutically acceptable carrier.
In another aspect, the invention relates to a dosage form for administration to the upper respiratory tract of a subject of a pseudotyped LV vector particle encoding a SARS-CoV-2 Spike (S) protein or a derivative or fragment thereof. In some embodiments the dosage form is for administration by aerosol inhalation. In some embodiments the dosage form is for administration by nasal instillation. In some embodiments the dosage form is for administration by nasal insufflation. In some embodiments the SARS-CoV-2 S protein derivative has an amino acid sequence at least 95% identical to SEQ ID NO: 1. In some embodiments the SARS-CoV-2 S protein derivative is expressed from a coding sequence having a nucleotide sequence at least 80% identical to SEQ ID NO: 2. In some embodiments the SARS-CoV-2 S protein fragment comprises a peptide selected from peptide 61-75 (NVTWFHAIHVSGTNG (SEQ ID NO: 15)), peptide 536-550 (NKCVNFNFNGLTGTG (SEQ ID NO: 16)) and peptide 576-590 (VRDPQTLEILDITPC (SEQ ID NO: 17)). In some embodiments the SARS-CoV-2 S derivative or fragment thereof comprises an amino acid modification relative to SEQ ID NO: 1, the modification selected from: (i) 986K→P and 987V→P, (ii) 681PRRARS686 (SEQ ID NO: 22)→681PGSAGS686 (SEQ ID NO: 23), and (iii) 986K→P, 987V→P, and 675QTQTNSPRRAR685 (SEQ ID NO: 24) deletion. Additional derivatives and fragments of the S protein are disclosed below along with various aspects of the invention.
In some embodiments the administered LV vector particle is integrative (ILV). In some embodiments the administered LV vector particle is nonintegrative (NILV). In some embodiments the NILV particle comprises a D64V mutation in an integrase coding sequence. In some embodiments the lentiviral vector particle is pseudotyped with Vesicular Stomatitis Virus envelop Glycoprotein (VSV-G).
In another aspect, a kit is provided. The kit may be suitable for use in practicing a method disclosed herein. In some embodiments the kit comprises a dosage form for administration to the upper respiratory tract of a subject of the pseudotyped LV vector particle encoding a SARS-CoV-2 S protein or a derivative or fragment thereof according to this disclosure. In some embodiments the applicator for administration is an applicator for aerosol inhalation. In some embodiments the applicator for administration to the upper respiratory tract of a subject is an applicator for nasal instillation. In some embodiments the applicator for administration to the upper respiratory tract of a subject is an applicator for nasal insufflation.
Also provided are novel and nonobvious pseudotyped LV vector particles encoding a SARS-CoV-2 Spike (S) protein or a derivative or fragment thereof. In some embodiments the pseudotyped LV vector particles are administered to the upper respiratory tract of a subject. In some embodiments the pseudotyped LV vector particles induce a protective immune response providing a reduced likelihood of developing SARS-CoV-2 infection-related inflammation following administration to the upper respiratory tract of a subject. In some embodiments the SARS-CoV-2 S protein derivative has an amino acid sequence at least 95% identical to SEQ ID NO: 1. In some embodiments the SARS-CoV-2 S protein derivative is expressed from a coding sequence having a nucleotide sequence at least 80% identical to SEQ ID NO: 2. In some embodiments the SARS-CoV-2 S protein fragment comprises a peptide selected from Peptide 61-75 (NVTWFHAIHVSGTNG—SEQ ID No.15), peptide 536-550 (NKCVNFNFNGLTGTG—SEQ ID No.16) and peptide 576-590 (VRDPQTLEILDITPC—SEQ ID No.17). In some embodiments the SARS-CoV-2 S derivative or fragment thereof comprises an amino acid modification relative to SEQ ID NO: 1, the modification selected from: (i) 986K→P and 987V→P, (ii) 681PRRARS686 (SEQ ID NO: 22)→681PGSAGS686 (SEQ ID NO: 23), and (iii) 986K→P, 987V→P, and 675QTQTNSPRRAR685 (SEQ ID NO: 24) deletion. In some embodiments the LV vector particle is integrative (ILV). In some embodiments the lentiviral vector particle is nonintegrative (NILV). In some embodiments the NILV particle comprises a D64V mutation in an integrase coding sequence. In some embodiments the LV vector particle is pseudotyped with Vesicular Stomatitis Virus envelop Glycoprotein (VSV-G). In some embodiments, the pseudotyped LV vector particle encodes a Spike glycoprotein, or fragment or derivative thereof, that has the same amino acid sequence as the spike protein, or fragment or derivative thereof, that is encoded by vector selected from:
pFlap-ieCMV-S2PAF-WPREm (also named pFlap-ieCMV-S2PdeltaF-WPREm) (CNCM I-5537), pFlap-ieCMV-S2P3F-WPREm (CNCM I-5538), pFlap-ieCMV-S2P-WPREm (CNCM I-5539), pFlap-ieCMV-SFL-WPREm (CNCM I-5540), pFlap-ieCMV-S-B1.1.7-WPREm (CNCM I-5708), pFlap-ieCMV-S-B351-WPREm (CNCM I-5709), pFlap-ieCMV-S-B351-2P-WPREm (CNCM I-5710), pFlap-ieCMV-SFL-D614G-WPREm (CNCM I-5711), and pFlap-ieCMV-S-P1-WPREm (CNCM I-5712).
Also provided is a vector selected from: pFlap-ieCMV-S2PAF-WPREm (also named pFlap-ieCMV-S2PdeltaF-WPREm) (CNCM I-5537), pFlap-ieCMV-S2P3F-WPREm (CNCM I-5538), pFlap-ieCMV-S2P-WPREm (CNCM I-5539), pFlap-ieCMV-SFL-WPREm (CNCM I-5540), pFlap-ieCMV-S-B1.1.7-WPREm (CNCM I-5708), pFlap-ieCMV-S-B351-WPREm (CNCM I-5709), pFlap-ieCMV-S-B351-2P-WPREm (CNCM I-5710), pFlap-ieCMV-SFL-D614G-WPREm (CNCM I-5711), and pFlap-ieCMV-S-P1-WPREm (CNCM I-5712).
Also provided is a host cell comprising a vector selected from: pFlap-ieCMV-S2PΔF-WPREm (CNCM I-5537), pFlap-ieCMV-S2P3F-WPREm (CNCM I-5538), pFlap-ieCMV-S2P-WPREm (CNCM I-5539), pFlap-ieCMV-SFL-WPREm (CNCM I-5540), pFlap-ieCMV-S-B1.1.7-WPREm (CNCM I-5708), pFlap-ieCMV-S-B351-WPREm (CNCM I-5709), pFlap-ieCMV-S-B351-2P-WPREm (CNCM I-5710), pFlap-ieCMV-SFL-D614G-WPREm (CNCM I-5711), pFlap-ieCMV-S-P1-WPREm (CNCM I-5712). In some embodiments the vector is stably integrated into the host cell genome, while in other embodiments it is not.
Also provided is a pseudotyped LV vector particle encoding a SARS-CoV-2 Spike (S) glycoprotein or a derivative or fragment thereof, wherein the pseudotyped LV vector particle is made by a method comprising co-transfection of a host cell with a vector selected from: pFlap-ieCMV-S2PΔF-WPREm (CNCM I-5537), pFlap-ieCMV-S2P3F-WPREm (CNCM I-5538), pFlap-ieCMV-S2P-WPREm (CNCM I-5539), pFlap-ieCMV-SFL-WPREm (CNCM I-5540), pFlap-ieCMV-S-B1.1.7-WPREm (CNCM I-5708), pFlap-ieCMV-S-B351-WPREm (CNCM I-5709), pFlap-ieCMV-S-B351-2P-WPREm (CNCM I-5710), pFlap-ieCMV-SFL-D614G-WPREm (CNCM I-5711), and pFlap-ieCMV-S-P1-WPREm (CNCM I-5712).
The sequences disclosed herein that are related to the transgene constructs are specified by their SEQ ID No. as follows:
The inventions described herein are based in part on the potent vaccination strategy demonstrated in the examples. The examples demonstrate the utility of the vaccine strategy, which is based in certain embodiments on lentiviral vectors (LVs), able to induce neutralizing antibodies specific to the Spike glycoprotein (S) of SARS-CoV-2, the etiologic agent of CoronaVirus Disease 2019 (COVID-19). Among several LV encoding distinct variants of S, one encoding the full-length, membrane anchored S (LV::SFL) and one encoding the mutated prefusion (an optionally stabilized) form such as in LV::SΔF2P (also designated LV::S2PΔF or LV::S2PDF or LV::S2PdeltaF) triggered high antibody titers in mice and hamsters, with substantial capacity to inhibit in vitro and in vivo viral invasion of host cells, expressing human Angiotensin-Converting Enzyme 2 (hACE2), the receptor for SARS-CoV-2 entry. S-specific T cells were also abundantly induced in LV::SFL- or LV::SΔF2P-vaccinated individuals. In mice, in which the expression of hACE2 was induced by transduction of the respiratory tract cells by an adenoviral type 5 (Ad5) vector or by transgenesis with hACE2 vectorized by LV vector (B6.K18-hACE2IP-THV mice), as well as in hamsters, substantial or full protective effect against pulmonary SARS-CoV-2 replication was afforded when LV::SFL or LV::SΔF2P was used in systemic prime immunization, followed by intranasal mucosal boost/target. The conferred protection avoided pulmonary inflammation and prevented tissue damage. Besides, in B6.K18-hACE2IP-THV mice with substantial brain permissibility to SARS-CoV-2 replication, protection was shown to extend to the brain and to CNS. The results presented demonstrate marked prophylactic effects of an LV-based vaccination strategy against SARS-CoV-2 in pre-clinical animal models and designate in particular the intranasal LV::SFL-based immunization as a vigorous and promising vaccine approach against COVID-19. The i.n. boost after a systemic prime with LV-based vaccine is required to reach full protection of CNS in the developed transgenic model, which is a stringent model of SARS-CoV-2 infection with particularly high permissibility of brain to SARS-CoV-2 replication.
Various aspects of this disclosure incorporate a SARS-CoV-2 S protein. In a preferred embodiment the SARS-CoV-2 S Protein comprises the following amino acid sequence (Genbank: YP_009724390.1; SEQ ID NO: 1):
In another preferred embodiment the SARS-CoV-2 S protein consists of the amino acid sequence (Genbank: YP_009724390.1; SEQ ID NO: 1).
It is pointed out that, unless it would appear technically not applicable to the person skilled in the art, the definitions provided herein for the SARS-CoV-2 S protein or the polynucleotide encoding the SARS-CoV-2 S protein similarly apply to the derivatives or to the fragments of the SARS-CoV-2 S protein defined with respect to the sequences of SEQ ID No. 1 or respectively SEQ ID No.2.
In some embodiments the SARS-CoV-2 S protein comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1. Such SARS-CoV-2 S protein may qualify as a SARS-CoV-2 S protein derivative and/or as a SARS-CoV-2 S protein fragment if the obtained sequence is shorter than SEQ ID NO: 1. It may also be a sequence of a SARS-CoV-2 S protein expressed by a different strain of the virus than the originally identified isolate Wuhan-Hu-1 (accession number MN908947).
In some embodiments the SARS-CoV-2 S protein consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1. Such SARS-CoV-2 S protein may qualify as a SARS-CoV-2 S protein derivative and/or as a SARS-CoV-2 S protein fragment if the obtained sequence is shorter than SEQ ID NO: 1. It may also be a sequence of a SARS-CoV-2 S protein expressed by a different strain of the virus than the originally identified isolate Wuhan-Hu-1 (accession number MN908947). In some embodiments, the SARS-CoV-2 S protein derivative has an amino acid sequence at least 95% identical or at least 99% identical to SEQ ID NO:1. In one embodiment the SARS-CoV-2 spike protein derivative or fragment has the amino acid sequence of SEQ ID No. 8, SEQ ID No. 11, SEQ ID No. 108, SEQ ID No. 111, SEQ ID No. 114, SEQ ID No. 117, or SEQ ID No. 120, or the SARS-CoV-2 S protein derivative has an amino acid sequence at least 95% identical or at least 99% identical to S SEQ ID No. 8, SEQ ID No. 11, SEQ ID No. 108, SEQ ID No. 111, SEQ ID No. 114, SEQ ID No. 117, or SEQ ID No. 120 or the SARS-CoV-2 spike protein fragment has the amino acid sequence of SEQ ID No. 14 or the SARS-CoV-2 S protein derivative has an amino acid sequence at least 95% identical or at least 99% identical to SEQ ID NO: 14.
In some embodiments the SARS-CoV-2 S protein comprises an amino acid sequence that has 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid changes relative to SEQ ID NO: 1. In some embodiments the SARS-CoV-2 S protein comprises of an amino acid sequence that has no more than 1, no more than 2, no more than 3, no more than 4, no more than 5, no more than 6, no more than 7, no more than 8, no more than 9 or no more than 10 amino acid changes relative to SEQ ID NO: 1. Such SARS-CoV-2 S protein may qualify as a SARS-CoV-2 S protein derivative and/or as a SARS-CoV-2 S protein fragment if the obtained sequence is shorter than SEQ ID NO: 1. It may also be a sequence of a SARS-CoV-2 S protein expressed by a different variant of the virus than the originally identified isolate Wuhan-Hu-1 (accession number MN908947).
In some embodiments the SARS-CoV-2 S protein consists of an amino acid sequence that has 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid changes relative to SEQ ID NO: 1. In some embodiments the SARS-CoV-2 S protein consist of an amino acid sequence that has no more than 1, no more than 2, no more than 3, no more than 4, no more than 5, no more than 6, no more than 7, no more than 8, no more than 9 or no more than 10 amino acid changes relative to SEQ ID NO: 1 in particular no more than 10 amino acid changes at a single location in the protein. In some embodiments the SARS-CoV-2 S protein harbors mutation(s) such as those of the nucleotide sequence encoding S2PΔF or S2P3F In some embodiments a SARS-CoV-2 Spike protein comprises mutation(s) in the Receptor Binding Domain of the protein. In some embodiments the SARS-CoV-2 Spike protein harbors a substitution at residue 614 such as D614G or comprises such substitution. In some embodiments the SARS-CoV-2 Spike protein harbors mutation(s) identified in so-called variant SARS-CoV-2 VUI 2020 12/01 S protein i.e., mutations by substitution or deletion of amino acid residues of the Spike protein such as deletion 69-70, deletion 144, N501Y, substitutions A570D, D614G, P681H, T716I, S982A and D1118H. In some embodiments the SARS-CoV-2 Spike protein harbors mutation(s) that are present in SEQ ID No. 108, SEQ ID No. 111, SEQ ID No. 114, SEQ ID No. 117, or SEQ ID No. 120.
