Patent Application
20230218745
This application includes an electronically submitted sequence listing in .txt format. The .txt file contains a sequence listing entitled “Sindbis_Coronavirus_ST25.txt” created on May 28, 2021 and is 460 bytes in size. The sequence listing contained in this .txt file is part of the specification and is hereby incorporated by reference herein in its entirety.
The present disclosure relates generally to alphavirus-based vaccines that are to stimulate immune responses against SARS-CoV-2. The vaccines express one or more SARS-CoV-2 antigens and can express additional immunomodulatory agents.
Traditionally, vaccines have been designed to induce antibody responses and have been licensed on their capacity to induce high titers of circulating antibody to the pathogen[1]. With increased knowledge of host-virus interactions, it has become clear that the cellular arm of the immune response is also crucial to the efficacy of vaccines against pathogens and to provide appropriate help for antibody induction. Various strategies have emerged that specialize in developing candidate vaccines that solely induce either cellular or humoral responses[1]. However, as most viruses and pathogens reside at some point during their infectious cycle in the extracellular as well as intracellular space, vaccines need to promptly elicit a strong T cell memory response against intracellular pathogens, so that, at the earliest stages of the infective process preventing disease can be addressed in coordination with antibodies, thus preventing disease.
An early CD4+ and CD8+ T cell response against SARS-CoV-2 is considered to be protective[2; 3]. However, this response can be difficult to generate because of efficient immune evasion mechanisms of SARS-CoV-2 in humans[4], especially in the elderly. Immune evasion by SARS-CoV-2 is likely exacerbated by reduced myeloid cell antigen presenting cell (APC) function and B cell decline in older adults. In such cases, late T cell and uncoordinated adaptive immune responses may instead amplify pathogenic inflammatory outcomes in the presence of sustained high viral loads in the lungs, leading to death.
In the ongoing COVID19 pandemic, vaccines play a key role in the strategy to bring SARS-CoV-2 transmission under control. Safety and eliciting a broad-spectrum immune response are paramount for coronavirus vaccine development. Data from vaccine clinical trials and real-world evidence show that available coronavirus vaccines are able to cut the risk of severe COVID19 disease and transmission. However, even with first generation vaccines currently being globally administered to reduce transmission and severity of the disease, the emergence of circulating variants has raised major concerns that challenge sustained vaccine efficacy, particularly in the face of waning immunity following vaccination[5; 6; 7; 8; 9; 10; 11]. Recent data have indicated that escape (appearance and spread of viral variants that can infect and cause illness in vaccinated hosts) protection by vaccines designed against the Wuhan-1 strain is inevitable[8].
The global COVID19 pandemic is unlikely to end until there is an efficient pan-global roll-out of SARS-CoV-2 vaccines. Though multiple vaccines are currently available, vaccine rollout and distribution at the time of writing this paper is quite incomplete. The three largest countries in the western hemisphere- US, Brazil, and Mexico - have vaccinated 32.7%, 7%, and 6.6% of their populations, respectively, compared to only 2.2% in India [12]. Vaccine distribution to date has been highly non-uniform among these and other countries around the globe, encountering many challenges. Unequal vaccine roll-out and the new B.1.617 variant are highly concerning. Major challenges have been supplies shortages, logistical problems, complex storage conditions, priced affordably, and safety[13]. Consequently, the pandemic is currently sweeping through India at a pace faster than ever before. The countries’ second wave became the worst COVID19 surge in the world, despite previous high infection rates in megacities that should have resulted in some immunity. More cost-effective and facilitated delivery of broad-spectrum SARS-CoV-2 vaccines would help improve wide and rapid distribution, which would in turn minimize vaccine-escape. Thus, there is an ongoing and unmet need for improved compositions and methods for combating SARS-CoV-2. The present disclosure is pertinent to these needs.
The present disclosure provides a modified Alphavirus and populations of the same for use in prophylaxis and/or therapy for SARS CoV-2 infection. The modified Alphavirus platform is illustrated by way of a novel modified Sindbis Virus (SV) vaccine encoding and transiently expressing the SARS-CoV-2 spike protein (SV.Spike), which induces a strong adaptive immunity that fully protects transgenic mice that express the SARS-CoV receptor (human angiotensin-converting enzyme 2 [hACE2]), K18-hACE2, against live SARS-CoV-2 virus infection. To additionally increase safety, the disclosure provides replication-deficient vectors that only transiently express the encoded antigen and other immunomodulatory proteins of interest. The disclosure demonstrates that a combination of the described vaccine with αOX40 agonistic antibodies significantly enhances the induction of immunity by the SV.Spike vector. Specifically, seroconversion and abundance of IgG neutralizing antibodies and T cell immunity through early initiation of Th1-type T cell polarization are markedly augmented to potentiate long-term immunity protective against SARS-CoV-2 infection in mice. The disclosure accordingly provides a safe and effective vaccine platform that provides humoral and cellular immunity to the SARS-CoV-2 spike. This platform has the potential to be applied to other emerging pathogens.
The disclosure includes compositions comprising the described modified alphaviruses, plasmids encoding the vaccine components, isolated polynucleotides, isolated viral particles, methods of making the described vaccines, and kits for producing the described vaccines.
Unless defined otherwise herein, all technical and scientific terms used in this disclosure have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains.
Every numerical range given throughout this specification includes its upper and lower values, as well as every narrower numerical range that falls within it, as if such narrower numerical ranges were all expressly written herein.
The disclosure includes all polynucleotide and amino acid sequences described herein expressly and by reference, and every polynucleotide sequence referred to herein includes its complementary sequence, and its reverse complement. All segments of polynucleotides from 10 nucleotides to the entire length of the polynucleotides, inclusive, and including numbers and ranges of numbers there between are included. DNA sequences includes the RNA equivalents thereof to the extent an RNA sequence is not given. Every DNA and RNA sequence encoding polypeptides disclosed herein is encompassed by this disclosure, including but not limited to sequences encoding all recombinant proteins that comprise any complete SARS-CoV-2 protein, or an antigenic segment thereof, any antibody or antigen binding segment thereof, and any other protein encoded by the described modified viruses. All of the amino acid sequences and nucleotide sequences associated with any accession numbers are incorporated herein by reference as they exist in the database as of the date of the filing of this application or patent. The disclosure includes all polynucleotide and protein sequences described herein expressly or by reference that are between 80.0% and 99.9% identical to the described sequences. The proteins may comprise one or more than one amino acid change. Such changes can comprise conservative or non-conservative amino acid substitutions, insertions, and deletions. Any one or combination of components can be omitted from the claims, including any polynucleotide sequence, any amino acid sequence, and any one or combination of steps.
The disclosure includes all immune responses described below and in the Examples, including but not necessarily limited to antibody responses and T cell responses, and all combinations thereof. The disclosure provides for eliciting a synergistic immunological response. A synergistic response includes but is not necessarily limited to a synergistic effect on stimulation of T cells, antibodies, and a combination of T cells and antibodies, and on the transcriptome profile of T cells. A synergistic effect stimulates an improved immune response relative to use of a modified virus encoding a spike protein alone, or an immunomodulatory agent alone.
In embodiments, the disclosure provides a modified Alphavirus and pluralities of modified Alphavirus particles that are modified for use in stimulating an immune response against SARS-CoV-2. In embodiments, the modified Alphavirus encodes one or more SARS-CoV-2 proteins or antigenic segments thereof that are selected from the SARS-CoV-2 spike glycoprotein (S), envelope protein (E), membrane protein (M), and nucleocapsid protein (N). In embodiments, the SARS-CoV-2 protein comprises all or a segment of the viral spike receptor binding domain (RBD).
