Patent Application
20240335531
The Sequence Listing associated with this application is provided electronically in.xml format and is hereby incorporated by reference into the specification. The Sequence Listing is provided as a file entitled 25133-100310_sl.xml, created Jan. 5, 2023 which is about 60 KB in size.
The present invention relates to compositions and methods for therapeutic immunization for treatment of chronic hepatitis B virus (CHB). Methods of the invention include a method generating a high titer hybrid-hepatitis B virus (HBV) vector, methods of treating and/or preventing HBV infection and/or CHB, and methods of inducing a memory T and B cell immune response against HBV infection in a subject administered the VLV composition produced thereby. Furthermore, the invention encompasses a pharmaceutical composition for vaccinating a subject to protect the subject against infection with HBV.
While current antiviral therapies for chronic hepatitis B virus (CHB) infection effectively reduce viremia, they rarely eliminate the virus. Thus, there remains a critical need for new treatment options for this serious disease. Because the human immune system can control HBV but often fails to do so, immunotherapies including therapeutic vaccination represent a promising approach to cure chronic CHB. However, although current HBV vaccine platforms generate potent antibody responses that prevent infection, they typically do not produce the broad CD8 T-cell responses needed to eliminate the virus after infection. Therefore, new vaccine delivery systems that can generate effective therapeutic immune responses to HBV are urgently needed. We have developed an immunotherapy based on our virus like-vesicle (VLV) platform for the treatment of patients with CHB infection.
We have established that an RNA replicon-based vector or VLV carrying RNA encoding one or more of the HBV major antigens [middle surface envelope glycoproteins (MHBs), hepatitis B core antigen (HBcAg), or polymerase] in a single open reading frame (CARG-101) in a polycistronic unit drives a broad multi-specific immune response that produces substantial clearance of HBV in the mouse liver. Treatment of mice chronically infected with adeno-associated virus (AAV)-HBV significantly reduces and, in some animals, eliminates serum HBV surface antigen (HBsAg), a surrogate biomarker for viral persistence in the liver. We have significantly enhanced overall gene expression which led to our next-generation clinical candidate, CARG-201, which induces both T-cell responses and antibodies in comparison to CARG-101. CARG-201 expressing MHBs and HBcAg under separate subgenomic promoters clears serum HBsAg completely in 100% of mice and reduces HBV RNA in the liver to undetectable levels in an AAV mouse model of CHB infection with low antigen burden (HBsAglow). However, in a more stringent AAV-HBV model (HBsAghigh), CARG-201 reduces HBsAg levels by only 80%. As high antigen burden is observed in many CHB patients and is associated with T-cell exhaustion or tolerance, successful immunotherapy should improve immunogenicity and overcome T-cell exhaustion and/or tolerance.
Modifications of CARG-201 in any one of, or one or more of three complementary approaches will enhance efficacy and lead to complete clearance of serum HBsAg levels in animals:
First, we have incorporated polymerase (Pol) antigen into CARG-201 to generate CARG-301 (expressing MHBs, HBcAg, plus Pol). A vaccine that generates multi-antigen specific T cells is better positioned to provide the desired therapeutic effect compared to one or two antigens.
Moreover, Pol is a highly immunogenic CD4 and CD8 T-cell target, and because of its high sequence conservation, it may prevent the generation of escape mutants in the T-cell epitope.
Second, we have engineered CARG-201 to incorporate human IgK signal sequence for the polymerase (pol) gene and VSV G glycoprotein signal sequence for the HBc gene. It is known that secreted proteins generally lead to the activation of dendritic cells, the enhancement of HBV antigen presentation, and the generation of new cytotoxic T-cell responses by epitope spreading. In this manner, the quality and quantity of the T-cell responses against HBV antigens may be further enhanced as compared to soluble and non-secreted counterparts. Secreted proteins also contribute to the adaptive immune responses by being taken up by antigen-presenting cells and processed via the major histocompatibility complex (MHC) class II pathway.