In a preferred embodiment the SARS-CoV-2 S protein is encoded by a nucleotide sequence that comprises nucleotides 21563 to 25384 of Genbank: NC_045512.2 (SEQ ID NO: 2):
In a preferred embodiment the SARS-CoV-2 S protein is encoded by a nucleotide sequence that consists of nucleotides 21563 to 25384 of Genbank: NC_045512.2 (SEQ ID NO: 2).
In some embodiments the SARS-CoV-2 S protein is encoded by a nucleotide sequence that is at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 2. Such SARS-CoV-2 S protein may qualify as a SARS-CoV-2 S protein derivative and/or as a SARS-CoV-2 S protein fragment if the nucleotide sequence having such defined percentage of identity is shorter than SEQ ID NO: 2. It may also be a sequence encoding a SARS-CoV-2 S protein which originates from a different strain of the virus than the originally identified isolate Wuhan-Hu-1 (accession number MN908947). In some embodiments the nucleotide sequence encoding the SARS-CoV-2 Spike protein harbors mutation(s) encompassing at least one non-synonymous mutation. In some embodiments the SARS-CoV-2 S protein is encoded by a nucleotide sequence that harbors mutation(s) such as those of the nucleotide sequence encoding S2PΔF or S2P3F. In some embodiments the nucleotide sequence encoding the SARS-CoV-2 Spike protein harbors a mutation at location 23403 in the sequence of SEQ ID No.2 wherein codon GGT is mutated, in particular substituted for codon GAT (corresponding to mutation at location 614, in particular to D614G substitution in the encoded protein). In some embodiments the nucleotide sequence is the sequence encoding the Spike protein of the so-called variant SARS-CoV-2 VUI 2020 12/01 wherein the Spike protein harbors multiple mutations by substitution or deletion of nucleotides wherein the mutations lead to the following changes in the amino acid residues of the encoded Spike protein: deletion 69-70, deletion 144, substitutions N501Y, A570D, D614G, P681H, T716I, S982A and D1118H.
In some embodiments the SARS-CoV-2 S protein is encoded by a nucleotide sequence that is codon-optimized, such as a codon optimized variant of SEQ ID NO: 2.
In some embodiments, the SARS-CoV-2 S protein comprises K986P and V987P amino acid substitutions.
In some embodiments, the SARS-CoV-2 S protein comprises a modification in which amino acids 681-686 are changed PRRARS (SEQ ID NO: 22) to PGSAGS (SEQ ID NO: 23).
In some embodiments, the SARS-CoV-2 S protein comprises a modification in which amino acids 675-685 (QTQTNSPRRAR (SEQ ID NO: 24)) are deleted.
Within the context of this invention, a “lentiviral vector” means a non-replicating vector for the transduction of a host cell with a transgene comprising cis-acting lentiviral RNA or DNA sequences, and requiring lentiviral proteins (e.g., Gag, Pol, and/or Env) that are provided in trans. The lentiviral vector lacks expression of functional Gag, Pol, and Env proteins. The lentiviral vector may be present in the form of an RNA or DNA molecule, depending on the stage of production or development of said retroviral vectors.
The lentiviral vector can be in the form of a recombinant DNA molecule, such as a plasmid. The lentiviral vector can be in the form of a lentiviral vector particle, such as an RNA molecule(s) within a complex of lentiviral other proteins. Typically, lentiviral particle vectors, which correspond to modified or recombinant lentivirus particles, comprise a genome which is composed of two copies of single-stranded RNA. These RNA sequences can be obtained by transcription from a double-stranded DNA sequence inserted into a host cell genome (proviral vector DNA) or can be obtained from the transient expression of plasmid DNA (plasmid vector DNA) in a transformed host cell.
The lentiviral vector particles may have the capacity for integration. As such, they contain a functional integrase protein. Alternatively, the lentiviral vector particles may have impaired or no capacity for integration. Non-integrating vector particles have one or more mutations that eliminate most or all of the integrating capacity of the lentiviral vector particles. For, example, a non-integrating vector particle can contain mutation(s) in the integrase encoded by the lentiviral pol gene that cause a reduction in integrating capacity. In contrast, an integrating vector particle comprises a functional integrase protein that does not contain any mutations that eliminate most or all of the integrating capacity of the lentiviral vector particles.
In some embodiments the lentiviral vector particles are integrative (ILV).
In some embodiments the lentiviral vector particles are non-integrative (NILV).
Lentiviral vectors derive from lentiviruses, in particular human immunodeficiency virus (HIV-1 or HIV-2), simian immunodeficiency virus (SIV), equine infectious encephalitis virus (EIAV), caprine arthritis encephalitis virus (CAEV), bovine immunodeficiency virus (BIV) and feline immunodeficiency virus (FIV), which are modified to remove genetic determinants involved in pathogenicity and introduce new determinants useful for obtaining therapeutic effects. Preferably lentiviral vectors derive from HIV-1.
Such vectors are based on the separation of the cis- and trans-acting sequences. In order to generate replication-defective vectors, the trans-acting sequences (e.g., gag, pol, tat, rev, and env genes) can be deleted and replaced by an expression cassette encoding a transgene.
Efficient integration and replication in non-dividing cells generally requires the presence of two cis-acting sequences at the center of the lentiviral genome, the central polypurine tract (cPPT) and the central termination sequence (CTS). These lead to the formation of a triple-stranded DNA structure called the central DNA “flap”, which acts as a signal for uncoating of the pre-integration complex at the nuclear pore and efficient importation of the expression cassette into the nucleus of non-dividing cells, such as dendritic cells.
In one embodiment, the invention encompasses a lentiviral vector comprising a central polypurine tract and central termination sequence referred to as cPPT/CTS sequence as described, in particular, in the European patent application EP 2 169 073.
Further sequences are usually present in cis, such as the long terminal repeats (LTRs) that are involved in integration of the vector proviral DNA sequence into a host cell genome. Vectors may be obtained by mutating the LTR sequences, for instance, in domain U3 of said LTR (AU3) (Miyoshi H et al, 1998, J Virol. 72(10):8150-7; Zufferey et al., 1998, J Virol 72(12):9873-80).
In some embodiments the vector does not contain an enhancer. In some embodiments the lentiviral vector comprises LTR sequences, preferably with a mutated U3 region (ΔU3) removing promoter and enhancer sequences in the 3′ LTR.
The packaging sequence ψ (psi) can also be incorporated to help the encapsidation of the polynucleotide sequence into the vector particles (Kessler et al., 2007, Leukemia, 21(9):1859-74; Paschen et al., 2004, Cancer Immunol Immunother 12(6): 196-203).
In some embodiments, the invention encompasses a lentiviral vector comprising a lentiviral packaging sequence ψ (psi).
Further additional functional sequences, such as a transport RNA-binding site or primer binding site (PBS) or a Woodchuck PostTranscriptional Regulatory Element (WPRE) wild type or mutated (WPREm) a mutation being introduced to the start codon of protein X in WPRE to avoid expression of X protein peptide, can also be included in the lentiviral vector polynucleotide sequence, which in some embodiments allows for a more stable expression of the transgene in vivo.
In some embodiments, the lentiviral vector comprises a PBS. In one embodiment, the invention encompasses a lentiviral vector comprising a WPRE and/or an IRES.
In some embodiments, the lentiviral vector comprises at least one cPPT/CTS sequence, one ψ sequence, one (preferably 2) LTR sequence, and an expression cassette including a transgene under the transcriptional control of a cytomegalovirus (CMV) immediate-early promoter, a β2m promoter or a class I MHC promoter.
Methods of producing lentiviral vector particles and lentiviral vector particles are also provided. A lentiviral vector particle (or lentiviral particle vector) comprises a lentiviral vector in association with viral proteins. The vector may be an integrating vector (IL) (in particular for the preparation of transgenic mice as illustrated below) or may be a non-integrating vector (NIL) in particular for administration to human subject.
In some embodiments, the lentiviral vector particles encode a SARS-CoV-2 S protein or a derivative or fragment thereof according to any of the embodiments disclosed herein.
In some embodiments, the lentiviral vector particles encode a SARS-CoV-2 S protein or a derivative or fragment thereof that comprises or consists of an amino acid sequence selected from SEQ ID NOS: 1, 5, 8, 11, 14, 108, 111, 114, 117, and 120.
In some embodiments, the lentiviral vector particles encode a SARS-CoV-2 S protein or a derivative or fragment thereof that comprises or consists of the amino acid sequence of SEQ ID NO: 1.
In some embodiments, the lentiviral vector particles encode a SARS-CoV-2 S protein or a derivative or fragment thereof that comprises or consists of the amino acid sequence of SEQ ID NO: 5.
In some embodiments, the lentiviral vector particles encode a SARS-CoV-2 S protein or a derivative or fragment thereof that comprises or consists of the amino acid sequence of SEQ ID NO: 8.
In some embodiments, the lentiviral vector particles encode a SARS-CoV-2 S protein or a derivative or fragment thereof that comprises or consists of the amino acid sequence of SEQ ID NO: 11.
In some embodiments, the lentiviral vector particles encode a SARS-CoV-2 S protein or a derivative or fragment thereof that comprises or consists of the amino acid sequence of SEQ ID NO: 14.
In some embodiments, the lentiviral vector particles encode a SARS-CoV-2 S protein or a derivative or fragment thereof that comprises or consists of the amino acid sequence of SEQ ID NO: 108.
In some embodiments, the lentiviral vector particles encode a SARS-CoV-2 S protein or a derivative or fragment thereof that comprises or consists of the amino acid sequence of SEQ ID NO: 111.
In some embodiments, the lentiviral vector particles encode a SARS-CoV-2 S protein or a derivative or fragment thereof that comprises or consists of the amino acid sequence of SEQ ID NO: 114.
In some embodiments, the lentiviral vector particles encode a SARS-CoV-2 S protein or a derivative or fragment thereof that comprises or consists of the amino acid sequence of SEQ ID NO: 117.
In some embodiments, the lentiviral vector particles encode a SARS-CoV-2 S protein or a derivative or fragment thereof that comprises or consists of the amino acid sequence of SEQ ID NO: 120.
In some embodiments, the lentiviral vector particles encode a SARS-CoV-2 S protein or a derivative or fragment thereof that consists of the amino acid sequence Genbank: YP_009724390.1 (SEQ ID NO: 1).
In some embodiments, the lentiviral vector particles encode a SARS-CoV-2 S protein or a derivative or fragment thereof that comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1. The specific embodiments of such protein S derivative or fragment are also encompassed within these embodiments of the lentiviral vector particles.
In some embodiments, the lentiviral vector particles encode a SARS-CoV-2 S protein or a derivative or fragment thereof that comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NOS: 5, 8, 11, 14, 108, 111, 114, 117, or 120. The specific embodiments of such protein S derivative or fragment are also encompassed within these embodiments of the lentiviral vector particles.
In some embodiments, the lentiviral vector particles encode a SARS-CoV-2 S protein or a derivative or fragment thereof that consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1. The specific embodiments of such protein S derivative or fragment disclosed herein are also encompassed within these embodiments of the lentiviral vector particles.
In some embodiments, the lentiviral vector particles encode a SARS-CoV-2 S protein or a derivative or fragment thereof that consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NOS: 5, 8, 11, 14, 108, 111, 114, 117, or 120. The specific embodiments of such protein S derivative or fragment are also encompassed within these embodiments of the lentiviral vector particles.
In some embodiments, the lentiviral vector particles encode a SARS-CoV-2 S protein or a derivative or fragment thereof that comprises an amino acid sequence that has 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid changes relative to SEQ ID NO: 1. In some embodiments the SARS-CoV-2 S protein comprises of an amino acid sequence that has no more than 1, no more than 2, no more than 3, no more than 4, no more than 5, no more than 6, no more than 7, no more than 8, no more than 9 or no more than 10 amino acid changes relative to SEQ ID NO: 1.
In some embodiments, the lentiviral vector particles encode a SARS-CoV-2 S protein or a derivative or fragment thereof that comprises an amino acid sequence that has 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid changes relative to SEQ ID NOS: 5, 8, 11, 14, 108, 111, 114, 117, or 120. In some embodiments the SARS-CoV-2 S protein comprises of an amino acid sequence that has no more than 1, no more than 2, no more than 3, no more than 4, no more than 5, no more than 6, no more than 7, no more than 8, no more than 9 or no more than 10 amino acid changes relative to SEQ ID NO: 1.
In some embodiments, the lentiviral vector particles encode a SARS-CoV-2 S protein or a derivative or fragment thereof that consists of an amino acid sequence that has 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid changes relative to SEQ ID NO: 1. In some embodiments the SARS-CoV-2 S protein consists of an amino acid sequence that has no more than 1, no more than 2, no more than 3, no more than 4, no more than 5, no more than 6, no more than 7, no more than 8, no more than 9 or no more than 10 amino acid changes relative to SEQ ID NO: 1.
In some embodiments, the lentiviral vector particles encode a SARS-CoV-2 S protein or a derivative or fragment thereof that consists of an amino acid sequence that has 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid changes relative to SEQ ID NOS: 5, 8, 11, 14, 108, 111, 114, 117, or 120. In some embodiments the SARS-CoV-2 S protein consists of an amino acid sequence that has no more than 1, no more than 2, no more than 3, no more than 4, no more than 5, no more than 6, no more than 7, no more than 8, no more than 9 or no more than 10 amino acid changes relative to SEQ ID NO: 1.
In some embodiments the lentiviral vector particles encode a SARS-CoV-2 Spike protein that harbors mutation(s) such as those contained in S2PΔF (S2PdeltaF) or S2P3F protein derivatives.