In embodiments, a modified Alphavirus of the disclosure encodes and expresses a spike protein that is expressed by the Wuhan-Hu-1 SARS-CoV2 virus, but the disclosure is not limited to this sequence and includes proteins and antigenic fragments thereof expressed by so-called SARS-CoV-2 variants, such variants including but not necessarily limited to variants currently referred to as variants of interest, variants of concern, and variants of high consequence. In embodiments, the described modified viruses encode spike protein or one or more antigenic fragments thereof from SARS-CoV-2 variants that include at least one of an L452R or E484K spike protein amino acid substitution. In embodiments, the spike protein or antigenic fragment of it is from SARS-CoV-2 variants currently referred to as B.1.1.7, B.1.351, P.1, P.2., B.1.427, B.1.429, or B.1.526.1. Any spike protein variant described herein may be compared to SEQ ID NO: 1 for reference to amino acid position.
In embodiments, an antigenic segment of a SARS-CoV-2 protein that is expressed by the described modified alphaviruses comprises or consists of the receptor binding domain (RBD) of the spike protein. In embodiments, the RBD comprises or consists of amino acids 333-527 of the spike protein. In embodiments, the antigenic segment of the spike protein comprises a receptor binding motif (RBF), which may include amino acids 438-506 of the spike protein.
The described modified alphaviruses may also encode and express one or more immunomodulating agents to generate effective anti-viral immune responses, including but not necessarily limited to T cell responses.
In embodiments, the described modified viruses are any type of alphavirus. In embodiments, the alphavirus that is modified according to the present disclosure comprises one or more modifications in the virus and/or plasmids that are used to make the modified viruses that are described in Current Opinion in Schlesinger and Dubensky, Biotechnology 1999,10:434-439, from which the description is incorporated herein by reference. In embodiments, the alphavirus of the disclosure comprises a modified Barmah Forest virus, Barmah Forest virus complex, Eastern equine encephalitis virus (EEEV), Eastern equine encephalitis virus complex, Middelburg virus, Middelburg virus complex, Ndumu virus, Ndumu virus complex, Semliki Forest virus, Semliki Forest virus complex, Bebaru virus, Chikungunya virus, Mayaro virus, Subtype Una virus, O′Nyong Nyong virus, Subtype Igbo-Ora virus, Ross River virus, Subtype Getah virus, Subtype Bebaru virus, Subtype Sagiyama virus, Subtype Me Tri virus, Venezuelan equine encephalitis virus (VEEV), VEEV complex, Cabassou virus, Everglades virus, Mosso das Pedras virus, Mucambo virus, Paramana virus, Pixuna virus, Western equine encephalitis virus (WEEV), Rio Negro virus, Trocara virus, Subtype Bijou Bridge virus, Western equine encephalitis virus complex, Aura virus, Babanki virus, Kyzylagach virus, Sindbis virus, Ockelbo virus, Whataroa virus, Buggy Creek virus, Fort Morgan virus, Highlands J virus, Eilat virus, Salmon pancreatic disease virus (SPDV), Southern elephant seal virus (SESV), Tai Forest virus, or Tonate virus. All alphavirus nucleotide and amino acid sequences, including all viral genomic sequences, expression vector and plasmid sequences, described in PCT publication WO/2021/007276 are incorporated herein by reference.
In embodiments, an immunomodulating agent that is encoded and expressed by a described modified virus, or is co-administered with a modified virus, comprises an antibody or antigen binding fragment thereof. In embodiments, the antibody or antigen binding fragment thereof has a receptor agonist function. In embodiments, the described immunomodulating agent comprises an anti-OX40 antibody. The amino acid sequences of suitable anti-OX40 antibodies are known in the art, and representative sequences are provided herein. In embodiments anti-OX40 antibody comprises complementarity determining regions (CDRs) and may include the complete heavy and light chain variable regions as described in PCT publication WO/2021/007276, from which all anti-OX40 antibody amino acid sequences and nucleotide sequences encoding them are incorporated herein by reference. The antibody may be of any isotype. In embodiments, the isotype is an IgG, which may be an IgG2a isotype.
In embodiments, the disclosure provides modified alphaviruses that co-express or are delivered in conjunction with an immunomodulating agent that may be distinct from an anti-OX40 antibody, representative examples of which include but are not limited to therapeutic proteins, such as cytokines, including but not necessarily limited to one or more interleukins (ILs). In an embodiment the IL is IL-12. In an embodiment, the IL is any IL described in PCT publication WO/2021/007276 from which the description of interleukins and their amino acid sequences is incorporated herein by reference.
In one embodiment, the present disclosure provides modified Sindbis virus (SV) vectors, which combine a SV-based approach of delivering the described SARS-CoV-2 proteins or antigenic segments thereof in combination with one or more immunomodulating agents. In this regard, SV is an RNA virus without replicative DNA intermediates and poses no risk of chromosomal integration or insertional mutagenesis. Hence, its presence within cells is transitory.
In non-limiting embodiments, the disclosure provides a therapeutically effective amount of one or more Sindbis viral vectors expressing a gene encoding a SARS-CoV-2 protein or antigenic segment of the protein, and (b) either independently or by expression of the viral vector an intact anti-OX40 monoclonal antibody, or an OX40-binding fragment thereof. In embodiments, the anti-OX40 binding fragment is any of OX40-binding (Fab) fragments, Fab′ fragments, (Fab′)2 fragments, Fd (N-terminal part of the heavy chain) fragments, Fv fragments (the two variable domains), dAb fragments, single domain fragments or single monomeric variable antibody domain (e.g., a nanobody), isolated CDR regions, single-chain variable fragment (scFv), and other antibody fragments or derivatives thereof, provided they bind with specificity to the Fc. In an embodiment, the antibody comprises an anti-SARS VHH single chain antibody.
In embodiments, the disclosure provides compositions and methods in which the Sindbis genome is split into two plasmids, one providing the replicon and the other providing the helper. This vector system may be used, for example, to electroporate in vitro transcribed viral RNA into a susceptible cell line to produce replicative defective Sindbis virus for use as a viral vector, wherein the viral vector contains, as a genome, the replicase RNA, but lacks Sindbis structural genes. Thus, in certain approaches, the Sindbis viral vector may be replication defective by way of deleting certain genes that are required to maintain its infectivity.
In embodiments, the disclosure provides modified Sindbis viral vectors, viral particles, pharmaceutical compositions comprising the viral particles, and methods comprising administering modified Sindbis-derived particles to individuals in need thereof.
In embodiments, the Sindbis virus vector or virus particle comprises a polynucleotide that encodes one or multiple (e.g., two or more) epitopes of one or more SARS-CoV-2 proteins. In certain embodiments, more than one SARS-CoV-2 protein or antigenic fragment may be included in the modified Sindbis virus, wherein each protein or antigenic fragment is separated by, for example, an enzyme cleavage site, or by a self-cleaving amino sequence. While T2A sequences are used in the Examples, other suitable ribosome skipping sequences may be used, and include but are not limited to P2A, E2A and F2A, the sequences of which are known in the art. In embodiments, the disclosure provides modified viruses that encode contiguously or separately non-overlapping spike protein epitopes to thereby reduce or prevent the formation of escape mutants.