Third, we have also targeted for disruption the programmed death-ligand 1 (PD-L1) immune checkpoint by short hairpin RNA (shRNA) to achieve sustained long-term viral suppression or complete elimination of the virus in the liver. Checkpoint inhibition can enhance ex vivo effector T-cell responses from patients with other chronic infections. We predict that disruption of the PD-1/PD-L1 pathway will re-invigorate the otherwise exhausted T-cell function
The combined effects of shRNA-mediated PD-L1 inhibition and the improved secretion of the HBV antigens as result of ER-targeting confer on these modified multivalent constructs a superior therapeutic index necessary to clear the virus and to halt disease progression and mortality in CHB patients.
A high titer hybrid-hepatitis B virus (HBV) vector comprising a DNA sequence comprising a promoter sequence operably linked to a DNA sequence encoding Semliki Forest virus (SFV) non-structural protein nucleotide sequences, operably linked to an SFV subgenomic RNA promoter, operably linked to DNA encoding an HBV antigen or fragment thereof, operably linked to a 2A DNA encoding a 2A peptide, which is in turn operably linked to a vesicular stomatitis virus (VSV) G DNA encoding a VSV G protein, wherein the SFV non-structural protein nucleotide sequences comprise at least two of the mutations selected from the group consisting of G-4700-A, A-5424-G, G-5434-A, T-5825-C, T-5930-C, A-6047-G, G-6783-A, G-6963-A, G-7834-A, T-8859-A, T-8864-C, G-9211-A, A-10427-G, G-11560-A, A-11871-G and T-11978-C, wherein the vector lacks nucleotide sequences which encode SFV structural proteins, further wherein when the vector is propagated in cell culture, titers of at least 107 plaque forming units (pfu) per ml of virus like vesicles (VLVs) are obtained.
In an embodiment, the present disclosure relates to a high-titer hybrid virus vector for treatment, prophylaxis or prevention of hepatitis B virus infections comprising the following operably linked sequence elements:
In some embodiments, the first DNA sequence comprising a DNA promoter sequence comprises a CMV promoter, optionally including a CMV enhancer. In some embodiments, the promoter and optional enhancer can be any effective promoter/enhancer. In some embodiments, the promoter and optional enhancer can be any construct that recruits RNA polymerase II in eukaryotic cells (preferably mammalian cells).
In some embodiments, the at least two alphavirus subgenomic promoters are synthesized on the negative strand of the RNA that is synthesized by the alphavirus non-structural protein polynucleotide sequences (such as SFVnsp1-4). In some embodiments, the subgenomic promoters are recognized by SFVnsp1-4. In some embodiments, the subgenomic promoters are recognized by the alphavirus non-structural protein to generate 26S subgenomic RNA. In some embodiments, the subgenomic promoters are recognized by SFVnsp1-4 to generate 26S subgenomic RNA.
In further embodiments, the alphavirus non-structural protein polynucleotide sequence is a semiliki forest virus sequence having at least 70% homology to SEQ ID NO: 2.
In further embodiments, the alphavirus non-structural protein polynucleotide sequence is a semiliki forest virus sequence having at least 80% homology to SEQ ID NO: 2.
In further embodiments, the alphavirus non-structural protein polynucleotide sequence is a semiliki forest virus sequence having at least 90% homology to SEQ ID NO: 2.
In further embodiments, the alphavirus non-structural protein polynucleotide sequence is a semiliki forest virus sequence having at least 95% homology to SEQ ID NO: 2.
In further embodiments, the alphavirus non-structural protein polynucleotide sequence is a semiliki forest virus sequence having at least 99% homology to SEQ ID NO: 2.
In further embodiments, the sequence domain encoding the HBV antigen is selected from a hepatitis B core antigen (HBcAg), a hepatitis B surface antigen (HBsAg), polymerase (Pol), and HBx, and combinations thereof.
In further embodiments, the hepatitis B core antigen (HBcAg) is a cysteine-modified HBcAg.
In further embodiments, the cysteine-modified HBcAg comprises a polynucleotide sequence having at least 70% homology to SEQ ID NO: 10.
In further embodiments, wherein the cysteine-modified HBcAg comprises a polynucleotide sequence having at least 80% homology to SEQ ID NO: 10.