In some embodiments the lentiviral vector particles encode a SARS-CoV-2 Spike protein that harbors a substitution at residue 614 such as D614G or that comprises such substitution. In some embodiments the lentiviral vector particles encode a SARS-CoV-2 Spike protein that harbors mutation(s) identified in so-called variant SARS-CoV-2 VUI 2020 12/01 S protein i.e., mutations by substitution or deletion of amino acid residues of the Spike protein such as deletion 69-70, deletion 144, N501Y, substitutions A570D, D614G, P681H, T716I, S982A and D1118H.
In some embodiments the lentiviral vector particles encode a SARS-CoV-2 S protein that is encoded by a nucleotide sequence that comprises SEQ ID NO: 2.
In some embodiments the lentiviral vector particles encode a SARS-CoV-2 S protein that is encoded by a nucleotide sequence that consists of nucleotides 21563 to 25384 of Genbank: NC_045512.2 (SEQ ID NO: 2).
In some embodiments the lentiviral vector particles comprise a nucleotide sequence that is at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 2.
In some embodiments the lentiviral vector particles encode a SARS-CoV-2 S protein that is encoded by the nucleotide sequence that harbors mutation(s) with respect to the sequence of SEQ ID NO: 2, wherein the mutation(s) encompass at least one non-synonymous mutation. In some embodiments the lentiviral vector particles encode a SARS-CoV-2 S protein whose nucleotide sequence harbors a mutation at location 23403 in the sequence of SEQ ID No.2 wherein codon GGT is mutated, in particular substituted for codon GAT (corresponding to mutation at location 614, in particular to D614G substitution in the encoded S protein of SEQ ID No.1). In some embodiments the lentiviral vector particles encode the Spike protein of the so-called variant SARS-CoV-2 VUI 2020 12/01 wherein the Spike protein harbors multiple mutations by substitution or deletion of nucleotides with respect to the sequence of SEQ ID No.2 and wherein the nucleotide mutations lead to the following changes in the amino acid residues of the encoded Spike protein: deletion 69-70, deletion 144, substitutions N501Y, A570D, D614G, P681H, T716I, S982A and D1118H.
In some embodiments the lentiviral vector particles comprise a nucleotide sequence that is codon-optimized, such as a codon optimized variant of SEQ ID NO: 2 or a codon optimized variant of the nucleotide sequence encoding the S2PΔF (S2PdeltaF) or the S2P3F derivatives.
In some embodiments the lentiviral vector particles comprise a nucleotide sequence that encodes a SARS-CoV-2 S protein that comprises K986P and V987P amino acid substitutions.
In some embodiments the lentiviral vector particles comprise a nucleotide sequence that encodes a SARS-CoV-2 S protein that comprises a modification in which amino acids 681-686 PRRARS (SEQ ID No.22) are changed to PGSAGS (SEQ ID No.23) such as in LV::S2P3F.
In some embodiments the lentiviral vector particles comprise a nucleotide sequence that encodes a SARS-CoV-2 S protein that comprises a modification in which amino acids 675-685 (QTQTNSPRRAR) (SEQ ID No.24) are deleted such as in LV::S2PΔF (LV::S2PdeltaF).
In some embodiments, the pseudotyped lentiviral vector particles comprise a polynucleotide selected from:
In some embodiments, the lentiviral vector particle comprises HIV-1 Gag and Pol proteins. In some embodiments, the lentiviral vector particle comprises subtype D, especially HIV-1NDK, Gag and Pol proteins.
According to some embodiments, the lentivector particles are obtained in a host cell transformed with a DNA plasmid.
Such a DNA plasmid can comprise:
Such a method allows producing a recombinant vector particle according to the invention, comprising the following steps of:
i) transfecting a suitable host cell with a lentiviral vector;
ii) transfecting said host cell with a packaging plasmid vector, containing viral DNA sequences encoding at least structural and polymerase (+ integrase) activities of a retrovirus (preferably lentivirus); Such packaging plasmids are described in the art (Dull et al., 1998, J Virol, 72(11):8463-71; Zufferey et al., 1998, J Virol 72(12):9873-80).
iii) culturing said transfected host cell in order to obtain expression and packaging of said lentiviral vector into lentiviral vector particles; and
iv) harvesting the lentiviral vector particles resulting from the expression and packaging of step iii) in said cultured host cells.
For different reasons, in particular for administration to a human subject, it may be helpful to pseudotype the obtained retroviral particles, i.e. to add or replace specific particle envelope proteins. In some embodiments pseudotyping extends the spectrum of cell types that may be transduced while avoiding being the target of pre-existing immunity in human populations.
In order to pseudotype the retroviral particles of the invention, the host cell can be further transfected with one or several envelope DNA plasmid(s) encoding viral envelope protein(s), preferably a VSV-G envelope protein.
An appropriate host cell is preferably a human cultured cell line as, for example, a HEK cell line, such as a HEK293T line.
Alternatively, the method for producing the vector particle is carried out in a host cell, which genome has been stably transformed with one or more of the following components: a lentiviral vector DNA sequence, the packaging genes, and the envelope gene. Such a DNA sequence may be regarded as being similar to a proviral vector according to the invention, comprising an additional promoter to allow the transcription of the vector sequence and improve the particle production rate.
In a preferred embodiment, the host cell is further modified to be able to produce viral particle in a culture medium in a continuous manner, without the entire cells swelling or dying. One may refer to Strang et al., 2005, J Virol 79(3):1165-71; Relander et al., 2005, Mol Ther 11(3):452-9; Stewart et al., 2009, Gene Ther, 16(6):805-14; and Stuart et al., 2011, Hum gene Ther, with respect to such techniques for producing viral particles.
An object of the present invention consists of a host cell transformed with a lentiviral particle vector.
The lentiviral particle vectors can comprise the following elements, as previously defined:
Preferably, the lentivector particles are in a dose of 106, 2×106, 5×106, 107, 2×107, 5×107, 108, 2×108, 5×108, or 109 TU.
This disclosure provides pseudotyped lentiviral vector particles bearing a SARS-CoV-2 S protein according to this disclosure. The lentivector can be integrative or non-integrative. The lentiviral vectors are pseudotyped lentiviral vectors (i.e. “lentiviral vector particles”) bearing a SARS-CoV-2 S protein.
The disclosure also provides an immunogenic composition comprising a lentiviral vector particle bearing a SARS-CoV-2 S protein according to this disclosure. All embodiments disclosed herein in relation to the lentiviral particles apply to the definition of the immunogenic composition.
In some embodiments, the immunogenic composition is for use in a method of prevention of infection of a human subject by SARS-CoV-2. In some embodiments, the immunogenic composition is for use in a method of protection against SARS-CoV-2 replication in a human subject at risk of being exposed to SARS-CoV-2 or infected by SARS-CoV-2. In some embodiments, the immunogenic composition is for use in a method of preventing development of symptoms or development of a disease associated with infection by SARS-CoV-2, such as COVID-19 in a human subject at risk of being exposed to SARS-CoV-2 or infected by SARS-CoV-2. In some embodiments, the immunogenic composition is for use in a method of preventing the onset of neurological outcome associated with infection by SARS-CoV-2 in a human subject at risk of being exposed to SARS-CoV-2 or infected by SARS-CoV-2. In some embodiments, the immunogenic composition is for use in a method of protecting the Central Nervous System (CNS) of a human subject at risk of being exposed to SARS-CoV-2 or infected by SARS-CoV-2. In some embodiments, in any of these applications for use in a method disclosed, the immunogenic composition may be administered to the subject as a prophylactic agent in an effective amount for elicitation of an immune response against SARS-CoV-2.
In some embodiment the immunogenic composition is for use in a method of protection of a human subject against SARS-CoV-2 infection or against development of the symptoms or the disease (COVID-19) associated with SARS-CoV-2 infection, wherein the subject is at risk of developing lung and/or CNS pathology. In particular the human subject is in need of immune protection of CNS from SARS-CoV-2 replication because he/she is affected with comorbid conditions, in particular comorbid conditions affecting the CNS.
The disclosure also provides a vaccine composition comprising a lentiviral vector particle bearing a SARS-CoV-2 S protein according to this disclosure and a carrier. In some embodiments the vaccine reduces the likelihood that a vaccinated subject, especially a human subject, will develop COVID-19. In some embodiments the reduction is by at least 30%, 40%, 50%, 60%, 70%, 80% or 90%. In some embodiments the vaccine reduces COVID-19 disease severity in a subject by at least 30%, 40%, 50%, 60%, 70%, 80%, or 90%. In some embodiments the reduction is by at least 30%, 40%, 50%, 60%, 70%, 80%, or 90%.
In some embodiments the vaccine provides protection against the infection by SARS-Cov-2, especially sterilizing protection. In some embodiments, the vaccine is for use in a method as disclosed herein in respect of the immunogenic composition.
The herein disclosed immunogenic composition and vaccine may be administered according to the administration route and administration regimen disclosed herein, in particular in accordance with the specific embodiments disclosed in C. below in particular in accordance with the illustrated embodiments.
Also provided are methods of inducing or activating a protective immune response against Severe Acute Respiratory Syndrome beta-coronavirus 2 (SARS-CoV-2), comprising administering to the upper respiratory tract of a subject an effective amount of an agent that induces a protective immune response against SARS-CoV-2. In certain embodiments the agent that induces a protective immune response against SARS-CoV-2 is a pseudotyped lentiviral vector particle encoding a Severe Acute Respiratory Syndrome beta-coronavirus 2 (SARS-CoV-2) spike (S) protein or a derivative or fragment thereof. The disclosure of the methods herein is similarly applicable to the immunogenic composition for use in a method as disclosed in the present disclosure or to the vaccine for use in a method as disclosed in the present disclosure.
In some embodiments the agent is administered by nasal inhalation.
As used herein, “administered to the upper respiratory tract” includes any type of administration that results in delivery to the mucosa lining of the upper respiratory tract and includes in particular nasal administration. Administration to the upper respiratory tract includes without limitation aerosol inhalation, nasal instillation, nasal insufflation, and all combinations thereof. In some embodiments the administration is by aerosol inhalation. In some embodiments the administration is by nasal instillation. In some embodiments the administration is by nasal insufflation.
In some embodiments the treatment course consists of a single administration to the upper respiratory tract. In some embodiments the treatment course comprises a plurality of administrations to the upper respiratory tract. In some embodiments the treatment course comprises at least one administration to the upper respiratory tract and at least one administration outside of the respiratory tract. In some embodiments the treatment course comprises at least one priming administration via route outside of the respiratory tract followed by at least one boosting administration to the upper respiratory tract. The administration outside of the respiratory tract may be intramuscular, intradermal or subcutaneous. In some embodiments the treatment course comprises at least a prime/boost or a prime/target administration. In some embodiments the administration regimen comprises or consists of a prime administration outside of the upper respiratory tract, such as systemic (in particular intramuscular) administration and a boost or a target administration to the upper respiratory tract. The administered doses of the agent may be identical or may be different in the prime and boost/target administration steps, in particular may be higher for the administration to the upper respiratory tract. Details for the administration to the upper respiratory tract are provided below.
In a particular embodiment the lentiviral vector particles are LV::SFL, in particular NILV::SFL and the administration regimen consists in a systemic, especially i.m. prime and a boost to the upper respiratory tract, in particular by i.n. boost.
In a particular embodiment the lentiviral vector particles are LV::Sprefusion, in particular NILV::Sprefusion, such as LV::S2PΔF or NILV::S2PΔF, or LV::S2P3F or NI LV::S2P3F and the administration regimen consists in a systemic, especially i.m. prime and a boost to the upper respiratory tract, in particular by i.n. boost.
In some embodiments, the lentiviral vector particles comprise a polynucleotide selected from:
In some embodiments the protective immune response comprises production of SARS-CoV-2 neutralizing antibodies in the subject. In some embodiments the neutralizing antibodies comprise IgG antibodies. In some embodiments the protective immune response comprises production of SARS-CoV-2 S-specific T cells in the subject. In some embodiments the SARS-CoV-2 S-specific T cells comprise CD4+ T cells. In some embodiments the SARS-CoV-2 S-specific T cells comprise CD8+ T cells. In some embodiments the SARS-CoV-2 S-specific T cells comprise CD4+ T cells and CD8+ T cells. In some embodiments the SARS-CoV-2 S-specific T cells comprise lung CD8+ T cells. In some embodiments the SARS-CoV-2 S-specific T cells comprise IFN-γ-producing T-cells. In some embodiments the SARS-CoV-2 S-specific T cells comprise T cells with an effector memory (Tem) and/or resident memory (Trm) phenotype. In some embodiments the SARS-CoV-2 S-specific T cells are recruited to the olfactory bulb. In some embodiments the protective immune response reduces the development of at least one symptom of a SARS-CoV-2 infection. In some embodiments the protective immune response reduces the time period during which an infected subject suffers from at least one symptom of a SARS-CoV-2 infection. In some embodiments the protective immune response reduces the likelihood of developing SARS-CoV-2 infection-related inflammation in the subject.
In various embodiments, the pseudotyped lentiviral vector particle may encode any Severe Acute Respiratory Syndrome beta-coronavirus 2 (SARS-CoV-2) spike (S) protein or a derivative or fragment thereof that is disclosed herein in the above embodiments relating to the description of the lentiviral vector particles.
In some embodiments the SARS-CoV-2 S protein derivative has an amino acid sequence at least 95% identical to SEQ ID NO: 1. In some embodiments the SARS-CoV-2 S protein derivative is expressed from a coding sequence having a nucleotide sequence at least 80% identical to SEQ ID NO: 2. In some embodiments the SARS-CoV-2 S protein fragment comprises a peptide selected from peptide 61-75 (NVTWFHAIHVSGTNG (SEQ ID NO: 15)), peptide 536-550 (NKCVNFNFNGLTGTG (SEQ ID NO: 16)) and peptide 576-590 (VRDPQTLEILDITPC (SEQ ID NO: 17)). In some embodiments the SARS-CoV-2 S derivative or fragment thereof comprises an amino acid modification relative to SEQ ID NO: 1, the modification selected from: (i) 986K→P and 987V→P, (ii) 681PRRARS686 (SEQ ID NO: 22)→681PGSAGS686 (SEQ ID NO: 23), and (iii) 986K→P, 987V→P, and 675QTQTNSPRRAR685 (SEQ ID NO: 24) deletion. In some embodiments the Severe Acute Respiratory Syndrome beta-coronavirus 2 (SARS-CoV-2) spike (S) protein or a derivative or fragment thereof comprises or consists of an amino acid sequence selected from SEQ ID NOS: 1, 5, 8, 11, 14, 108, 111, 114, 117, and 120.