In embodiments the disclosure provides isolated polynucleotides. By “isolated polynucleotide” is meant a nucleic acid (e.g., a DNA or RNA molecule) that is free of the genes which, in the naturally-occurring genome of the organism from which the nucleic acid molecule of the invention is derived, flank the gene. The term therefore includes, for example, a recombinant DNA that is incorporated into a vector; into an autonomously replicating plasmid or a described virus; or that exists as a separate molecule (for example, a cDNA or a genomic or cDNA fragment produced by PCR or restriction endonuclease digestion) independent of other sequences. In addition, the term includes an RNA molecule that is transcribed from a DNA molecule, as well as a recombinant DNA that is part of a hybrid gene encoding additional polypeptide sequence.
In embodiments the polynucleotides and/or viral particles produced using the described modified Sindbis or other alphavirus vectors are introduced into a subject as a component of a pharmaceutical composition. Suitable pharmaceutical compositions can be prepared by mixing one described modified alphaviruses described herein with a pharmaceutically acceptable additive, such as a pharmaceutically acceptable carrier, diluent or excipient, and suitable such components are well known in the art. Some examples of such carriers, diluents and excipients can be found in: Remington: The Science and Practice of Pharmacy 23rd edition (2020), the disclosure of which is incorporated herein by reference. In embodiments, the pharmaceutical formulation does not comprise a cell culture, or a cell culture media. In embodiments, the pharmaceutical formulation is free from any cell culture media. In embodiments, the pharmaceutical formulation is free from any mammalian cells or mammalian cell culture. In embodiments, the pharmaceutical formulation is free from Lipid inorganic nanoparticles (LIONs).
The described modified viruses and pharmaceutical formulations comprising them can be administered using any suitable route and method. In embodiments, the amount of agent includes an effective amount of the SARS-CoV-2 protein(s) and one or more immunomodulatory agents to achieve a desired result. The desired result can comprise a prophylactic effect, or a therapeutic effect. In embodiments, sufficient viral particles are introduced such that a cell mediated immune response is mounted against the one or more SARS-CoV-2 proteins, and wherein such cell mediated immune response, which may be accompanied by a humoral response, is therapeutic in an individual who is infected by SARS-CoV-2, and wherein the individual may or may not exhibit COVID-19 infection symptoms. Thus, in embodiments, a composition of the disclosure is administered to an individual who is infected with SARS-CoV-2, or is suspected of having a SARS-CoV-2 infection. In embodiments, the composition is administered to an individual who is at risk for contracting a SARS-CoV-2 infection. In embodiments, the individual is of an age wherein such risk is heightened, such as any individual over the age of 50 years. In embodiments, the individual has an underlying condition wherein the risk of developing severe symptoms of COVID-19 infection is increased, including but not necessarily limited to any respiratory condition.
In embodiments, an effective amount of a composition is administered to an individual. An effective amount means an amount of the described modified virus that will elicit the biological or medical response by a subject that is being sought by a medical doctor or other clinician. In embodiments, an effective amount means an amount sufficient to prevent, or reduce by at least about 30 percent, or by at least 50 percent, or by at least 90 percent, any sign or symptom of viral infection, e.g., any sign or symptom of COVID-19. In embodiments, fever is prevented or is less severe than if the presently described vaccine had not been administered. In embodiments, viral pneumonia is inhibited or prevented. In an embodiment, administration of a described vaccine prevents a SARS-CoV-2 infection in an individual who is exposed to SARS-CoV-2. In embodiments, an effective amount comprises 106 - 109 transducing units (TU)/mL. In embodiments, about 107 TU are administered.
In embodiments, an effective amount is provided a single time and provides a therapeutic or prophylactic effect. In embodiments, a dose is administered at least one time, at least two times, at least three times, at least four times or at least five times. In embodiments, a prime-boost dosage approach is used. The described approaches may be sufficient to provide a durable immune response that is protective against SARS-CoV-2 infection, and further described herein.
Administration of formulations comprising the modified Sindbis or other alphavirus vectors and/or viral particles as described herein can be performed using any suitable route of administration, including but not limited to parenteral, intraperitoneal, and oral administration. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, and subcutaneous administration. In one embodiment, a composition comprising the described modified viruses is provided in a form suitable for inhalation, including but not necessarily limited to an aerosol. In embodiments, a composition of the disclosure is lyophilized and is suitable for reconstitution in a liquid or aerosolizable form. In embodiments, a composition of the disclosure is administered to the lungs and/or gastrointestinal track of an individual. The compositions can be administered to humans, and are also suitable for use in a veterinary context and accordingly can be given to non-human animals, including non-human mammals such as canines, felines, and equine animals. In embodiments the disclosure provides a pancornavirus vaccine. In embodiments, in addition for the described uses related to SARS-CoV-2 and its variants, the described compositions and methods can be used for prophylaxis or therapy for any infectious member of the virus family Coronaviridae. Non-limiting examples of such viruses include any Coronavirus that that causes any of severe acute respiratory syndrome (SARS), Middle East respiratory syndrome (MERS), and feline Coronavirus (FCoV) that can lead to the development of feline infectious peritonitis (FIP), and any variants thereof.
In embodiments, a described composition may further comprise, or may be administered concurrently or consecutively with any anti-viral compound or other agent, non-limiting examples of which include Lopinavir, Ritonavir, one or more protease inhibitors, nucleoside derivatives such as Romidepsin and Ribavirin, Favipiravir, interferons, and therapeutic and prophylactic antibodies, such as antibodies identified or derived from convalescent patient plasma. In embodiments, the described modified viruses potentiate the described anti-viral compounds or antibodies, and may result in a synergistic anti-viral effect.
In embodiments, viral particle preparations can be produced for use in pharmaceutical preparations by adapting previous approaches so that the modified SV vectors that express the SARS-CoV-2 protein or antigenic binding fragment thereof, optionally along with the anti-OX40 and/or IL component, are produced. In embodiments, plasmids encoding the replicon or helper RNA are linearized and transcribed in vitro.
In more detail, in certain embodiments, a replicon plasmid encoding the Sindbis replicase genes (nsP1-nsP4) and a helper plasmid, encoding the viral structural genes (capsid protein C, E1, E2, E3, and 6K), are transcribed in vitro. To limit viral replication in vivo, the replicon genes are separated from the structural genes, which additionally contain a mutated packaging signal to prevent incorporation into virus particles. Virus particles are produced by transient transfection of baby hamster kidney (BHK) cells with in vitro synthesized Sindbis replicon RNA and helper RNA transcripts. Within the cell, genomic RNA is replicated by the Sindbis replicase and expressed from the capped replicon RNA transcript. Structural proteins are expressed from the helper RNA transcript. Only the replicon RNA is packaged into the capsid to form the nucleocapsid, which then associates with the viral glycoproteins E1 and E2 and buds out of the cell. The resulting virions contain the capped SV single-stranded RNA message for nsP1-nsP4 genes, which encode the viral replicase, and may include a subgenomic promoter (Psg).
The viral particles can be purified using any suitable technique to any desired degree of purity, and combined with one or more pharmaceutically acceptable excipients, carriers and the like, as described above.
In certain embodiments, the SV vectors used in this disclosure are generated from the Sindbis strain AR339, the genomic and protein sequence of which is known are the art, and which does not cause disease in humans. In embodiments, the SV AR339 has the sequence as available under GenBank: MH212167.1, from which all of the amino acid and nucleotide sequences from which are incorporated herein by reference as of the effective filing date of this application or patent. Nonetheless, to limit even transient adverse effects, in embodiments, the presently provided vectors are attenuated as discussed above by splitting the SV genome and removing the packaging signal from the genomic strand that encodes the structural genes. Thus, the described vectors cannot propagate beyond the cells they initially infect.
In certain embodiments, to further increase safety, the disclosure provides for utilization of only the replicon strand to produce vaccine formulations. Accordingly, in certain embodiments, no SV structural protein coding sequences are present in the described vaccines.