In further embodiments, the cysteine-modified HBcAg comprises a polynucleotide sequence having at least 90% homology to SEQ ID NO: 10.
In further embodiments, the cysteine-modified HBcAg comprises a polynucleotide sequence having at least 95% homology to SEQ ID NO: 10.
In further embodiments, the cysteine-modified HBcAg comprises a polynucleotide sequence having at least 99% homology to SEQ ID NO: 10.
In further embodiments, the hepatitis B surface antigen (HBsAg) is selected from middle (M), large (L), and small(S) hepatitis B surface antigens.
In further embodiments, the polymerase (Pol) comprises a truncated and modified polynucleotide sequence.
In further embodiments, the polymerase (Pol) comprises a polynucleotide sequence having at least 70% homology to SEQ ID NO: 12.
In further embodiments, the polymerase (Pol) comprises a polynucleotide sequence having at least 80% homology to SEQ ID NO: 12.
In further embodiments, the polymerase (Pol) comprises a polynucleotide sequence having at least 90% homology to SEQ ID NO: 12.
In further embodiments, the polymerase (Pol) comprises a polynucleotide sequence having at least 95% homology to SEQ ID NO: 12.
In further embodiments, the polymerase (Pol) comprises a polynucleotide sequence having at least 99% homology to SEQ ID NO: 12.
In further embodiments, the heterologous secretion signal sequence is a human IgK secretion signal sequence or a VSV G secretion signal sequence.
In further embodiments, the human IgK secretion signal sequence comprises a polynucleotide sequence having at least 70% homology to SEQ ID NO: 8.
In further embodiments, the human IgK secretion signal sequence comprises a polynucleotide sequence having at least 80% homology to SEQ ID NO: 8.
In further embodiments, the human IgK secretion signal sequence comprises a polynucleotide sequence having at least 90% homology to SEQ ID NO: 8.
In further embodiments, the human IgK secretion signal sequence comprises a polynucleotide sequence having at least 95% homology to SEQ ID NO: 8.
In further embodiments, the human IgK secretion signal sequence comprises a polynucleotide sequence having at least 99% homology to SEQ ID NO: 8.
In further embodiments, the VSV G secretion signal sequence comprises a polynucleotide sequence having at least 70% homology to SEQ ID NO: 6.
In further embodiments, the VSV G secretion signal sequence comprises a polynucleotide sequence having at least 80% homology to SEQ ID NO: 6.
In further embodiments, the VSV G secretion signal sequence comprises a polynucleotide sequence having at least 90% homology to SEQ ID NO: 6.
In further embodiments, the VSV G secretion signal sequence comprises a polynucleotide sequence having at least 95% homology to SEQ ID NO: 6.
In further embodiments, the VSV G secretion signal sequence comprises a polynucleotide sequence having at least 99% homology to SEQ ID NO: 6.
In further embodiments, the sequence domain encoding an HBV antigen is a cysteine-modified hepatitis B core antigen (HBcAg) comprising a polynucleotide sequence having at least 70% homology to SEQ ID NO: 10, and wherein the heterologous secretion signal sequence is a VSV G secretion signal sequence comprising a polynucleotide sequence having at least 70% homology to SEQ ID NO: 6.
In further embodiments, the sequence domain encoding an HBV antigen is a polymerase (Pol) gene comprising a polynucleotide sequence having at least 70% homology to SEQ ID NO: 12, and wherein the heterologous secretion signal sequence is a human IgK secretion signal sequence comprising a polynucleotide sequence having at least 70% homology to SEQ ID NO: 6.
In further embodiments, the sequence domain encoding a human short hairpin RNA (shRNA) targets PD-L1.
In further embodiments, the sequence domain encoding the shRNA comprises a polynucleotide sequence having at least 70% homology to SEQ. ID NO: 13.
In further embodiments, the sequence domain encoding the shRNA comprises a polynucleotide sequence having at least 80% homology to SEQ. ID NO: 13.
In further embodiments, the sequence domain encoding the shRNA comprises a polynucleotide sequence having at least 90% homology to SEQ. ID NO: 13.