In some embodiments the administered lentiviral vector particle is integrative. In some embodiments the administered lentiviral vector particle is nonintegrative. In some embodiments the administered nonintegrative lentiviral particle comprises a D64V mutation in an integrase coding sequence. In some embodiments the administered lentiviral vector particle is pseudotyped with Vesicular Stomatitis Virus envelop Glycoprotein (VSV-G). In some embodiments the lentiviral vector particle is administered as a vaccine formulation comprising the lentiviral vector particle and a pharmaceutically acceptable carrier.
In some embodiments, the lentivector contains a promoter that drives high expression of the Severe Acute Respiratory Syndrome beta-coronavirus 2 (SARS-CoV-2) spike (S) protein or a derivative or fragment thereof, and drives expression in sufficient quantity for elimination by the induced immune response. In some embodiments, the promoter lacks an enhancer element to avoid insertional effects.
In some embodiments, at least 95%, 99%, 99.9%, or 99.99% of the lentiviral DNA integrated in cells of a mouse or hamster animal model at day 4 after administration is eliminated by day 21 after administration.
In some embodiments, the lentivector particles are in a dose of 106, 2×106, 5×106, 107, 2×107, 5×107, 108, 2×108, 5×108, or 109 TU.
The immune response induced by the lentiviral vector can be a B cell response, a CD4+ T cell response, and/or a CD8+ T cell response.
The present invention thus provides vectors that are useful as a medicament or vaccine, particularly for administration to the upper respiratory tract.
The disclosed lentiviral vectors have the ability to induce, improve, or in general be associated with the occurrence of a B cell response, a CD4+ T cell response, and/or a CD8+ T cell response, including a memory CTL response.
In some embodiments the lentiviral vector is used in combination with adjuvants, other immunogenic compositions, and/or any other therapeutic treatment.
According to some embodiments the immunogenic compositions as defined or illustrated herein are for use to induce a protective immune response against SARS-CoV-2 in the upper respiratory tract and/or in the brain against SARS-CoV-2 of a subject.
According to some embodiments the immunogenic compositions are for use to induce a cross protective immune response of lungs and brain against ancestral including SARS-CoV-2 selected from the group of SARS-CoV-2 Wuhan strain, SARS-CoV-2 D614G strain and SARS-CoV-2 B1.117 strain and against emerging SARS-CoV-2 variants such as SARS-CoV-2 P.1 variant, by eliciting B and T cell-responses.
According to some embodiments the immunogenic compositions are for use as defined herein and are characterized in that the dosage form or the pseudotyped lentiviral particle comprises pseudotyped lentiviral particles as defined herein wherein the pseudotyped lentiviral particles are non-integrative.
In some embodiments, these immunogenic compositions are for use to elicit a protective immune response against SARS-CoV-2 wherein the response elicits SARS-CoV-2 S-specific T cells, in particular SARS-CoV-2 S-specific T cells that comprise lung CD8+ T cells and/or IFN-γ-producing T-cells.
According to some embodiments the immunogenic compositions are for use to elicit a protective immune response against SARS-CoV-2 wherein the response elicits CD8+ T cells that comprise T cells with an effector memory (Tem) and/or resident memory (Trm) phenotype.
According to some embodiments the immunogenic compositions are for use as defined herein, the SARS-CoV-2 S-specific T cells are recruited to the olfactory bulb.
According to some embodiments the immunogenic compositions for use according to the invention are characterized in that the Severe Acute Respiratory Syndrome beta-coronavirus 2 (SARS-CoV-2) spike (S) protein or a derivative or fragment thereof comprises or consists of an amino acid sequence selected from SEQ ID NOS: 1, 5, 8, 11, 14, 108, 111, 114, 117, and 120.
According to some embodiments the immunogenic compositions are for use to prevent or to alleviate SARS-CoV-2 infection-related inflammation in the subject.
The immunogenic compositions of the disclosure may be provided in a dosage form suitable for administration to the upper respiratory tract of a subject. Appropriate formulations are known in the art. In some embodiments the dosage form is adapted for aerosol inhalation. In some embodiments the dosage form is adapted for nasal instillation. In some embodiments the nasal dosage form is adapted for nasal insufflation. In some embodiments the dosage form is aliquoted in a single dose. In some embodiments the dosage form is packaged in a single dose.
Also provided are kits suitable for use in practicing a method disclosed herein. In some embodiments the kit comprises a dosage form for administration to the upper respiratory tract of a subject of the pseudotyped lentiviral vector particle encoding a SARS-CoV-2 S protein or a derivative or fragment thereof according to this disclosure, and an applicator. In some embodiments the applicator is an applicator for aerosol inhalation. In some embodiments the applicator is an applicator for nasal instillation. In some embodiments the applicator is an applicator for nasal insufflation. Suitable examples of each are known in the art and may be used.
Also provided are novel and nonobvious lentiviral vectors and plasmids for creating the same. The LV and the plasmids encode a Severe Acute Respiratory Syndrome beta-coronavirus 2 (SARS-CoV-2) spike (S) protein or a derivative or fragment thereof.
Having thus described different embodiments of the present invention, it should be noted by those skilled in the art that the disclosures herein are exemplary only and that various other alternatives, adaptations, and modifications may be made within the scope of the present invention. Accordingly, the present invention is not limited to the specific embodiments as illustrated herein.
1. 1.1 Construction of Transfer pFLAP Plasmids Coding SFL, S1-S2, or S1 Derived from SCoV-2.
codon-optimized full-length S (1-1273) sequence was amplified from pMK-RQ_S-2019-nCoV and inserted between BamHI and XhoI sites of pFlap-ieCMV-WPREm. Sequences encoding for S1-S2 (1-1211) or S1 (1-681) were amplified by PCR from the pFlap-ieCMV-SFL-WPREm plasmid and sub-cloned into pFlap-ieCMV-WPREm between the BamHI and XhoI restriction sites. Each of the PCR products were inserted between the native human ieCMV promoter and a mutated WPRE (Woodchuck Posttranscriptional Regulatory Element) sequence, where a mutation was introduced to the start codon of protein X in WPRE to avoid expression of X protein peptide. Plasmids were amplified in Escherichia coli DH5a in Lysogeny Broth (LB) supplemented with 50 μg/ml of kanamycin, purified using the NucleoBond Xtra Maxi EF Kit (Macherey Nagel) and resuspended in Tris-EDTA Endotoxin-Free (TE-EF) buffer overnight. The plasmid was quantified with a NanoDrop 2000c spectrophotometer (Thermo Scientific), adjusted to 1 μg/μl in TE-EF buffer, aliquoted and stored at −20° C. The plasmid DNA was verified by (i) diagnostic check with restriction digestion, and (ii) sequencing the region proximal to the transgene insertion sites.
1. 1.2 Production and Titration of LV Vectors
Non-replicative integrative LV vectors were produced in Human Embryonic Kidney (HEK)-293T cells, as previously detailed (Zennou et al., 2000). 6×106 cells/Petri dish were cultured in DMEM and were co-transfected in a tripartite fashion with 1 ml of a mixture of: (i) 2.5 μg/ml of the pSD-GP-NDK packaging plasmid, coding for codon-optimized gag-pol-tat-rre-rev, (ii) 10 μg/ml of VSV-G Indiana envelop plasmid, and (iii) 10 μg/ml of transfer pFLAP plasmid in Hepes 1× containing 125 mM of Ca(ClO3)2 Supernatants were harvested at 48h post transfection, clarified by 6-minute centrifugation at 2500 rpm at 4° C., then treated for 30 min with benzonase 10 U/ml final concentration at 37° C. in Hepes-buffered solution, containing MgCl2 (2 mM) final to eliminate residual DNA. LV vectors were aliquoted and conserved at −80° C. To determine the titers of LV preparations, HEK-293T were distributed at 4×105 cell/well in flat-bottom 6-well-plates in complete DMEM in the presence of 8 μM aphidicolin (Sigma) which blocks the cell proliferation. The cells were then transduced with serial dilutions of LV preparations. The titer, proportional to the efficacy of nuclear gene transfer, is determined as Transduction Unit (TU)/ml by qPCR on total lysates at day 3 post transduction, by use of forward 5′-TGG AGG AGG AGA TAT GAG GG-3′ (SEQ ID NO: 100) and reverse 5′-CTG CTG CAC TAT ACC AGA CA-3′ (SEQ ID NO: 101) primers, specific to pFLAP plasmid and forward 5′-TCT CCT CTG ACT TCA ACA GC-3′ (SEQ ID NO: 102) and reverse 5′-CCC TGC ACT TTT TAA GAG CC-3′ (SEQ ID NO: 103) primers specific to the host housekeeping gene gadph, as described elsewhere (Iglesias et al., 2006).
1. 1.3 Mouse Studies
Female C57BL/6J mice (Janvier, Le Genest Saint Isle, France) were used between the age of 6 and 10 weeks. Male Mesocricetus auratus golden hamsters (Janvier, Le Genest Saint Isle, France) were purchased mature, i.e. 80-90 gr weight. At the beginning of the immunization regimen they weigh between 100 and 120 gr. Experimentation on animals was performed in accordance with the European and French guidelines (Directive 86/609/CEE and Decree 87-848 of 19 Oct. 1987) subsequent to approval by the Institut Pasteur Safety, Animal Care and Use Committee, protocol agreement delivered by local ethical committee (CETEA #DAP20007) and Ministry of High Education and Research APAFIS #24627-2020031117362508 v1. Mice were vaccinated with the indicated TU of LV via intraperitoneal (i.p.) injection. Sera were collected at various time points post immunization to monitor binding and neutralization activities.
1. 1.4 SARS-CoV-2 Inoculation
Ad5::hACE2-pretreated mice or hamsters were anesthetized by peritoneal injection of mixture Ketamine and Xylazine, transferred into a PSM-III where they were inoculated with 1×105 TCID50 of a SARS-CoV-2 clinical isolate amplified in VeroE6 cells, provided by the Centre National de Reference des Virus Respiratoires, France. The viral inoculum was contained in 20 μl for mice and in 50 μl for hamsters. Animals were then housed in an isolator in BSL3 animal facilities of Institut Pasteur. The organs and fluids recovered from the infected mice, with live SARS-CoV-2 were manipulated following the approved standard operating procedures of the BioSafety Level BSL3 facilities.
1. 1.5 Recombinant SCoV-2 Protein Variants
Codon-optimized nucleotide fragments encoding a stabilized foldon-trimerized version of the SARS-CoV-2 S ectodomain (a.a. 1 to 1208), the S1 monomer (a.a. 16 to 681) and the RBD subdomain (amino acid 331 to 519) both preceded by a murine IgK leader peptide, followed by an 8×His Tag (SEQ ID NO: 104) were synthetized and cloned into pcDNA™3.1/Zeom expression vector (Thermo Fisher Scientific). Proteins were produced by transient co-transfection of exponentially growing Freestyle™ 293-F suspension cells (Thermo Fisher Scientific, Waltham, Mass.) using polyethylenimine (PEI)-precipitation method as previously described (Lorin and Mouquet, 2015). Recombinant SCoV-2 proteins were purified by affinity chromatography using the Ni Sepharose® Excel Resin according to manufacturer's instructions (Thermo Fisher Scientific). Protein purity was evaluated by in-gel protein silver-staining using Pierce Silver Stain kit (Thermo Fisher Scientific) following SDS-PAGE in reducing and non-reducing conditions using NuPAGE™ 3-8% Tris-Acetate gels (Life Technologies). Purified proteins were dialyzed overnight against PBS using Slide-A-Lyzer® dialysis cassettes (10 kDa MW cut-off, Thermo Fisher Scientific). Protein concentration was determined using the NanoDrop™ One instrument (Thermo Fisher Scientific).
1. 1.6 ELISA
Ninety-six-well Nunc Polysorp plates (Nunc, Thermo Scientific) were coated overnight at 4° C. with 100 ng/well of purified tri-S proteins in carbonate buffer pH 9.6. After washings with PBS containing 0.1% Tween 20 (PBST), plate wells were blocked with PBS containing 1% Tween20 and 10% FBS for 2 h at room temperature. After PBST washings, 1:100-diluted sera in PBST containing 10% FBS and 4 consecutive 1:10 dilutions were added and incubated during 2h at 37° C. After PBST washings, plates were incubated with 1,000-fold diluted peroxydase-conjugated goat anti-mouse IgG/IgM (Jackson ImmunoResearch Europe Ltd, Cambridgeshire, United Kingdom) for 1 h. Plates were revealed by adding 100 μl of TMB chromogenic substrate (TMB, Eurobio Scientific) after PBST washings. Optical densities were measured at 450 nm/620 nm on a PR3100 reader following a 30 min incubation.
1. 1.7 nAb Detection
Serial dilutions of plasma were assessed for nAbs via an inhibition assay which uses Human Embryonic Kidney (HEK) 293-T cells transduced to express stably human ACE2, and safe, non-replicative SCoV-2 pseudo-typed LV particles which harbor the reporter luciferase firefly gene, allowing quantitation of the host cell invasion by mimicking fusion step of native SARS-CoV-2 virus (Sterlin et al.). First, 1.5×102 TU of SCoV-2 pseudo-typed LV were pre-incubated, during 30 min at room temperature, in U-bottom plates, with serial dilutions of each serum in a final volume of 50 μl in DMEM, completed with 10% heat-inactivated FCS and 100 U/ml penicillin and 100 μg/ml streptomycin. The samples were then transferred into clear-flat-bottom 96-well-black-plates, and each well received 2×104 hACE2+ HEK293-T cells contained in 50 μl. After 2 days incubation at 37° C. 5% CO2, the transduction efficiency of hACE2+ HEK293-T cells by pseudo-typed LV particles was determined by measuring the luciferase activity, using the Luciferase Assay System Kit with Reporter Lysis Buffer (Promega). To do so, the supernatants were completely removed from the culture wells, 40 μl of Reporter Lysis Buffer 1× and 50 μl of Luciferase Assay Reagent (Luciferase FireFly) were sequentially added to each culture well. The bioluminescent signal was quantified using an LB 960 plate reader (Berthold).