In one non-limiting approach, the SV replicon strand is used to express the spike protein isolated from the plasmid NR-52310, encoding the SARS-CoV-2, Wuhan-Hu-1 spike glycoprotein gene. In a non-limiting approach, expression of the spike glycoprotein (gp) or a derivative thereof from the replicon is achieved by transfection into Baby Hamster Kidney (BHK) cells or other suitable mammalian cells, followed by collection of the supernatant within a suitable period of time, such as about 48 hours after transfection. Western blot and Elisa using a commercially available antibody, such as ProSci catalog numbers 3223 and 3525, or any other suitable antibody, can be used to confirm expression if desired. Representative and non-limiting examples of the described approaches are provided in the Examples, which demonstrate production of high affinity antibodies and metabolic reprogramming of T cells that is expected to confer long-term memory to one or more SARS-CoV-2 antigens.
In embodiments, use of the compositions and methods of this disclosure stimulates production of SARS-CoV-2 protective antibodies, which may include neutralizing antibodies. The term “neutralizing antibody” refers to an antibody or a plurality of antibodies that inhibits, reduces or completely prevents viral infection.
The disclosure provides for measuring antibody and T cell responses to the spike protein, and/or to any other SARS-CoV-2 proteins that are included in the described vaccines. Analysis of the antibody responses can be performed using any suitable control, such as the NR-52306 spike glycoprotein RBD recombinant protein from SARS-CoV-2, Wuhan-Hu-1 produced from HEK293T cells. In embodiments, this spike protein is as available under NCBI Reference Sequence: NC_045512.2, from which all amino acid and nucleotide sequences are incorporated herein by reference as they exist on the effective filing date of this application or patent.
To determine T cell responses, and as further illustrated by the Examples, the disclosure includes use of mouse models, such as BALB/c derived CT26-Luc-NR-52310 spike glycoprotein cells. The disclosure includes safety and pharmacokinetic analysis. For example, cytotoxicity can be determined in vitro by diminution of luciferase signal. The disclosure includes in vivo analysis, such as by injecting the described cells into BALB/c mice and measuring the ability of the elicited T cells to suppress their growth as compared to CT26-Luc cells. It is expected that positive results will be obtained, and any other assays that directly impact COVID-19 infection can be performed to advance human implementations, and may include virus pseudotypes to preclude safety concerns.
In embodiments, the disclosure provides kits and articles of manufacture. Either may contain one or more sealed containers that contain one or more plasmids for producing the described modified viruses. The kit or article of manufacture may comprise a form of the vaccine that is suitable for injection or oral delivery, including by inhalation. The kit or article of manufacture may further include cell culture media, and/or cells suitable for producing the described modified viruses. The kit or article of manufacture may include printed material, such as a label or product insert that provides an indication that the contents of the container is for use in prophylaxis and/or therapy for a SARS-CoV-2 infection.
A representative and non-limiting example of a DNA sequence that corresponds to the modified viruses as further described herein is provided in SEQ ID NO:5. Within SEQ ID NO:5, the following proteins are encoded: Anti-OX40 IgG2a Heavy Chain, nucleotides 7661-9085; Anti-OX40 IgG2a Light Chain, nucleotides 9270-9992; IL- 12, nucleotides 10056-11675; SARS CoV2 Spike protein, nucleotides 11751-15599. The plasmids or other expression vectors and the RNA transcribed from them can, as further described herein, include other features. For instance, in SEQ ID NO:5 includes a 2Psg sequence at nucleotides 9086-9209 and T2A coding sequences at nucleotides 9993-10055 and 11676-11740. The disclosure includes all RNA equivalents of SEQ ID NO:5 (e.g., where U is substituted for T).
A representative and non-limiting example of an Anti-OX40 IgG2a Heavy Chain protein sequence is provided as SEQ ID NO:2:
A representative and non-limiting example of an Anti-OX40 IgG2a light chain protein sequence is provided as SEQ ID NO:3:
A representative and non-limiting example of an IL-12 amino acid sequence is provided is SEQ ID NO:4:
A representative and non-limiting example of a SARS CoV2 Spike protein sequence is provided as SEQ ID NO: 1:
The following examples are intended to illustrate but not limit the disclosure.
This Example demonstrates a Sindbis alphavirus vector (SV), transiently expressing the SARS-CoV-2 spike protein (SV.Spike), combined with the OX40 immunostimulatory antibody (αOX40) as a novel, highly effective vaccine approach.
Construction and characterization of Sindbis carrying the SARS-CoV-2-spike.
We designed and generated a Sindbis alphavirus replicon carrying the SARS-CoV-2 spike mRNA. SV vectors are generated from two plasmids: a replicon and helper (
The combination of SV vectors encoding a selected antigen with immunomodulatory antibodies makes them far more effective than they are alone[40; 41; 42]. In particular we have found that combining SV vectors expressing specific antigens with αOX40 generates very potent immune responses capable of eradicating tumors in multiple murine models and conferring long-term protection against tumor recurrences or rechallenges[40].
The overall design in the production of Sindbis SARS-CoV-2 Spike (SV.Spike) is illustrated in
The immune responses induced by the Sindbis SARS-CoV-2 Spike (SV.Spike) vaccine candidate were analyzed in C57BL/6J mice. Groups of mice (n = 5) were immunized by intraperitoneal (i.p.) route, by prime-boost vaccine strategy with SV.Spike and/or αOX40, with 14 days difference between the two doses (
Sindbis vaccine-elicited antibodies to SARS-CoV-2 spike.
Serum IgM, IgG and IgA responses to SV.Spike, SV.Spike+αOX40, injections were measured on days 21, 75 and 100 days after vaccination by enzyme-linked immunosorbent assay (ELISA) against recombinant SARS CoV2 spike protein[3; 4]. Sera from all of mice tested showed reactivity to recombinant SARS-CoV-2 spike protein and, as might be expected, levels of antibodies varied based on the experimental group and time point. Consistent with previous reports[43; 44; 45], levels of IgM and IgG measured at day 21 and 75 post injection (p.i.) were significantly higher in the mice vaccinated with SV.Spike and combination of SV.Spike+□OX40 than in the mice who had received □OX40 alone or the naive group (
Anti-SARS-CoV-2 spike neutralizing antibodies induced in Sindbis vaccinated mice block the SARS-CoV-2 spike protein from binding to hACE2 receptor proteins.
Immediately after SARS-CoV-2 was identified as the causative agent of the COVID-19 outbreak, it was shown that human ACE2 (hACE2) is the main functional receptor for viral entry[46]. We hypothesized that the virus-receptor binding can be mimicked in vitro via a protein-protein interaction using purified recombinant hACE2 and the Spike of the SARS-CoV-2 protein. This interaction can be blocked by virus naturalizing antibodies (NAbs) present in the test serum of vaccinated mice.