In further embodiments, the sequence domain encoding the shRNA comprises a polynucleotide sequence having at least 95% homology to SEQ. ID NO: 13.
In further embodiments, the sequence domain encoding the shRNA comprises a polynucleotide sequence having at least 99% homology to SEQ. ID NO: 13.
In further embodiments, the DNA sequence encoding a vesiculovirus glycoprotein encodes a New Jersey (NJ) serotype vesiculovirus glycoprotein.
In further embodiments, the NJ serotype vesiculovirus glycoprotein comprises a polynucleotide sequence having at least 70% homology to SEQ ID NO: 15.
In further embodiments, the NJ serotype vesiculovirus glycoprotein comprises a polynucleotide sequence having at least 80% homology to SEQ ID NO: 15.
In further embodiments, the NJ serotype vesiculovirus glycoprotein comprises a polynucleotide sequence having at least 90% homology to SEQ ID NO: 15.
In further embodiments, the NJ serotype vesiculovirus glycoprotein comprises a polynucleotide sequence having at least 95% homology to SEQ ID NO: 15.
In further embodiments, the NJ serotype vesiculovirus glycoprotein comprises a polynucleotide sequence having at least 99% homology to SEQ ID NO: 15.
In further embodiments, the sequence domain encoding an HBV antigen is linked to the sequence encoding a vesiculovirus glycoprotein by a sequence comprising 2A ribosome skipping sequence.
In further embodiments, the 2A ribosome skipping sequence is a Thosea asigna virus 2A (T2A) sequence.
In further embodiments, the T2A sequence comprises a polynucleotide sequence having at least 70% homology to SEQ ID NO: 4.
In further embodiments, the T2A sequence comprises a polynucleotide sequence having at least 80% homology to SEQ ID NO: 4.
In further embodiments, the T2A sequence comprises a polynucleotide sequence having at least 90% homology to SEQ ID NO: 4.
In further embodiments, the T2A sequence comprises a polynucleotide sequence having at least 95% homology to SEQ ID NO: 4.
In further embodiments, the T2A sequence comprises a polynucleotide sequence having at least 99% homology to SEQ ID NO: 4.
In an embodiment, the vector (or plasmid) comprises the following operably linked sequence elements:
In further embodiments, the recited homologies are each at least 90% homology.
In further embodiments, titers of at least 1×1010 plaque forming units (pfu) per mL of virus like vesicles (VLVs) are obtained.
In an embodiment, the present disclosure provides for a high-titer hybrid virus vector for generating virus-like vesicles (VLVs) for treatment, prophylaxis or prevention of hepatitis B virus infections.
In an embodiment, the present disclosure provides for Virus-like vesicles (VLVs) containing replicon RNA generated by a high-titer hybrid-virus vector.
In an embodiment, the present disclosure provides for a composition comprising virus-like vesicles (VLVs) produced by a high-titer hybrid virus vector.
In an embodiment, the vector is a plasmid comprising a polynucleotide sequence having at least 70% homology to SEQ ID NO: 16 or SEQ ID NO: 17.
In an embodiment, the vector is a plasmid comprising a polynucleotide sequence having at least 80% homology to SEQ ID NO: 16 or SEQ ID NO: 17.
In an embodiment, the vector is a plasmid comprising a polynucleotide sequence having at least 90% homology to SEQ ID NO: 16 or SEQ ID NO: 17.
In an embodiment, the vector is a plasmid comprising a polynucleotide sequence having at least 95% homology to SEQ ID NO: 16 or SEQ ID NO: 17.
In an embodiment, the vector is a plasmid comprising a polynucleotide sequence having at least 97% homology to SEQ ID NO: 16 or SEQ ID NO: 17.
In an embodiment, the vector is a plasmid comprising a polynucleotide sequence having at least 98% homology to SEQ ID NO: 16 or SEQ ID NO: 17.
In an embodiment, the vector is a plasmid comprising a polynucleotide sequence having at least 99% homology to SEQ ID NO: 16 or SEQ ID NO: 17.