1. 1.8 SFS T-Cell Epitope Mapping
In order to map the immuno-dominant epitopes, peptides spanning the whole spike protein were pooled in ten pools, each containing 15 amino-acid residues overlapping by ten amino acids. Synthetic peptides were purchased from Mimotopes (Australia). IFN-g ELISpot assay was performed as previously described (Dion et al, 2013). These different sets of pooled peptides were used in a matrix assay to map by ICS the epitope responses induced by each construct. Peptides were dissolved in DMSO at a concentration of 2 mg/ml and diluted before use at 1 μg/ml and 2-5 μg/mL with culture medium before their use in ELISpot and ICS assays, respectively. Responses in ELISpot were considered positive if the median number of spot-forming cells in triplicate wells was at least twice that observed in control wells and at least 50 spot-forming cells per million splenocytes were detected after subtraction of the background.
1. 1.9 Generation of Ad5 Gene Transfer Vectors and Intranasal Pretreatment of Mice
The Ad5 gene transfer vectors were produced by use of ViraPower Adenoviral Promoterless Gateway Expression Kit (Thermo Fisher Scientific, France). The pCMV-BamH1-Xho1-WPRE sequence was PCR amplified from the pTRIPΔU3CMV plasmid, by use of: (i) forward primer, encoding the attB1 in the 5′ end, and (ii) reverse primer, encoding both the attB2 and SV40 polyA signal sequence in the 5′ end. The attb-PCR product was cloned into the gateway pDORN207 donor vector, via BP Clonase reaction, to form the pDORN207-CMV-BamH1-Xho1-WPRE-SV40 polyA. The hACE2 was amplified from a plasmid derivative of hACE2-expressing pcDNA3.11 (generous gift from Nicolas Escriou) while egfp was amplified from pTRIP-ieCMV-eGFP-WPRE2. The amplified PCR products were cloned into the pDORN207-CMV-BamH1-Xho1-WPRE-SV40 polyA plasmid via the BamH1 and Xho1 restriction sites. To obtain the final Ad5 plasmid, the pDORN207 vector, harboring hACE2 or gfp genes, was further inserted into pAd/PL-DEST™ vector via LR Clonase reaction.
The Ad5 virions were generated by transfecting the E3-transcomplementing HEK-293A cell line with pAd CMV-GFP-WPRE-SV40 polyA or pAd CMV-hACE2-WPRE-SV40 polyA plasmid followed by subsequent vector amplification, according to the manufacturer's protocol (ViraPower Adenoviral Promoterless Gateway Expression Kit, Thermo Fisher Scientific). The Ad5 particles were purified using Adeno-X rapid Maxi purification kit and concentrated with the Amicon Ultra-4 10k centrifugal filter unit. Vectors were resuspended and stocked à −80° C. in PIPES buffer pH 7.5, supplemented with 2.5% glucose. Ad5 were titrated using qRT-PCR protocol, as described by Gallaher et al3, adapted to HEK-293T cells.
Four days before the challenge, mice were instilled i.n. with 2.4×109 IGU of Ad5::hACE2, Ad5::GFP or control empty vector resuspended in 15 μl of PBS, under general anesthesia, obtained by i.p. injection of a mixture of Ketamine (Imalgene, 100 mg/kg) and Xylazine (Rompun, 10 mg/kg).
1. 1.10 Western Blot
Expression of hACE2 in the lungs of Ad5::hACE2-transduced mice was assessed by Western Blotting. One ×106 cells from lung homogenate were resolved on 4-12% NuPAGE Bis-Tris protein gels (Thermo Fisher Scientific, France), then transferred onto a nitrocellulose membrane (Biorad, France). The nitrocellulose membrane was blocked in 5% non-fat milk in 0.5% Tween PBS (PBS-T) for 2 hours at room temperature and probed overnight with goat anti-hACE2 primary Ab at 1 μg/mL (AF933, R&D systems). Following three washing intervals of 10 minutes with PBS-T, the membrane was incubated for 1 hour at room temperature with HRP-conjugated anti-goat secondary Ab and HRP-conjugated anti-β-actin (ab197277, Abcam). The membrane was washed with PBS-T thrice before visualization with enhanced chemiluminescence via the super signal west femto maximum sensitivity substrate (ThermoFisher, France) on ChemiDoc XRS+ (Biorad, France). PageRuler Plus prestained protein ladder was used as size reference.
1. 1.11 Determination of SARS-CoV-2 Viral Loads in the Lungs
Half of each lung lobes were removed aseptically and were frozen at −80° C. Organs were thawed and homogenized twice for 20 s at 4.0 m/s, using lysing matrix D (MP Biomedical) in 500 μl of ice-cold PBS. The homogenization was performed in an MP Biomedical Fastprep 24 Tissue Homogenizer. Particulate viral RNA was extracted from 70 μl of lung homogenate using QIAamp Viral RNA Mini Kit (Qiagen) according to the manufacturer's procedure. Viral load was determined following reverse transcription and real-time TaqMan® PCR essentially as described by Corman et al. (Corman et al., 2020) using SuperScript™ II Platinum One-Step Quantitative RT-PCR System (Invitrogen) and primers and probe (Eurofins) targeting SARS-CoV-2 envelope (E) gene as listed in (Table 1). In vitro transcribed RNA derived from plasmid pCI/SARS-CoV E was synthesized using T7 RiboMAX Express Large Scale RNA production system (Promega), then purified by phenol/chloroform extractions and two successive precipitations with ethanol. RNA concentration was determined by optical density measurement, then RNA was diluted to 10 genome equivalents/μL in RNAse-free water containing 100 μg/mL tRNA carrier, and stored in single-use aliquots at −80° C. Serial dilutions of this in vitro transcribed RNA were prepared in RNAse-free water containing 10 μg/ml tRNA carrier and used to establish a standard curve in each assay. Thermal cycling conditions were: (i) reverse transcription at 55° C. for 10 min, (ii) enzyme inactivation at 95° C. for 3 min, and (iii) 45 cycles of denaturation/amplification at 95° C. for 15 s, 58° C. for 30 s. Products were analyzed on an ABI 7500 Fast real-time PCR system (Applied Biosystems).
1. 1.12 Cytometric Analysis of Lung Innate Immune Cells
Lungs from individual mice were treated with collagenase-DNAse-I for 30-minute incubation at 370 C and homogenized by use of GentleMacs. Cells were and filtered through 100 μm-pore filters and centrifuged at 1200 rpm during 8 minutes. Cells were then treated with Red Blood Cell Lysing Buffer (Sigma), washed twice in PBS. Cells were then stained as following. (i) To detect DC, monocytes, alveolar and interstitial macrophages: Near IR Live/Dead (Invitrogen), FcγII/III receptor blocking anti-CD16/CD32 (BD Biosciences), BV605-anti-CD45 (BD Biosciences), PE-anti-CD11b (eBioscience), PE-Cy7-antiCD11c (eBioscience), BV450-anti-CD64 (BD Biosciences), FITC-anti-CD24 (BD Biosciences), BV711-anti-CD103 (BioLegend), AF700-anti-MHC-II (BioLegend), PerCP-Cy5.5-anti-Ly6C (eBioscience) and APC anti-Ly-6G (Miltenyi) mAbs, (ii) to detect neutrophils or eosinophils: Near IR DL (Invitrogen), FcγII/III receptor blocking anti-CD16/CD32 (BD Biosciences), PerCP-Vio700-anti-CD45 (Miltenyi), APC-anti-CD11 b (BD Biosciences), PE-Cy7-anti-CD11c (eBioscience), FITC-anti-CD24 (BD Biosciences), AF700-anti-MHC-II (BioLegend), PE-anti-Ly6G (BioLegend), BV421-anti-Siglec-F (BD Biosciences), (iii) to detect mast cells, basophils, NK: Near IR DL (Invitrogen), BV605-anti-CD45 (BD Biosciences), PE-anti-CD11b (eBioscience), eF450-anti-CD11c (eBioscience), PE-Cy7-anti-CD117 (BD Biosciences), APC-anti-FcER1 (BioLegend), AF700-anti-NKp46 (BD Biosciences), FITC-anti-CCR3 (BioLegend), without FcγII/III receptor blocking anti-CD16/CD32. Cells were incubated with appropriate mixtures for 25 minutes at 4° C. Cells were then washed twice in PBS containing 3% FCS and then fixed PFA 4% and overnight incubation at 4° C. The cells were acquired in an Attune NxT cytometer system (Invitrogen) and data were analyzed by FlowJo software (Treestar, OR, USA).
1.1.13 qRT-PCR Detection of Inflammatory Cytokines and Chemokines in the Lungs
Lung samples were added to lysing matrix D (MP Biomedical) containing 1 mL of TRIzol reagent and homogenized during 30 seconds at 6.0 m/s, twice using MP Biomedical Fastprep 24 Tissue Homogenizer. Total RNA was extracted using TRIzol reagent (ThermoFisher Scientific, France), according to the manufacturer's procedure. cDNA was synthesized from 4 μg of RNA in the presence of 2.5 μM of oligo(dT) 18 primers (SEQ ID NO: 105), 0.5 mM of deoxyribonucleotides, 2.0 U of RNase Inhibitor and SuperScript IV Reverse Transcriptase (ThermoFisher Scientific, France) in 20 μl reaction. The real-time PCR was performed on QuantStudio™ 7 Flex Real-Time PCR System (ThermoFisher Scientific, France). Reactions were performed in triplicates in a final reaction volume of 10 μl containing 5 μl of iQ™ SYBR® Green Supermix (Biorad, France), 4 μl of cDNA diluted 1:15 in DEPC-water and 0.5 μl of each forward and reverse primers at a final concentration of 0.5 μM (Table 2). The following thermal profile was used: a single cycle of polymerase activation for 3 min at 95° C., followed by 40 amplification cycles of 15 sec at 95° C. and 30 sec 60° C. (annealing-extension step). The average CT values were calculated from the technical replicates for relative quantification of target cytokines/chemokines. The differences in the CT cytokines/chemokines amplicons and the CT of the reference β-globin, termed ACT, were calculated to normalized for differences in the quantity of nucleic acid. The ACT of experimental condition were compared relatively to the PBS-treated mice using the comparative ΔΔCT method. The fold change in gene expression was further calculated using 2-ΔΔCT.
To develop a vaccine candidate able to induce nAbs specific to SCoV-2, we generated LV encoding, under the transcriptional control of the cytomegalovirus (CMV) immediate-early promoter, for codon-optimized sequences of: (i) full-length, membrane anchored form of S (LV::SFL), (ii) S1-S2 ecto-domain, without the transmembrane and C-terminal short internal tail (LV::S1-S2), or (iii) S1 alone (LV::S1), which all harbor the RBD (
Sera were then evaluated for their capacity to neutralize SARS-CoV-2, using a reliable neutralization assay based on nAb-mediated inhibition of hACE2+ cell invasion by non-replicative LV particle surrogates, pseudo-typed with SCoV-2 (Sterlin et al.). Such SCoV-2 pseudo-typed LV particles, harbor the reporter luciferase gene, which allows quantitation of the hACE2+ host cell invasion, inversely proportional to the neutralization efficiency of nAbs possibly contained in the biological fluids. Analysis of 50% Effective Concentrations (EC50) of the sera from the LV::SFL-, LV::S1-S2- or LV::S1-immunized mice clearly established that LV::SFL was the most potent vector at inducing SCoV-2-specific nAbs (
In order to potentially increase the immunogenicity of LV::S vectors at inducing neutralizing Abs, we generated LV vectors coding for stabilized pre-fusion SCoV-2, engineered as follows:
(i) SCoV-2 with prospective increased stability, harboring two 986K→P and 987V→P consecutive a.a. substitution. It is indeed established that the a.a substitution toward the rigid proline residue increases the protein stability by decreasing the conformational entropy.
(ii) SCoV-2 with the 681 PRRARS686 (SEQ ID NO: 22)→681PGSAGS686 (SEQ ID NO: 23) a.a. substitution at the furin cleavage site, thereby unrecognizable by this proteolytic enzyme.
(iii) SCoV-2 harboring the 986K→P and 987V→P consecutive a.a. substitutions, and deleted for the 675 QTQTNSPRRAR 685 (SEQ ID NO: 24), encompassing the furin cleavage site.
The nucleotide sequence of pFlap-ieCMV-SFL-WPREm is shown in
The nucleotide sequence of pFlap-ieCMV-S2P-WPREm is shown in
The nucleotide sequence of pFlap-ieCMV-S2P3F-WPREm is shown in
The nucleotide sequence of pFlap-ieCMV-S2PdeltaF-WPREm is shown in
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The following materials were deposited on Jul. 15, 2020: pFlap-ieCMV-S2PdeltaF-WPREm (CNCM I-5537), pFlap-ieCMV-S2P3F-WPREm (CNCM I-5538), pFlap-ieCMV-S2P-WPREm (CNCM I-5539), and pFlap-ieCMV-SFL-WPREm (CNCM I-5540). Deposit receipts are filed herewith.
The following materials were deposited on Jul. 6, 2021 at the CNCM: pFlap-ieCMV-S-B1.1.7-WPREm (CNCM I-5708), pFlap-ieCMV-S-B351-WPREm (CNCM I-5709), pFlap-ieCMV-S-B351-2P-WPREm (CNCM I-5710), pFlap-ieCMV-SFL-D614G-WPREm (CNCM I-5711), pFlap-ieCMV-S-P1-WPREm (CNCM I-5712). Deposit receipts are filed herewith.