A competition ELISA assay was developed to detect whether SARS-CoV-2 Spike-specific antisera from mice immunized with αOX40, SV.Spike and SV.Spike+αOX40 could block the interaction between SARS-CoV-2 Spike and hACE2. Our assay demonstrated that the specific Spike-hACE2 binding can be neutralized by SV.Spike or SV.Spike+αOX40 sera in a dose-dependent manner, but not by sera from αOX40 alone or naïve groups (
To investigate whether the neutralizing antibody response in immunized mice could maintain a high level for a longer period of time, we tested the neutralization activity of mice sera at 75 days post-immunization. The results showed that, although the overall antibody neutralizing capacity decreased compared to day 21, antibodies from SV.Spike and SV.Spike+αOX40 groups still significantly competed for the binding of the SARS-CoV-2 spike and hACE2 (
Next, we investigated if the serum from mice immunized with SV.Spike could inhibit the cell membrane fusion process for viral entry[47; 48; 49]occurring upon the binding of SARS-CoV-2 Spike Receptor Binding Domain (RBD) fragment to the ACE2 receptor on target cells. To establish an assay for measuring SARS-CoV-2-Spike-mediated cell-cell fusion, we employed HEK293T cells (a highly transfectable derivative of human embryonic kidney 293 cells, that contain the SV40 T-antigen) expressing both SARS-CoV-2 Spike and enhanced green fluorescent protein (EGFP) as effector cells and 293T cells stably expressing the human ACE2 receptor (ACE2/293T) as target cells. Notably, when the effector cells and the target cells were co-cultured at 37° C. for 6 h and 24 h, the two types of cells started to fuse at 6 h, exhibiting a much larger size and multiple nuclei compared to the unfused cells. These changes were more significant at 24 h, resulting in hundreds of cells fused as one large syncytium with multiple nuclei that could be easily seen under both light and fluorescence microscopy (
To determine whether the serum of mice immunized with SV.Spike can block Spike protein-mediated cell-cell fusion, we incubated the effector cells with serum from Naive, SV.Spike and/or αOX40 mice (diluted 1:100) at 37° C. for 1 h and then we co-cultured them with the ACE2/293T target cells. We found that not only were fewer fusing cells observed, but also the size of fused cells were visually smaller in the groups of SARS-CoV-2-Spike/293T effector cells pre-incubated SV.Spike with or without αOX40 sera compared to controls (
The interference of immunized sera NAbs with SARS-CoV-2-hACE2 binding was also determined by immunofluorescence experiments performed by culturing ACE2/293T cells with recombinant SARS-CoV-2 Spike previously incubated with serum from naive and SV.Spike and αOX40 immunized mice. The binding between Spike and hACE2 expressed on the cell surface was subsequently visualized via confocal fluorescence microscopy (
Taken together, these data demonstrate that SV.Spike alone, and to a greater extent SV.Spike+αOX40 sera, can neutralize SARS-CoV-2 Spike-hACE2 interaction and in turn counteract virus entry mediated by cell-membrane fusion.
This Example demonstrates that SV.Spike vaccine prevents infection of SARS-CoV-2 in hACE2 transgenic mice.
The neutralizing activity of serum from vaccinated mice was determined using Luciferase-encoding SARS-CoV-2 Spike pseudotyped lentivirus[50; 51] [52] (
Recently, hACE2 transgenic (B6(Cg)-Tg(K18-ACE2)2Prlmn/J or hACE2-Tg) mice were used for the development of an animal model of SARS-CoV-2 infection[53]. In order to test pseudotyped lentivirus infectivity rate in vivo, we produced a nLacZ-encoding lentivirus expressing SARS-CoV-2 spike protein (
In order to investigate the protective effects of SV.Spike vaccination in vivo, we subsequently immunized hACE2-Tg mice with the same strategy as used for the C57BL/6J mice (
This Example demonstrates that SV.Spike in combination with αOX40 metabolically reprograms and activates T cells shortly after prime vaccine doses.
Analysis of SARS-COV-2 specific adaptive immune responses during acute COVID-19 identified coordination between SARS-COV-2-specific CD4+ T cells and CD8+ T cells in limiting disease severity[54]. We analyzed vaccine elicited T cell responses in the spleen 7 days after mice received prime doses of SV.Spike and/or αOX40 and compared the initial T cell response to naïve mice (
For a successful vaccine-elicited immune response, differentiation of virus-specific T cells from the naive to the effector state requires a change in the metabolic pathways utilized for energy production[55]. Therefore, metabolic profiles of vaccine-induced T cells are of interest and correlate to vaccine-mediated immunity[56].
We performed metabolic analysis of isolated T cells from spleens in an Extracellular Flux Analyzer XFe24 (Seahorse Bioscience) to investigate metabolic changes of T cells. We found, that combining our SV.Spike vaccine with agonistic αOX40 antibody metabolically rewires T cells in vivo shortly after initial vaccine doses (
This Example demonstrates that SV.Spike+αOX40 vaccinated mice are characterized by a unique T cell transcriptome signature profile after prime vaccine doses.
To reveal the molecular profile of SV.Spike vaccine induced T cell responses, we isolated T cells 7 days after prime vaccine doses from spleens of mice from SV.Spike and/or αOX40 vaccinated groups and naive group. We then performed mRNA deep sequencing (RNAseq) and network analysis (
We next identified the top 10 hub GO terms by employing the Maximal Clique Centrality (MCC) for SV.Spike (
In conclusion, these findings indicate that synergistic SV.Spike+αOX40 vaccine combination successfully changes the transcriptome profile of T cells that is indispensable for building up humoral and T cell immunity.
This Example demonstrates that CD4+ T cell help promotes effector differentiation of cytotoxic T cells.
SARS-CoV-2-specific T cells are associated with protective immune responses[54]. Th1- type differentiated effector CD4+ T helper cells promote the development of CD8+ T cells into anti-viral cytotoxic T lymphocytes (CTLs) and functional memory T cells that can be quickly mobilized to directly kill SARS-CoV-2 early on upon re-infection preventing disease in coordination with SARS-CoV-2 specific humoral immune responses. CD4+ T helper cells are critical for success of vaccines and generally work by providing cytokines. We performed flow cytometry analysis to investigate CD4+ T helper differentiation, formation and antiviral cytotoxic effector T cell differentiation in T cells from SV.Spike and/or αOX40 immunized animals (
The predominant pathway used by human and murine CD8+ T cells to kill virus-infected cells is granule exocytosis, involving the release of perforin and GrB. It is known from influenza vaccine research that GrB correlates with protection and enhanced CTL response to influenza vaccination in older adults[60]. We looked at CTLs after day 7 of prime doses and found that combination immunization significantly increased differentiation of CTLs indicated by GrB expression (
Interestingly, it has been reported that cytotoxic CD4+ T cells can compensate for age related decline of immune cell protection such as B cell loss and a less robust antibody response[61]. Strikingly, we found in SV.Spike+αOX40 immunized mice showed a significant increase of cytotoxic CD4+ T cells indicating that the presently described vaccine not only induced Th1-type CD4+ T helper functions but has the potential to improve direct CD4+ T cell mediated virus-killing, thus, adding an extra layer to immune protection against SARS-CoV-2 in more vulnerable older populations. One important early feature of response to the SV.Spike+αOX40 immunization is a strong interferon-gamma (IFNγ) secretion (
This Example demonstrates that SV.Spike in combination with αOX40 drives metabolic activation of B cells and T cell dependent B cell support.
Almost all durable neutralizing antibody responses as well as affinity matured B cell memory depend on CD4+ T cell helper. GSEA of RNAseq data between T cells from the SV.Spike+αOX40 vaccinated and naive group one week after prime vaccine doses revealed selective enrichment of the gene set characteristic for activation of B cells (
This Example demonstrates that a combination of SV.Spike and αOX40 promotes robust T cell specific immune response in lungs.
Most vaccines for airborne infectious diseases are designed for delivery via the muscle or skin for enhanced protection in the lung. We investigated if SV.Spike vaccine-induced T cells can readily home most efficiently to the lungs prior to and shortly after pathogen exposure. To address the immune responses in the lungs, we immunized mice with SV.Spike and/or αOX40 and excised PBS-perfused lungs one week after booster doses for single cell suspensions and performed flow cytometry staining (
This Example demonstrates that SV.Spike and αOX40 promotes CD4+ T cell memory formation and long-term protection upon re-challenge with SARS-CoV-2 Spike antigen.