LV::SFL-immunized C57BL/6 mice (n=3) also displayed strong anti-SCoV-2 T-cell responses, as detected at week 2 post immunization by IFNγ ELISPOT-based epitope mapping, applied to splenocytes stimulated with distinct pools of 15-mer peptides spanning the full-length SCoV-2 (
As SCoV-2 does not interact efficaciously with murine ACE2, wild-type laboratory mice are not permissive to replication of SARS-CoV-2 clinical isolates. Due to unavailability of hACE2 transgenic mice in Europe during the progression of the present study, to evaluate the LV::SFL vaccine efficacy, we sought to elaborate a murine model in which the hACE2 expression is induced in the respiratory tracts and pulmonary mucosa. To do so, we generated an Ad5 gene delivery vector able to vehicle in non-integrating episomes, the gene coding for hACE2 under the transcriptional control of CMV promoter (Ad5::hACE2). We first checked in vitro the potential of the Ad5::hACE2 vector to transduce HEK293T cells by RT-PCR (
To evaluate the permissibility of such hACE2-transduced mice to SARS-CoV-2 infection, 4 days after i.n. pretreatment with either Ad5::hACE2 or an empty control Ad5 vector, C57BL/6 mice were inoculated i.n. with 1×105 TCID50 of a SARS-CoV-2 clinical isolate, which was isolated in February 2020 from a COVID-19 patient by the National Reference Centre for Respiratory Viruses (Institut Pasteur, France). The lung viral loads, determined at 2 days post inoculation (dpi), were as high as (4.4±1.8)×109 copies of SARS-CoV-2 RNA/mouse in Ad5::hACE2-pretreated mice, compared to only (6.2±0.5)×105 copies/mouse in empty Ad5-pretreated, or (4.0±2.9)×105 copies/mouse in un-pretreated mice (
Ad5::hACE-2 i.n. instillation induced CD45+ cell recruitment to the lungs, however, this effect was reduced with decreasing vector doses, as determined at day 4 post instillation. The dose of 4×108 IGU/mouse did not cause CD45+ cell recruitment, as compared to the PBS-treated controls (
To investigate the prophylactic potential of LV::SFL against SARS-CoV-2, C57BL/6 mice (n=4/group) were injected i.p. with a single dose of 1×107 TU/mouse of LV::SFL or a negative control LV (sham). At week 6 post immunization, the mice were pretreated with Ad5::hACE2, and 4 days later, they were inoculated i.n. with 1×105 TCID50 of SARS-CoV-2 (
To further improve the prophylactic effect, we evaluated the prime-boost or prime-target approaches. C57BL/6 mice (n=4-5/group) were primed i.p. with 1×107 TU of LV::SFL or a control LV at week 0, and then boosted at week 3 with: (i) 1×107 TU of the same LV via the i.p. route (“LV::SFL i.p.-i.p.”, prime-boost), or (ii) with 3×107 TU via the i.n. route (“LV::SFL i.p.-i.n.”, prime-target) to attract the mediators of systemic immunity to the lung mucosa (
All mice were then pretreated with Ad5::hACE2 and challenged i.n. with 0.3×105 TCID50 of SARS-CoV-2 at week 4 post prime. At 3 dpi, the lung viral loads were significantly lower in LV::SFL i.p.-i.p. immunized mice, i.e., mean±SD (2.3±3.2)×108, than in sham-vaccinated mice (13.7±7.5)×108 copies of SARS-CoV-2 RNA, (
Based on the compelling evidences of innate immune hyperactivity in the acute lung injury in COVID-19 (Vabret et al., 2020), we investigated the possible variations of the lung innate immune cell subsets (
Outbred Mesocricetus auratus, so-called golden hamsters, provide a suitable pre-clinical model to study the COVID-19 pathology, as the ACE2 ortholog of this species interacts efficaciously with SCoV-2, whereby host cell invasion and viral replication (Sia et al., 2020). We thus investigated the prophylactic effect of LV::SFL vaccination on SARS-CoV-2 infection in this pertinent model. Although integrative LV vectors are largely safe and passed successfully a phase 1 clinical trial (2011-006260-52 EN), in addition to the integrative LV::SFL, we also evaluated an integrase deficient, non-integrative version of LV::SFL with the prospect of application un future clinical trials.
To assess the prophylactic effect of vaccination following prime-boost/target regimen, M. auratus hamsters (n=6/group) were: (i) primed i.p. with the low dose of 1×106 TU of integrative LV::SFL and boosted i.n. at week 4 with 3×107 TU of integrative LV::SFL, (“int LV::SFL i.p.-i.n. Low”), (ii) primed i.p. with the high dose of 1×107 TU of integrative LV::SFL and boosted i.n. at week 4 with 3×107 TU of integrative LV::SFL (“int LV::SFL i.p.-i.n. High”), or (iii) primed intramuscularly (i.m.) with 1×108 TU of non-integrative LV::SFL and boosted i.n. at week 4 with 3×107 TU of non-integrative LV::SFL (“non int LV::SFL i.m.-i.n.”) (
In an additional experiment (
Sterilizing protection in hamster model by a single i.n. NILV::SΔF2P administration We generated LV encoding a prefusion form of SCoV-2 under transcriptional control of the cytomegalovirus promoter. This prefusion SCoV-2 variant (SΔF2P) has a deletion of 675QTQTNSPRRAR685 (SEQ ID NO: 24) sequence, encompassing the polybasic RRAR (SEQ ID NO: 99) furin cleavage site, at the boundary of S1/S2 subunits, and harbors K986P and V987P consecutive proline substitutions in S2, within the hinge loop between heptad repeat 1 and the central helix (
We also assessed the prophylactic effect of vaccination with only a single i.n. administration of NILV::SΔF2P in the hamster model.
Hamsters (n=6/group) were: (i) primed i.m. at wk 0 with 1×108 TU of NILV::SΔF2P and boosted i.n. at wk 5 with the same amount of the vector, as a positive protection control, (ii) immunized i.n. with a single injection of 1×108 TU of NILV::SΔF2P at wk 0, or (iii) at wk 5 (
At wk 7, all animals were challenged i.n. with 0.3×105 TCID50 of a SARS-CoV-2. At 4 days post inoculation (dpi), only 2-3% weight loss was detected in the NILV::SΔF2P-vaccinated groups, compared to 12% in sham-vaccinated hamsters (
At 4 dpi, as evaluated by qRT-PCR in total lung homogenates, substantially decreased inflammation was detected in NILV::SΔF2P-vaccinated hamsters compared to their sham-vaccinated counterparts, regardless of the immunization regimen, i.e., i.m.-i.n. prime-boost or single i.n. injection given at wk 0 or 5 (
These data collectively indicated that a single i.n. administration of NILV::SΔF2P was as protective as a systemic prime and i.n. boost regimen, conferred sterilizing pulmonary immunity against SARS-CoV-2 and readily prevented lung inflammation and pathogenic tissue injury in the susceptible hamster model.
Altogether, based on a complete set of virological, immunological and expected histopathological data (the latter in progress), the LV::SFL vector elicits SCoV-2-specific nAbs and T-cell responses, correlative with substantial level of protection against SARS-CoV-2 infection in two pertinent animal models, and notably upon mucosal i.n. administration.
Prophylactic strategies are necessary to control SARS-CoV-2 infection which, 6 months into the pandemic, still continue to spread exponentially without sign of slowing down. It is now demonstrated that primary infection with SARS-CoV-2 in rhesus macaques leads to protective immunity against re-exposure (Chandrashekar et al., 2020). Numerous vaccine candidates, based on naked DNA (Yu et al., 2020) or mRNA, recombinant protein, replicating or non-replicating viral vectors, including adenoviral Ad5 vector (Zhu et al., 2020), or alum-adjuvanted inactivated virus (Gao et al., 2020) are under active development for COVID-19 prevention. Our immunologic rationale for selecting LV vector to deliver gene encoding SCoV-2 antigen is based on the insights obtained on the efficacy of heterologous gene expression in situ, as well as the longevity and composite nature of humoral and cell-mediated immunity elicited by this immunization platform. Unique to LV is the ability to transduce proliferating and resting cells (Esslinger et al., 2002; He et al., 2005), thereby LV serves as a powerful vaccination strategy (Beignon et al., 2009; Buffa et al., 2006; Coutant et al., 2012; Gallinaro et al., 2018; Iglesias et al., 2006) to provokes strong and long-lasting adaptive responses. Notably, in net contrast to many other viral vectors, LV vectors do not suffer from pre-existing immunity in populations, which is linked to their pseudo-typing with the glycoprotein envelop from Vesicular Stomatitis Virus, in which humans are barely exposed. We recently demonstrated that a single injection of a LV expressing Zika envelop provides a rapid and durable protection against Zika infection (Ku et al., 2020). Our recent comprehensive systematic comparison of LV to the gold standard Ad5 immunization vector also documented the superior ability of LV to induce multifunctional and central memory T cells in the mouse model, and stronger immunogenicity in outbred rats (Ku et al., 2021 (PMID: 33357418), underlining the largely adapted properties of LV for vaccinal applications.
We evaluated the efficacy of LV each encoding one of the variants of S, i.e., full-length, membrane anchored (LV::SFL), S1-S2 ecto-domain, devoid of the transmembrane and C-terminal short internal tail (LV::S1-S2), or S1 alone (LV::S1). Even though a single administration of each of these LV was able to induce high anti-SCoV-2 Ab titers, only LV::SFL was able to induce highly functional nAbs. Such single-injection of LV-based vaccine induced a neutralizing activity, which on average was comparable to those found in a cohort of SARS-CoV-2 patients manifesting mild symptoms. This finding predicted a protective potential of the humoral responses induced by the LV::SFL vector. In parallel, S-specific CD4+ and CD8+ T-cell responses were also observed in the spleen of mice as early as 2 weeks after a single LV::SFL injection, as detectable against numerous MHC-I- or -II-restricted immunogenic regions that we identified in C57BL/6 (H-2b) mice.
Linked to the absence of permissibility of laboratory mice to SARS-CoV-2 replication and the current unavailability of hACE2 transgenic mice in Europe, we set up an in vivo-infection murine model in which the hACE2 expression is induced in the respiratory tracts by an i.n. Ad5::hACE2 pretreatment prior to SARS-CoV-2 inoculation. This approach renders mice largely permissive to SARS-CoV-2 replication in the lungs and allows assessment of vaccine or drug efficacy against this virus. This method has also been successfully used to establish the expression of human DPP4 for the study of mouse infection with MERS-CoV (Zhao et al., 2014). Even though the Ad5::hACE2 model may not fully mimic the physiological ACE2 expression profile and thus may not reflect all the aspects of the pathophysiology of SARS-CoV-2 infection, it provides a pertinent model to evaluate in vivo the effects of anti-viral drugs, vaccine candidates, various mutations or genetic backgrounds on the SARS-CoV-2 replication. By using a low dose of Ad5::hACE2/mouse, no particular CD45+ cell recruitments were detectable at day 4 post instillation, indicative of an absence of Ad5-related inflammation before the inoculation of SARS-CoV-2.
In the transduced mouse model which allows high rate of SARS-CoV-2 replication, vaccination by a single i.p. administration of 1×107 TU of LV::SFL, 6 weeks before the virus inoculation, was sufficient to inhibit the viral replication by ˜1 log10. Further boosting via the systemic route did not afford improved protection rate when compared to a single administration. However, priming by systemic route and boosting via mucosal route efficiently inhibited viral replication and avoided lung inflammation. Such protection was correlated with high titers of anti-SCoV-2 IgG and a strong neutralization activity in sera. S-specific T-cell responses were also detected in the spleen of LV::SFL-immunized mice, as assessed by ELISPOT followed by stimulation of splenocytes with pools of overlapping 15-mer peptides. Much longer termed experiments in appropriate KO mice or adoptive immune cell transfer approaches are necessary to identify the immunological pathways that contribute to disease severity or protection against SARS-CoV-2. Both nAbs and cell-mediated immunity, together very efficaciously induced with the LV-based vaccine candidate, synergize to inhibit infection and viral replication.
Substantial degrees of protection against SARS-CoV-2 infection, accompanied by drastic reduction in mucosal inflammation and lung tissue damage, were observed in Mesocricetus auratus Golden hamsters immunized following prime-boost/target regimen with either integrative or non-integrative LV::SFL. Confirmation of the protection results in this highly sensitive species further favors the LV::SFL vaccine candidate, especially under its non-integrative variant, for future introduction into clinical trials.
Ab-Dependent Enhancement (ADE) of coronavirus entry to the host cells has been evoked as a mechanism which could be an obstacle in vaccination against coronaviruses. With DNA (Yu et al., 2020) or inactivated SARS-CoV-2 virus (Gao et al., 2020) vaccination in macaques, no immunopathological exacerbation has been observed but could not be excluded. Long term observation even after decrement in Ab titer could be necessary to exclude such hypothesis. In the case of MERS-CoV, it has been reported that one particular RBD-specific neutralizing monoclonal Ab (Mersmab1), by mimicking the viral receptor human DPP4 and inducing conformational rearrangements of SMERS, can mediate in vitro ADE of MERS-CoV into the host cells (Wan et al., 2020). We believe that it is difficult to compare the polyclonal Ab response with its paratope repertoire complexity with the singular properties of a monoclonal Ab which cannot be representative of the polyclonal response induced by a vaccine. In addition, very contradictory results from the same team reported that a single-dose treatment with a humanized version of Mersmab1 afforded complete protection of a human transgenic mouse model from lethal MERS challenge (Qiu et al., 2016). Therefore, even with an Ab which could facilitate the cell host invasion in vitro in some conditions, not only there is no exacerbation of the infection in vivo, but also there is a notable protection. Indeed, to affirm that Abs could cause ADE in vivo, it is necessary, by large scale B-cell fusions, until they have made to estimate the probability of generation of such Ab.
Prophylactic vaccination is the most cost-effective and efficient strategy against infectious diseases and notably against emerging coronaviruses in particular. Our results provide strong evidences that the LV vector coding for SFS protein of SARS-CoV-2 used via the mucosal route of vaccination represent a promising vaccine candidate against COVID-19.
To date several Transgenic (Tg) mice of different strains expressing the hACE2 gene under distinct transcription and expression control sequences have been provided, some of them originating from developments performed to fulfil needs that arose when on emergence of SARS-CoV epidemic in 2003. These earlier developed Tg mice and further models have been assessed for the study and understanding of the pathogenesis of SARS-CoV and have shown to be permissible to viral replication and sometimes to some degree of disease symptom or clinical illness but the observed various clinical profiles in Tg mice inoculated with SARS-CoV-2 have not yet provided proved suitable to reproduce all aspects of the outcome of the infection, in particular have not adequately shown virus spread as observed in human patients, in particular spread beyond the airways and the pulmonary tract, such as spread to the brain. Also the available Tg mice have not shown all the consistent disease symptoms that would reproduce the symptoms observed in human patients.
A B6.K18-ACE22PrImn/JAX mouse strain has been previously deposited at JAX Laboratories (Jackson Laboratories, Bar Harbor, Me.). However, the new B6.K18-hACE2IP-THV transgenic mice that the inventors generated according to the present invention display distinctive characteristics identified following SARS-CoV-2 intranasal (i.n.) inoculation. In fact, in addition to the large permissibility of their lungs to SARS-CoV-2 replication and viral dissemination to peripheral organs, B6.K18-hACE2IP-THV mice surprisingly allow substantial viral replication in the brain, which is ≈4 log10 higher than the replication range observed in the previously available B6.K18-ACE22PrImn/JAX strain (McCray et al., 2007). This new mouse model, not only has broad applications in the study of COVID-19 vaccine or COVID-19 therapeutics efficacy, but also provides an experimental model to elucidate COVID-19 immune/neuro-physiopathology. Neurotropism of SARS-CoV-2 has been demonstrated and some COVID-19 human patients present symptoms like headache, confusion, anosmia, dysgeusia, nausea, and vomiting (Bourgonje et al., 2020). Olfactory transmucosal SARS-CoV-2 invasion is also very recently described as a port of central nervous system entry in human individuals with COVID-19 (https://doi.org/10.1038/s41593-020-00758-5). Since coronaviruses can infect the central nervous system (Bergmann et al., 2006), the B6.K18-hACE2IP-THV small rodent experimental model represents an invaluable pre-clinical or co-clinical animal model of major interest for: (i) investigation of immune protection of the brain and (ii) exploration of COVID-19-derived neuropathology.
1. Construction of the Human Keratin 18 Promoter
The human K18 promoter (GenBank: AF179904.1 nucleotides 90 to 2579) was amplified by nested PCR from A549 cell lysate, as described previously (Chow et al., 1997; Koehler et al., 2000). The “i6x7” intron (GenBank: AF179904.1 nucleotides 2988 to 3740) was synthesized by Genscript. The “K18i6x7PA” promoter, previously used to generate B6.K18-ACE22PrImn/JAX strain, includes the K18 promoter, the “i6x7” intron at 5′ and an enhancer/polyA sequence (PA) at 3′ of the hACE2 gene. TheK18 IP-ThV promoter used here contains, instead of PA, the stronger wild-type Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element (WPRE) at 3′ of the hACE2 gene. In contrast to K18i6x7PA construct which harbors the 3′ regulatory region containing a polyA sequence, the K18IP-ThV construct takes benefice of the polyA sequence already present within the 3′ Long Terminal Repeats (LTR) of the pFLAP LV plasmid, used for transgenesis. The i6x7 intronic part was modified to introduce a consensus 5′ splicing donor and a 3′ donor site sequence. The AAGGGG (SEQ ID No.97) donor site was further modified for the AAGTGG (SEQ ID No.95) consensus site. Based on a consensus sequence logo (Dogan et al., 2007), the poly-pyrimidine tract preceding splicing acceptor site (TACAATCCCTC (SEQ ID No.82) in original sequence GenBank AF179904.1 and TTTTTTTTTTT (SEQ ID No.83) in K18JAX) was replaced by CTTTTTCCTTCC (SEQ ID No.96) to limit incompatibility with the reverse transcription step during transduction. Moreover, original splicing acceptor site CAGAT was modified to correspond to the consensus sequence CAGGT (SEQ ID No.84). As a construction facility, a ClaI restriction site was introduced between the promoter and the intron. The construct was inserted into a pFLAP plasmid between the MluI and BamHI sites. The hACE2 cDNA was introduced between the BamHI and XhoI sites by restriction/ligation. Integrative LV::K18-hACE2IP-THV was produced as described elsewhere (Sayes et al., 2018) and concentrated by two cycles of ultracentrifugation at 22,000 rpm for 1 h at 4° C.
2. Transgenesis
High tittered (8.32×109 TU/ml) integrative LV::K18-hACE2IP-THV was micro-injected into the pellucid area of fertilized eggs which were transplanted into pseudo-pregnant B6CBAF1 females (Charles Rivers). The NO mice were investigated for integration and copy number of hACE2 gene per genome by using hACE2-forward: 5′-TCC TAA CCA GCC CCC TGT T-3′ (SEQ ID No.85) and hACE2-reverse: 5′-TGA CAA TGC CAA CCA CTA TCA CT-3′ (SEQ ID No.86) primers in PCR applied on genomic DNA prepared from the tail biopsies. Toward stabilization of the progeny, transgene positive males were then crossed to WT C57BL/6 females (Charles Rivers). Transgene transfer by microinjection of integrative LV::K18-hACE2IP-THV into the nucleus of fertilized eggs was particularly efficient. At the NO generation, 11% of the mice obtained, i.e., 15 out of 139, had at least one copy of the transgene per genome. Eight NO males carrying the transgene were crossed with female C57BL/6 WT mice (Janvier, Le Genest Saint Isle, France). At the N1 generation, ≈62% of the mice obtained, i.e., 91 out of 147, had at least one copy of the transgene per genome. 10 N1 males carrying the transgene were further crossed with female C57BL/6 WT mice.
During the immunization period female or male transgenic mice were housed in individually-ventilated cages under specific pathogen-free conditions. Mice were transferred into individually filtered cages in isolator for SARS-CoV-2 inoculation at the Institut Pasteur animal facilities. Prior to i.n. injections, mice were anesthetized by i.p. injection of Ketamine (Imalgene, 80 mg/kg) and Xylazine (Rompun, 5 mg/kg).
3. Genotyping and Quantitation of hACE2 Gene Copy Number/Genome in Transgenic Mice
Genomic DNA (gDNA) from transgenic mice was prepared from the tail biopsies by phenol-chloroform extraction. A 60 ng of gDNA were used as a template of qPCR with SyBr Green using specific primers listed in Table 3. Using the same template and in the similar reaction plate, mouse PKD1 (Polycystic Kidney Disease 1) and GAPDH were also quantified. All samples were run in quadruplicate in 10 μl reaction as follows: 10 in at 95° C., 40 cycles of 15 s at 95° C. and 30 sec at 60° C. To calculate the transgene copy number, the 2−ΔΔCt method was applied using the PKD1 as a calibrator and GAPDH as a endogenous control. The 2−ΔΔCt provides the fold change in copy number of the hACE2 gene relative to PKD1 gene.
4. K18-hACE2IP-THV permissibility to SARS-CoV-2 replication
The permissibility of N1 mice to SARS-CoV-2 replication was evaluated in the sampled individuals from the progeny. N1 females with varying number of transgene copies per genome were sampled (
The organs recovered from the animals infected with live SARS-CoV-2 were manipulated following the approved standard operating procedures of these facilities.
At 3 days post-inoculation (dpi) the Mean±SD of lung viral loads were as high as (3.3±1.6)×1010 copies of SARS-CoV-2 RNA/mouse in the permissive mice (
5. Comparison of B6.K18-ACE22PrImn/JAX and K18-hACE2IP-THV Strains in Terms of Permissibility to SARS-CoV-2 Replication
We further comparatively evaluated SARS-CoV-2 replication in lungs and brain and dissemination to various organs in B6.K18-hACE2IP-THV and B6.K18-ACE22PrImn/JAX mice (
Correlative with the brain viral loads, much higher inflammation was detected by qRT-PCR in the brain of B6.K18-hACE2IP-THV mice compared to B6.K18-ACE22PrImn/JAX mice, at 3 dpi, showing an immunological/inflammatory symptom in the central nervous system of the former, but not in the latter (
Therefore, large permissibility to SARS-CoV-2 replication at both lung and CNS, marked brain inflammation and rapid lethal disease are major distinctive features of this new B6.K18-hACE2IP-THV transgenic model.
Ethical Approval of Animal Studies
In all Examples, experimentation on mice and hamsters was realized in accordance with the European and French guidelines (Directive 86/609/CEE and Decree 87-848 of 19 Oct. 1987) subsequent to approval by the Institut Pasteur Safety, Animal Care and Use Committee, protocol agreement delivered by local ethical committee (CETEA #DAP20007, CETEA #DAP200058) and Ministry of High Education and Research APAFIS #24627-2020031117362508 v1.
Here, we generated a new hACE2 transgenic mouse strain with unprecedent permissibility of the brain to SARS-CoV-2 replication. By use of this unique preclinical animal model, we demonstrated the importance of i.n. booster immunization with this LV-based vaccine candidate to reach full protection of not only lungs but also CNS against SARS-CoV-2. Our results indicate that i.n. vaccination step with non-cytopathic and non-inflammatory LV, appears to be a performant and safe strategy to elicit sterilizing immunity in the main anatomical sites affected by COVID-19.
Methods
Construction and production of LV::SΔF2P
A codon-optimized SΔF2P sequence (1-1262) (SEQ ID No. 14). was amplified from pMK-RQ_S-2019-nCoV and inserted into pFlap by restriction/ligation between BamHI and XhoI sites, between the native human ieCMV promoter and a mutated Woodchuck Posttranscriptional Regulatory Element (WPRE) sequence. The atg starting codon of WPRE was mutated (mWPRE) to avoid transcription of the downstream truncated “X” protein of Woodchuck Hepatitis Virus for safety concerns (
Mice
Transgenic mice were generated as disclosed in detail in Example 2.
Humoral and T-Cell Immunity, Inflammation
As recently detailed elsewhere (Ku et al., 2021), T-splenocyte responses were quantitated by IFN-g ELISPOT and anti-S IgG or IgA Abs were detected by ELISA by use of recombinant stabilized SCoV-2. NAb quantitation was performed by use of SCoV-2 pseudo-typed LV, as recently described (Anna et al., 2020; Sterlin et al., 2020). The qRT-PCR quantification of inflammatory mediators in the lungs and brain of hamsters and mice was performed in total RNA extracted by TRIzol reagent, as detailed in Example 1.
SARS-CoV-2 Inoculation
Hamsters or transgenic B6.K18-hACE2IP-THV or B6.K18-ACE22PrImn/JAX were anesthetized by i.p. injection of mixture Ketamine and Xylazine, transferred into a biosafety cabinet 3 and inoculated i.n. with 0.3×105 TCID50 of the BetaCoV/France/IDF0372/2020 SARS-CoV-2 clinical isolate (Lescure et al., 2020). This clinical isolate was a gift of the National Reference Centre for Respiratory Viruses hosted by Institut Pasteur (Paris, France), headed by Pr. van der Werf. The human sample from which this strain was isolated has been provided by Dr. Lescure and Pr. Yazdanpanah from the Bichat Hospital, Paris, France. The viral inoculum was contained in 20 μl for mice and in 50 μl for hamsters. Animals were housed in an isolator in BioSafety Level 3 animal facilities of Institut Pasteur. The organs recovered from the infected animals were manipulated according to the approved standard procedures of these facilities.
Determination of Viral Loads in the Organs
Organs from mice or hamsters were removed aseptically and immediately frozen at −80° C. RNA from circulating SARS-CoV-2 was prepared from lungs as recently described (Ku et al). Briefly, lung homogenates were prepared by thawing and homogenizing of the organs using lysing matrix M (MP Biomedical) in 500 μl of ice-cold PBS in an MP Biomedical Fastprep 24 Tissue Homogenizer. RNA was extracted from the supernatants of lung homogenates centrifuged during 10 min at 2000 g. Alternatively, total RNA was prepared from lungs or other organs by addition of lysing matrix D (MP Biomedical) containing 1 mL of TRIzol reagent and homogenization at 30 s at 6.0 m/s twice using MP Biomedical Fastprep 24 Tissue Homogenizer. Total RNA was extracted using TRIzol reagent (ThermoFisher). SARS-CoV-2 E gene (Corman et al., 2020) or E sub-genomic mRNA (sgmRNA) (Wolfel et al., 2020), was quantitated following reverse transcription and real-time quantitative TaqMan® PCR, using SuperScript™ Ill Platinum One-Step qRT-PCR System (Invitrogen) and specific primers and probe (Eurofins) (Table 4). The standard curve of EsgmRNA assay was performed using in vitro transcribed RNA derived from PCR fragment of “T7 SARS-CoV-2 E-sgmRNA”. The in vitro transcribed RNA was synthesized using T7 RiboMAX Express Large Scale RNA production system (Promega) and purified by phenol/chloroform extraction and two successive precipitations with isopropanol and ethanol. Concentration of RNA was determined by optical density measurement, diluted to 109 genome equivalents/μL in RNAse-free water containing 100 μg/mL tRNA carrier, and stored at −80° C. Serial dilutions of this in vitro transcribed RNA were prepared in RNAse-free water containing 10 μg/ml tRNA carrier to build a standard curve for each assay. PCR conditions were: (i) reverse transcription at 55° C. for 10 min, (ii) enzyme inactivation at 95° C. for 3 min, and (iii) 45 cycles of denaturation/amplification at 95° C. for 15 s, 58° C. for 30 s. PCR products were analyzed on an ABI 7500 Fast real-time PCR system (Applied Biosystems).
Cytometric Analysis of Immune Lung and Brain Cells
Isolation and staining of lung innate immune cells were largely detailed in Example 1. Cervical lymph nodes, olfactory bulb and brain from each group of mice were pooled and treated with 400 U/ml type IV collagenase and DNase I (Roche) for a 30-minute incubation at 37° C. Cervical lymph nodes and olfactory bulbs were then homogenized with glass homogenizer while brains were homogenized by use of GentleMacs (Miltenyi Biotech). Cell suspensions were then filtered through 100 μm-pore filters, washed and centrifuged at 1200 rpm during 8 minutes. Cell suspensions from brain were enriched in immune cells on Percoll gradient after 25 min centrifugation at 1360 g at RT. The recovered cells from lungs were stained as recently described elsewhere (Ku et al., 2021). The recovered cells from brain were stained by appropriate mAb mixture as follows. (i) To detect innate immune cells: Near IR Live/Dead (Invitrogen), FcγII/III receptor blocking anti-CD16/CD32 (BD Biosciences), BV605-anti-CD45 (BD Biosciences), PE-anti-CD11b (eBioscience), PE-Cy7-antiCD11c (eBioscience), (ii) to detect NK, neutrophils, Ly-6C+/− monocytes and macrophages: Near IR DL (Invitrogen), FcγII/III receptor blocking anti-CD16/CD32 (BD Biosciences), BV605-anti-CD45 (BD Biosciences), PE-anti-CD11b (eBioscience), LPE-Cy7-antiCD11c (eBioscience), APC-anti-Ly6G (Miltenyi), BV711-anti-Siglec-F (BD), AF700-anti-NKp46 (BD Biosciences), FITC-anti-Ly6C (Abcam) (iii) To detect adaptive immune cells: Near IR Live/Dead (Invitrogen), FcγII/III receptor blocking anti-CD16/CD32 (BD Biosciences), APC-anti-CD45 (BD), PerCP-Cy5.5-anti-CD3 (eBioscience), FITC-anti-CD4 (BD Pharmingen), BV711-anti-CD8 (BD Horizon), BV605-anti-CD69 (Biolegend), PE-anti-CCR7 (eBioscience) and VioBlue-Anti-B220 (Miltenyi).Cells were incubated with appropriate mixtures for 25 minutes at 4° C., washed in PBS containing 3% FCS and fixed with Paraformaldehyde 4% by an overnight incubation at 4° C. Samples were acquired in an Attune NxT cytometer (Invitrogen) and data analyzed by FlowJo software (Treestar, OR, USA).