Boosting both, local and systemic memory T cell response is a useful strategy to achieve long term immunity. We analyzed development of T cell memory in spleens fourteen weeks after initial prime vaccine doses of SV.Spike and/or αOX40 prime-boost immunized mice by flow cytometry. We found that mice in the SV.Spike+αOX40 combination group developed significant effector CD4+ T memory indicated by CD44+ CD62L+ double-positive CD4+ T cells (
To further explore the long-term protection efficacy of our SV.Spike vaccine against SARS-CoV-2 virus challenge, C57BL/6J mice (n = 5 each group) received prime and boost immunizations of αOX40, SV.Spike and/or αOX40 and placebo (naive group) via the i.p. route. At day 100 post-immunization, we additionally administered one dose of SV.Spike, to recapitulate Spike antigen endogenous entry through SV vector injection (
Antibody response analysis showed that immunization with SV.Spike or SV.Spike+αOX40 followed by Spike antigen injection induced strong production of IgM antibodies compared to the mice which did not received the antigen and the Naive groups, and that was particularly evident in mice vaccinated with SV.Spike (
Despite promising results of early clinical trials of several vaccine candidates against SARS-CoV-2, there are still concerns regarding both safety and durability of the immune responses. Consequently, the Examples above demonstrate development of an improved vaccine. This vaccine is considered, without intending to be bound by any particular theory, to be effective after one or two immunizations, conferring long-term protection to target populations such as the elderly or immunocompromised individuals, and reducing onward transmission of the virus to contacts[65].
It is expected, based on the foregoing description, that new constructs can be made rapidly with synthetic design of the insert, and readily adapted to SARS-CoV-2 variants. Moreover, when new virus species emerge, the described vaccine platform can be rapidly adapted to combat emerging viruses.
Sindbis virus and other alphaviruses have a natural tropism for lymphatic tissues and dendritic cells, relative resistance to interferon, high expression levels, lack of pre-existing anti-vector immunity in most human and animal populations, and efficient production of methodology in cell lines, with an accepted regulatory pedigree[72].
Neutralizing antibodies (NAbs) have conventionally been the desired outcome of vaccination, as they are capable of intercepting and neutralizing microbes and their components, as well as eliciting destructive anti-microbial innate immune responses[73]. Nonetheless, humoral immunity can decline over time and, as seen with influenza, can only last as short as one season. Many newer vaccines and vaccines in development are also designed to generate T cell responses that have the potential to help the antibody response, promote long-term immune memory, have direct effector functions themselves, or activate innate effector cells such as macrophages and neutrophils[45; 74].
The present disclosure provides a Sindbis-based Spike-encoding RNA vaccine against SARS-CoV-2 and demonstrates that immunization with SV vector expressing SARS-CoV-2 Spike along with a costimulatory agonistic αOX40 antibody induced a synergistic T cell and antibody response and provided complete protection against authentic SARS-CoV-2 challenge in hACE2 transgenic mice. It is expected that this approach will boost tissue specific immunity and immune memory against the described viruses and could protect for several seasons or years. As a viral vector, we found that a Sindbis vector expressing SARS-CoV-2 Spike antigen in combination with αOX40 markedly improves the initial T cell priming, compared with the viral vector alone, which results in a robust CD4+ and CD8+ T cell response and stable SARS-CoV-2 specific neutralizing antibodies. The vaccine efficiently elicits effector T cell memory in respiratory tissues with a potential for long lasting protection against COVID19, which might extend for several years, through multiple beneficial mechanisms. It protects against infection with authentic, SARS-CoV-2 preventing morbidity and mortality.
αOX40 controls survival of primed CD8+ T cells and confers CTL-mediated protection[31; 75]. CTLs are a critical component of the adaptive immune response but during aging, uncoordinated adaptive responses have been identified as potential risk factors that are linked to disease severity for the outcome of COVID19 patients. It is known from influenza vaccine research that Granzyme B correlates with protection and enhanced CTL response to influenza vaccination in older adults. We looked at cytotoxic T cells (CTLs) and found that combination vaccination significantly increased CD8+ cytotoxic T cells indicated by granzyme B and perforin upregulation. Almost all durable neutralizing antibody responses as well as affinity matured B cell memory depend on CD4+ T helper cells. We found in combination vaccinated mice a significant increase of cytotoxic CD4+ T cells indicating that the presently described vaccine not only induced CD4+ T helper functions but has the potential to improve direct CD4+ T mediated virus-killing adding an extra layer to long-term immunity/protection in more vulnerable older populations.
Virus-specific CTLs are quickly recruited to influenza-infected lungs by a Th1 response, specifically due to the production of IFNγ[59]. IFNγ regulates various immune responses that are critical for vaccine-induced protection and has been well studied[76; 77]. In a clinical trial of the now approved BNT162b1, IFNγ secreting T cells increased in participants 7 days after boost [45]. In this regard, one important early feature of the response to the SV.Spike+αOX40 immunization is a strong interferon-gamma (IFNγ) secretion. We found a significant increase of CXCR3 and CX3CR1 positive expressing CD4+ T cells, indicating effective recruitment and mobility of generated effector Th1 type T cells in mice. This recruitment positively correlates with vaccine induced long-term immune protection and generation of neutralizing antibodies against SARS-CoV-2.
Both humoral and cell-mediated immune responses have been associated with vaccine-induced protection against challenge or subsequent re-challenge after live SARS-CoV-2 infection in recent rhesus macaque studies [78; 79] and there is mounting evidence that T-cell responses play an important role in COVID-19 mitigation[3; 80; 81]. We demonstrated that two doses of SV.Spike with or without αOX40 candidate vaccines induced neutralizing antibody titers in all immunized mice, with a strong IgG response in the mice receiving combination vaccination. Moreover, the Examples show that SV.Spike+αOX40 skewed Tfh cells toward CXCR5+ Tfh differentiation, which positively correlated with the magnitude of IgG isotype response. These findings indicate that the induction of CXCR5+ Tfh cell differentiation through vaccination may be beneficial for eliciting broad and specific NAb responses. Importantly, the synergistic activity of combination vaccination elicited antibodies that were able to efficiently neutralize SARS-CoV-2 pseudotyped lentivirus in all the mice tested. In addition, we show SV-Spike-based re-challenge in mice immunized with combination vaccination led to enhanced cytotoxic reactivation of T cells and increased IgG seroconversion and response, and provided protection against re-challenge, reiterating the importance of the involvement of both humoral and cellular immune responses in SARS-CoV-2-mediated immunity.
The described SV.Spike platform has the advantage that it is inexpensive, stable, and easy to produce when given the benefit of the present disclosure . Cost projections based on using the described processes for production of a SV based vaccine are in line with or below costs per dose for other vaccines in use today. Moreover, unlike other mRNA vaccine candidates this viral platform does not require a cold-chain during transportation and storage. It can be easily reconstituted after lyophilization process and is suitable for rapid adaptation such that potential new viruses/threats in an emerging outbreak can be rapidly targeted[82]. Thus, for emerging pathogens like SARS-CoV-2, the described SV platform can be an efficient and cost-effective alternative to the traditional large-scale antigen production or technology platforms that require extended time for implementation.
As shown in this disclosure, SV.Spike can be applied alone or can be combined with immunomodulatory reagents like αOX40 in a remarkably efficient prime-boost regimen. The disclosure includes a combined SV.Spike + αOX40 coding single vector.
Baby hamster kidney (BHK) and HEK293T cell lines were obtained from the American Type Culture Collection (ATCC). 293T-ACE2 cell line was obtained from BEI Resources.
BHK cells were maintained in minimum essential α-modified media (α-MEM) (Corning CellGro) with 5% fetal bovine serum (FCS, Gibco) and 100 mg/ml penicillin-streptomycin (Corning CellGro). 293T and 293T-ACE2 cells were maintained in Dulbecco’s modified Eagles medium containing 4.5 g/l Glucose (DMEM, Corning CellGro) supplemented with 10% FCS, 100 mg/ml penicillin-streptomycin. All cell lines were cultured at 37° C. and 5% CO2.
SV.Spike expressing vector was produced as previously described[38; 39; 83; 84]. Briefly, plasmids carrying the replicon (pT7-SV-Spike) orT7-DMHelper RNAs were linearized with XhoI. In vitro transcription was performed using the mMessage mMachine RNA transcription kit (Invitrogen Life Sciences). Helper and replicon RNAs were then electroporated into BHK cells and incubated at 37° C. in αMEM supplemented with 10% FCS. After 12 hours, the media was replaced with OPTI-MEM (GIBCO-BRL) supplemented with CaCl2 (100 mg/l) and cells were incubated at 37° C. After 24 hours, the supernatant was collected, centrifuged to remove cellular debris, and frozen at -80° C. Vectors were titrated as previously described [85].
SARS CoV-2 pseudotyped lentiviruses were produced by transfecting the HEK293T cells with the pLenti-Puro vectors (Addgene) expressing Luciferase or β-Galactosidase, with pcDNa3.1 vector expressing SARS-CoV-2 Spike (BEI repository) and the helper plasmid pSPAX2 (Addgene). The VSV-G and empty lentiviruses were produced by replacing pcDNA3.1-Spike with pcDNA3.1-VSV-G or pcDNA3.1 empty vector, respectively (Addgene). The transfections were carried out using the Polyethylenimine (PEI) method with the ratio at PEI:pLenti:pcNDA3.1-Spike:pSPAX2 = 14:2:2:1 or PEI:pLenti:pVSV-G/pcNDA3. 1:pSPAX2 = 10:1:0.5:3. The virus-containing medium was harvested 72 hours after transfection and subsequently pre-cleaned by centrifugation (3,000 g) and a 0.45 µm filtration (Millipore). The virus-containing medium was concentrated by using a LentiX solution (TakaraBio) a 10:1 v/v ratio and centrifuged at the indicated RCF at 4° C. After centrifugation, the supernatant was carefully removed and the tube was drained on the tissue paper for 3 minutes. Dulbecco’s modified Eagles medium containing 4.5 g/l Glucose (DMEM) was added to the semi-dried tube for re-suspension and then stored at -80° C.
Luciferase- and nLacZ-encoding SARS CoV-2 Spike or VSV-G pseudotyped lentivirus titers were determined making serial dilutions of the vectors in DMEM and infect 293T/ACE2 cells pre-plated in 96-well culture plates (104 cells/well) and 24 h later, fresh media was added. For Luciferase-encoding pseudotype, cells were lysed 72 h later using cell lysis buffer and lysates were transferred into fresh 96-well luminometer plates, where luciferase substrate was added (Thermo Fisher), and relative luciferase activity was determined (
All experiments were performed in accordance with protocols approved by the Institutional Animal Care and Use Committee of New York University Grossman School of Medicine. Six to 12-week old female C57BL/6J albino mice (B6(Cg)-Tyr<c-2J>/J,Cat#000058) and mice expressing the human ACE2 receptor2 : B6(Cg)-Tg(K18-ACE2)2Prlmn/J Hemizygous or non-carrier controls (Cat#034860) were purchased from Jackson Laboratory.
Three weeks after prime and boost vaccination doses, B6(Cg)-Tg(K18-ACE2)2Prlmn/J and non-carrier control mice were challenged with 104 particles of SARS-CoV-2 Coronavirus via the intranasal (i.n.) route (
Mice were i.p. immunized with SV.Spike (107 TU/ml) in a total volume of 500 µl was injected i.p. into the left side of the animal. The immunostimulatory αOX40 antibody (clone OX-86, BioXCell) was injected i.p. into the left side of the animal at a dose of 250 µg per injection. Mice were boosted once at 2 weeks. Sera were collected at 7 days post-2nd vaccination and used to detect neutralizing activity.
Therapeutic efficacy of vaccines was monitored in two ways: vaccinated B6(Cg)-Tg(K18-ACE2)2Prlmn/J mice that were challenged with SARS-CoV-2 Coronavirus in BSL3 were tested for survival compared to their non immunized control group. Survival was monitored and recorded daily.
Isoflurane-anesthetized 4-week-old young adult B6(Cg)-Tg(K18-ACE2)2Prlmn/J (hACE2-Tg) mice were dosed intranasally with a 70-µl volume of nLacZ-encoding lentiviral vector (titer 5.18×103 TU/ml). Isoflurane anesthesia (2.5% isoflurane/1.51 oxygen per minute) and dosing of animals was carried out in a vented BSL-2 biological safety cabinet. For processing of mouse lungs for X-Gal staining of intact tissue, lungs were inflated through the trachea with OCT embedding as described previously[86]. Intact airways were submerged in 0.5% glutaraldehyde for 2 hat 4° C., washed in PBS/1 mM MgCl2 and stained for 16 h at 37° C. with X-Gal Solution [1 mg/ml X-Gal in PBS (pH 7.0) containing 20 mM potassium ferricyanide, 20 mM potassium ferrocyanide and 1 mM MgCl2].
To measure protective NAbs, COVID-19 convalescent plasma was diluted (1:10) and incubated with recombinant SARS-CoV-2 full-length Spike (BPS Bioscience) for 1 h at 37° C. prior to adding to an ACE2 pre-coated ELISA plates. The NAb levels were calculated based on their inhibition extents of Spike and hACE2 interactions according to the following equation: [(1-OD value of samples/OD value of negative control) × 100%]. A neutralizing antibody against SARS-CoV-2 Spike (Bio Legend) was used as a positive control.
Pseudotyped lentivirus inhibition assay was established to detect neutralizing activity of vaccinated mouse sera and inhibitory ability of antiviral agents against infection of SARS-CoV-2 Spike pseudotyped lentivirus in target cells. Briefly, pseudotyped virus containing supernatants were respectively incubated with serially diluted mouse sera at 37° C. for 1h before adding to target cells pre-plated in 96-well culture plates (104 cells/well). 24 h later, fresh media was added and cells were lysed 72 h later using cell lysis buffer. Lysates were transferred into fresh 96-well luminometer plates. Luciferase substrate was added (Promega), and relative luciferase activity was determined. The inhibition of SARS-COV-2 Spike pseudotype lentivirus was presented as % inhibition.
The establishment and detection of several cell-cell fusion assays are as previously described [47]. In brief, 293T/ACE2 cells were used as target cells. For preparing effector cells expressing SARS-CoV-2 Spike, 293T cells were transiently co-transfected with pcDNA3.1-Spike and pMAX-GFP or with pMAX-GFP only as control, and applied onto 293T/ACE2 cells after 48 h. Effector and target cells were cocultured in DMEM plus 10% FBS for 6 h. After incubation, five fields were randomly selected in each well to count the number of fused and unfused cells under an inverted fluorescence microscope (Nikon Eclipse Ti-S).