Results
New hACE2 Transgenic Mice with Substantial Brain Permissibility to SARS-CoV-2 replication
B6.K18-hACE2IP-THV mice were generated as disclosed in Example 2. The permissibility of these mice to SARS-CoV-2 replication was evaluated and it was determined that large permissibility to SARS-CoV-2 replication at both lung and CNS, marked brain inflammation and rapid lethal disease are major distinctive features of this new B6.K18-hACE2IP-THV transgenic model.
Full Protection of Lungs and Brain in LV::SΔF2P-Immunized B6.K18-hACE2IP-THV Mice
We then evaluated the vaccine efficacy of LV::SΔF2P in B6.K18-hACE2IP-THV mice. Individuals (n=6/group) where primed i.m. with 1×107 TU/mouse of LV::SΔF2P or an empty LV (sham) at wk 0 and then boosted i.n. at wk 3 with the same dose of the same vectors (
At 3 dpi, cytometric investigation of the lung innate immune cell subsets (
Therefore, an i.m.-i.n. prime-boost with NILV::SΔF2P prevents SARS-CoV-2 replication in both lung and CNS anatomical areas and inhibits virus-mediated lung pathology and neuro-inflammation.
Requirement of i.n. Boost for Full Protection of Brain in B6.K18-hACE2IP-THV Mice
To go further in characterization of the protective properties of LV, in the following experiments in B6.K18-hACE2IP-THV mice, similar to the hamster model, we used the non-integrative version of LV. The observed protection of brain against SARS-CoV-2 may reflect the benefits of i.n. route of LV administration against this respiratory and neurotropic virus. To address this hypothesis, B6.K18-hACE2IP-THV mice were vaccinated with NILV::SΔF2P: (i) i.m. wk 0 and i.n. wk5, as a positive control, (ii) i.n. wk 0, or (iii) i.m. wk 5. Sham-vaccinated controls received i.n. an empty NILV at wks 0 and 5 (
As analyzed by cytometry, composition of innate and adaptive immune cells in the cervical lymph nodes were unchanged in NILV::SΔF2P i.m.-i.n. protected group, sham i.m.-i.n. unprotected group and untreated controls (data not shown). Notably, we detected increased proportion of CD8+ T cells in the olfactory bulb of NILV::SΔF2P i.m.-i.n. protected group compared to unprotected group (
Collectively, our data generated in the highly stringent B6.K18-hACE2IP-THV mouse model support the advantage of NILV::SΔF2P i.n. boost in the immune protection of CNS from SARS-CoV-2 replication and the resulting infiltration and neuro-inflammation. The local induction and/or activation of mucosal immune response in nasal cavity and olfactory bulbs, i.e. the entry point for the virus, is a performant strategy.
Discussion
LV-based platform emerges as a powerful vaccination approach against COVID-19, notably when used in systemic prime followed by mucosal i.n. boost, able to induce sterilizing immunity against lung SARS-CoV-2 infection in preclinical animal models. We first demonstrate that a single i.n. administration of an LV encoding the SΔF2P prefusion form of SCoV-2 confers, as efficiently as an i.m.-i.n. prime-boost regimen, full protection of respiratory tracts in the highly susceptible hamster model, as evaluated by virological, immunological and histopathological parameters. The hamster ACE2 ortholog interacts efficaciously with SCoV-2, which readily allows host cell invasion by SARS-CoV-2 and its high replication rate. With rapid weight loss and development of severe lung pathology subsequent to SARS-CoV-2 inoculation, this species provides a sensitive model to evaluate the efficacy of drug or vaccine candidates, for instance compared to Rhesus macaques which develop only a mild COVID-19 pathology (Munoz-Fontela et al., 2020; Sia et al., 2020). The fact that a single i.n. LV administration, either seven or two weeks before SARS-CoV-2 challenge, elicits sterilizing protection in this susceptible model is valuable in setting the upcoming clinical trials with this LV-based vaccine and could provide remarkable socio-economic advantages for mass vaccination.
To further investigate the efficacy of our vaccine candidates, we generated a new transgenic mouse model, by use of an LV-based transgenesis approach (Nakagawa and Hoogenraad, 2011). The ILV used in this strategy encodes for hACE2 controlled by cytokeratin K18 promoter, i.e., the same promoter as previously used by Perlman's team to generate B6.K18-ACE22PrImn/JAX mice (McCray et al., 2007), with a few adaptations to the lentiviral FLAP transfer plasmid. However, the new B6.K18-hACE2IP-THV mice have certain distinctive features, as they express much higher levels of hACE2 mRNA in the brain and display markedly increased brain permissibility to SARS-CoV-2 replication, in parallel with a substantial brain inflammation and development of a lethal disease in <4 days post infection. These distinct characteristics can result from differential hACE2 expression profile due to: (i) alternative insertion sites of ILV into the chromosome compared to naked DNA, and/or (ii) different effect of the Woodchuck Posttranscriptional Regulatory Element (WPRE) versus the alfalfa virus translational enhancer (McCray et al., 2007), in B6.K18-hACE2IP-THV and B6.K18-ACE22PrImn/JAX animals, respectively. Other reported hACE2 humanized mice express the transgene under: (i) murine ACE2 promoter, without reported hACE2 mRNA expression in the brain (Yang et al., 2007), (ii) “hepatocyte nuclear factor-3/forkhead homologue 4” (HFH4) promoter, i.e., “HFH4-hACE2” C3B6 mice, in which lung is the principal site of infection and pathology (Jiang et al., 2020; Menachery et al., 2016), and (iii) “CAG” mixed promoter, i.e. “AC70” C3H×C57BL/6 mice, in which hACE2 mRNA is expressed in various organs including lungs and brain (Tseng et al., 2007). Comparison of AC70 and B6.K18-hACE2IP-THV mice may be informative to assess similarities and distinctions of these two models. However, here we report much higher brain permissibility of B6.K18-hACE2IP-THV mice to SARS-CoV-2 replication, compared to B6.K18-ACE22PrImn/JAX mice. The B6.K18-hACE2IP-THV murine model not only has broad applications in COVID-19 vaccine studies, but also provides a unique rodent model for exploration of COVID-19-derived neuropathology. Based on the substantial permissibility of the brain to SARS-CoV-2 replication and development of a lethal disease by these transgenic mice, this pre-clinical model can be considered as more stringent than the golden hamster model.
In this study, the use of the highly stringent B6.K18-hACE2IP-THV mice demonstrated the importance of i.n. booster immunization for the induction of sterilizing protection of CNS by the LV-based vaccine candidate developed against SARS-CoV-2. Olfactory bulb may control viral CNS infection through the action of local innate and adaptive immunity (Durrant et al., 2016), and we observed increased frequencies of CD8+ T cells at this anatomically strategic area in i.m.-i.n. vaccinated and protected mice. Substantial reduction in the inflammation mediators was also demonstrated in the brain of these vaccinated and protected mice, together with decrease in the neutrophils and inflammatory monocytes in the olfactory bulbs and brain, respectively.
The source of neurological manifestations associated with COVID-19 in patients with comorbid conditions can be: (i) direct impact of SARS-CoV-2 on CNS, (ii) infection of brain vascular endothelium and, (iii) uncontrolled anti-viral immune reaction inside CNS. ACE2 is expressed in human neurons, astrocytes and oligodendrocytes, located in middle temporal gyrus and posterior cingulate cortex, which may explain the brain permissibility to SARS-CoV-2 in patients (Song et al., 2020; Hu et al., 2020). Viruses can invade the brain through neural dissemination or hematogenous route (Bohmwald et al., 2018; Desforges et al., 2019, 2014). The olfactory system establishes a direct connection to the CNS via frontal cortex (Mori et al., 2005). Neural transmission of viruses to the CNS can occur as a result of direct neuron invasion through axonal transport in the olfactory mucosa. Subsequent to intraneuronal replication, the virus spreads to synapses and disseminate to anatomical CNS zones receiving olfactory tract projections (Koyuncu et al., 2013; Zubair et al., 2020; Berth, 2009; Koyuncu et al., 2013; Roman et al., 2020). However, the detection of viral RNA in CNS regions without connection with olfactory mucosa suggests existence of another viral entry into the CNS, including migration of SARS-CoV-2-infected immune cells crossing the hemato-encephalic barrier or direct viral entry pathway via CNS vascular endothelium (Meinhardt et al., 2020). Although at steady state, viruses cannot penetrate to the brain through an intact blood-brain barrier (Berth, 2009), inflammation mediators which are massively produced during cytokine/chemokine storm, notably TNF-α and CCL2, can disrupt the integrity of blood-brain barrier or increase its permeability, allowing paracellular blood-to-brain transport of the virus or virus-infected leukocytes {Aghagoli, 2020 #77; Hu, 2011 #15}. Regardless of the mechanism of the SARS-CoV-2 entry to the brain, we provide evidence of the full protection of the CNS against SARS-CoV-2 by i.n. booster immunization with NILV::SΔF2P.
We reported results in Example 1 demonstrating the strong prophylactic capacity of LV::SFL at inducing sterilizing protection in the lungs against SARS-CoV-2 infection. In the present study, moving toward clinical assay, we used LV encoding stabilized prefusion SΔF2P forms of SCoV-2 as an additional form of the S protein exhibiting vaccinal interest. This choice was based on data indicating that stabilization of viral envelop glycoproteins at their prefusion forms improve the yield of their production as recombinant proteins in industrial manufacturing of subunit vaccines, and the efficacy of nucleic acid-based vaccines by raising availability of the antigen under its optimal immunogenic shape (Hsieh et al., 2020). The prefusion stabilization approach has been so far applied to S protein of several coronaviruses, including HKU1-CoV, SARS-CoV, and MERS-CoV. Stabilized SMERS-CoV has been shown to elicit much higher NAb responses and protection in pre-clinical animal models (Hsieh et al., 2020).
The sterilizing protection of the lungs conferred by a single i.n. administration and the full protection of CNS conferred by i.n. boost is an asset of primary importance. The non-cytopathic and non-inflammatory LV encoding either full length, or stabilized forms of SCoV-2, from either ancestral or emerging variants of SARS-CoV-2 provides a promising COVID-19 vaccine candidate of second generation. Protection of the brain, so far not directly addressed by other vaccine strategies, has to be taken into account, considering the multiple and sometimes severe neuropathological manifestations associated with COVID-19.
A critical issue regarding the COVID-19 vaccines currently in use is the protective potency against emerging variants. To assess this question with the NILV::SCoV-2 Wuhan vaccine candidate, B6.K18-hACE2IP-THV transgenic mice were primed i.m. (wk0) and boosted i.n. (wk5) with NILV::SCoV-2 or sham (
This drastically reduced protective B-cell response despite the remarkable protection, raised the possibility of T-cell involvement in this NILV::SCoV-2 Wuhan-mediated full protection. To evaluate this possibility, we vaccinated following the same protocol (
This is consistent with: (i) strong CD8+ T-cell responses induced by NILV::SCoV-2 Wuhan at the systemic level (
Remarkably, all murine and human CD8+ T-cell epitopes identified on SCoV-2 Wuhan sequence are preserved in the mutated SCoV-2 Manaus P.1 (Table 5). These observations indicate the strong potential of NILV at inducing full protection of lungs and brain against ancestral and emerging SARS-CoV-2 variants by eliciting marked B and T cell-responses. In contrast to the B-cell epitopes which are targets of NAbs (Hoffmann et al., 2021), the so far identified T-cell epitopes have not been impacted by mutations accumulated in the SCoV-2 of the emerging variants.
As demonstrated in Example 4, we showed that NI LV::SCoV-2 Wuhan largely protects the strongly susceptible B6. K18-hACE2IP-THV transgenic mice against both the ancestral Wuhan and the most genetically distant Manaus P.1 SARS-CoV-2 variants. For the establishment of a therapeutic, to further improve the antigen, the use of the most suitable Spike variant, which can best consider the dynamics of the virus propagation of the known variants was considered.
To identify the most cross-protective Spike variant, we primed and boosted C57BL/6 mice with LV encoding each Spike of interest (
(ii) sera from mice immunized with LV::SCoV-2 P.1 neutralized at high EC50 pseudo-viruses harboring SCoV-2 P.1 and LV::SCoV-2 B1.351, but poorly pseudo-viruses harboring SCoV-2 Wuhan and LV::SCoV-2 B1.1.7.
(iii) sera from mice immunized with LV::SCoV-2 B1.351 not only neutralized at high EC50 pseudo-viruses carrying SCoV-2 P.1 and LV::SCoV-2 B1.351 but also pseudo-viruses harboring SCoV-2 Wuhan and LV::SCoV-2 B1.1.7.
These results designate the Spike sequence from the B1.351 (South African or β) variant as the most cross-reactive immunogen in terms of neutralizing antibodies.
Furthermore, we showed that in the context of LV, Spike stabilization by K986P-V987P substitutions (2P) considerably improves the (cross) neutralizing antibody activity (
Therefore, our future lead antigen candidate is the full-length Spike from the B1.351 (South African or β) variant with 2P.
| Number | Date | Country | Kind |
|---|---|---|---|
| PCT/IB2021/000293 | Feb 2021 | WO | international |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2021/069890 | 7/15/2021 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63052264 | Jul 2020 | US | |
| 63130202 | Dec 2020 | US |