The inhibitory activity of neutralizing antibodies from immunized mice sera on a SARS-CoV-2-Spike-mediated cell-cell fusion was assessed as previously described[49; 87].
Briefly, a total of 2 × 104 target cells/well (293T/ACE2) were incubated for 5 h. Afterwards, medium was removed and 104 effector cells/well (293T/Spike/GFP) were added in the presence of serum from C57BL/6J immunized mice at 1:100 dilution in medium at 37° C. for 2 h. The fusion rate was calculated by observing the fused and unfused cells using fluorescence microscopy.
Cell immunocytochemistry was performed as described previously[88]. Briefly, cells were fixed with 4% paraformaldehyde (PFA) for 20 min at room temperature and then the membrane was permeabilized with 0.1% (vol/vol) Triton X-100 (Fisher Scientific). Incubation with blocking solution (5% normal goat serum) was performed at room temperature for 45 min. Anti-mouse SARS-CoV-2-Spike (GTX, 1:100) and anti-rabbit hACE2 (Thermo Fisher, 1: 100) were applied overnight at 4° C. followed by incubation of appropriate secondary antibodies conjugated with fluorophores. Confocal images were captured using the Zeiss LSM-800 system.
For flow cytometry analysis, spleens were harvested from mice and processed as previously described[39]. Extracted lungs were chopped in small pieces and incubated with a digestive mix containing RPMI with collagenase IV (50 µg/ml) and DNAseI (20 U/ml) for 30 min at 37° C. Spleens and lungs were mashed through a 70-µm strainer before red blood cells were lysed using ammonium-chloride-potassium (ACK) lysis (Gibco). Cells were washed with PBS containing 1% FCS and surface receptors were stained using various antibodies. Fluorochrome-conjugated antibodies against mouse CD3, CD4, CD44, CD38, ICOS, OX40, CD62L, Perforin, Granzyme B and Tbet, CXCR5 were purchased from Biolegend. Fluorochrome-conjugated antibodies against mouse CD8a were purchased from BD Biosciences. Fluorochrome-conjugated antibodies against CXCR3 and Ki67 were purchased from Thermofisher. Stained cells were fixed with PBS containing 4% Formaldehyde. For intracellular staining, the forkhead box P3 (FOXP3) staining buffer set was used (eBioscience). Flow cytometry analysis was performed on a LSR II machine (BD Bioscience) and data were analyzed using FlowJo (Tree Star).
Total T cells were freshly isolated with the EasySep™ mouse T Cell Isolation Kit. Total B cells were freshly isolated with the EasySep™ mouse B Cell Isolation Kit. Isolation of T and B cells were performed according to the manufacturer’s protocols (Stemcell Technologies).
Enzyme-linked immunospot was performed as previously described[39]. Mouse IFNγ ELISPOT was performed according to the manufacturer’s protocol (BD Bioscience). Freshly isolated (1 × 105) T cells were directly plated per well overnight in RPMI supplemented with 10% FCS. No in vitro activation step was included. As positive control, cells were stimulated with 5 ng/ml PMA+1 µg/ml Ionomycin.
T cells (8 × 105 /mL) from C57BL/6J immunized splenocytes were co-cultured with 293T/ACE2 cells (2 × 104 /mL), previously infected with 3×105 TU of SARS-CoV-2 Luc-SARS-CoV-2 Spike pseudotyped lentivirus. Cells were co-cultured in a 24-well plate for 2 days in 1 mL of RPMI 1640 supplemented with 10% FCS, washed with PBS and lysed with 100 µL of M-PER mammalian protein extraction reagent (ThermoFisher) per well. Cytotoxicity was assessed based on the viability of 293T/ACE2 cells, which was determined by measuring the luciferase activity in each well. Luciferase activity was measured by adding 100 µL of Steady-Glo reagent (Promega) to each cell lysate and measuring the luminescence using a GloMax portable luminometer (Promega).
Total RNA was extracted from freshly isolated T cells on day 7 of treatment from spleens using RNeasy Kit (Qiagen). For each group, 5 C57BL/6J mice were used for biological repeats. RNA-seq was done by NYUMC Genome Center. RNA quality and quantity were analyzed. RNAseq libraries were prepared and loaded on the automated Illumina NovaSeq 6000 Sequencing System (Illumina). 1x S1 100 Cycle Flow Cell v1.5, 30 automated stranded RNA-seq library prep polyA selection, per sample.
RNA-seq data were analyzed by sns rna-star pipeline (github.com/igordot/sns/blob/master/routes/rna-star.md). Sequencing reads were mapped to the reference genome (mm 10) using the STAR aligner (v2.6.1d)[89]. Alignments were guided by a Gene Transfer Format (GTF) file. The mean read insert sizes and their standard deviations were calculated using Picard tools (v.2.18.20) (broadinstitute.github.io/picard). The read count tables were generated using subread (v1.6.3)[90], (normalized based on their library size factors using DEseq2[91], and differential expression analysis was performed. To compare the level of similarity among the samples and their replicates, we used principal-component analysis. All the downstream statistical analyses and generating plots were performed in R environment (v4.0.3) (www.r-project.org/). The results of gene set enrichment analysis were generated by GSEA software[92; 93]. The network of Gene Ontology terms was generated by Enrichment Map in Cytoscape. Additional protein-protein functional associations used in this disclosure for bar graphs were retrieved from STRING (/www.string-db.org/, version 11)[94], a well-known public database on several collected associations between proteins from various organisms.
T and B cell metabolic output was measured by Seahorse technology as previously described[95]. Purified T cells from C57BL/6J immunized or control mice were plated at 6x105 cells/well in a Seahorse XF24 cell culture microplate. Oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were measured using an Agilent Seahorse XFe24 metabolic analyzer following the procedure recommended by the manufacturer (Agilent). For the mitochondrial stress test, 1) oligomycin (1 µM), 2) FCCP (1.5 µM) and 3) rotenone (100 nM) and antimycin A (1 pM) were injected sequentially through ports A, B and C.
Western blot was performed to detect SARS-CoV-2 spike protein in HEK293T cells infected with SV.Spike and in the generated pseudotyped lentivirus. Cells were lysed in M-PER® Mammalian Protein Extraction Reagent (Thermo Fisher) according to the manufacturer’s protocol. Lysates were separated by SDS-PAGE on 4-15% Bio-Rad gels, transferred to polyvinylidene difluoride (PVDF) membranes, blocked in 5% milk in TBS buffer with 0.1% Tween-20 (TBST). Primary antibodies to SARS-CoV-2 Spike (GTX, 1:1000) and p24 (Abcam, 1:1000) were added overnight at 4° C. HRP-conjugated secondary antibodies were added in 5% milk in TBST for 1 h at room temperature. BioRad Imaging System was used for visualization.
Statistical analysis was performed using GraphPad Prism 7.0 as described in figure legends. All data are shown as mean ± SEM. Figures were prepared using GraphPad Prism 7, Adobe Photoshop and ImageJ Software. Treated groups were compared using a one-way analysis using Prism7 (GraphPad Software) to naive mice. Differences with a P value of <0.05 were considered significant: *P<0.05; **P<0.005; ***P<0.001.
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While the disclosure has been particularly shown and described with reference to specific embodiments, it should be understood by those having skill in the art that various changes in form and detail may be made therein without departing from the spirit and scope of the present disclosure as disclosed herein.
This application claims priority to U.S. Provisional Application No. 63/034,791 filed on Jun. 4, 2020.
This invention was made with government support under grant number 5R44CA206606 awarded by the National Institutes of Health. The government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2021/034842 | 5/28/2021 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63034791 | Jun 2020 | US |