In an embodiment, the vector is a plasmid comprising a polynucleotide sequence consisting of SEQ ID NO: 16 or SEQ ID NO: 17.
In an embodiment, the vector is a plasmid consisting essentially of the polynucleotide sequence of SEQ ID NO: 16 or SEQ ID NO: 17.
In an embodiment, the vector is a plasmid consisting of the polynucleotide sequence of SEQ ID NO: 16 or SEQ ID NO: 17.
In an embodiment, the present disclosure provides for an isolated plasmid comprising a polynucleotide sequence having at least 70% homology to SEQ ID NO: 16 or SEQ ID NO: 17.
In an embodiment, the present disclosure provides for an isolated plasmid consisting essentially of the polynucleotide sequence of SEQ ID NO: 16 or SEQ ID NO: 17.
In an embodiment, the present disclosure provides for an isolated plasmid consisting of the polynucleotide sequence of SEQ ID NO: 16 or SEQ ID NO: 17.
In an embodiment, the present disclosure provides for a method of treating and preventing hepatitis B virus infections in a mammalian subject, the method comprising administering a therapeutically effective amount of a VLV composition a mammalian subject in need thereof.
In an embodiment, the present disclosure provides for a method of immunizing a mammalian subject against hepatitis B virus infections, the method comprising administering a therapeutically effective amount of a VLV composition to a mammalian subject in need thereof.
In an embodiment, the present disclosure provides for a method of downregulating genes associated with hepatitis B virus infections, the method comprising administering a therapeutically effective amount of a VLV composition to a mammalian subject in need thereof.
In some embodiments, the mammalian subject is a human or animal.
In further embodiments, the present disclosure provides for a use of a VLV composition in the manufacture of a medicament for the treatment, prophylaxis, or prevention of hepatitis B virus infections in a mammalian subject in need thereof.
In further embodiments, the mammalian subject is a human or animal.
In further embodiments, the present disclosure provides for a method of producing virus-like vesicles (VLVs) for treatment, prophylaxis, or prevention of hepatitis B virus infections comprising the steps of:
These and other aspects of the present invention will become apparent from the disclosure herein.
Aspects and advantages of the present disclosure will become apparent from the following exemplary embodiments taken in conjunction with the accompanying drawings, of which:
The present disclosure generally relates to compositions and methods for therapeutic immunization for treatment of chronic hepatitis B virus (CHB). Methods of the invention include a method generating a high titer hybrid-hepatitis B virus (HBV) vector, methods of producing related VLVs, methods of treating and/or preventing HBV infection and/or CHB, and methods of inducing a memory T and B cell immune response against HBV infection in a subject administered the VLV composition produced thereby.
HBV: Significance of the Problem. HBV infection is a major global public health problem. Worldwide, approximately 2 billion people are infected with hepatitis B virus (HBV) during their lifetime, and >240 million have current HBV infection, and about 600,000 people die from HBV-related liver disease every year. Patients with chronic HBV (CHB) infection, including inactive carriers of HBV, have an increased risk of developing liver cirrhosis, hepatic failure, and hepatocellular carcinoma (HCC). Although most of these patients will not develop HBV-related complications. 15-40% will develop serious complications during their lifetime. CHB has various clinical stages defined by HBV DNA titer, presence of hepatitis B e antigen (HBeAg, a secreted form of the core protein) and the presence or absence of liver inflammation measured by liver transaminase levels. CHB infection occurs as a result of continuous interaction between the viral replication and immune responses. T cells are exhausted by the persistent antigen exposure, which contribute to the persistence of HBV infection. When T cells encounter HBV antigens presented by the intrahepatic antigen presenting cells (APCs), such as the dendritic cells (DCs) and Kupffer cells, the costimulatory signals received by T cells are very weak. This result in immune tolerance rather than functional activation. In addition, the immunosuppressive microenvironment is formed in the liver of patients with CHB with high proportion of regulatory T cells (Tregs) and myeloid-derived suppressor cells (MDSCs). These provide T cells with inhibitory signals and disturb T cell-mediated anti-HBV functions.
Limitations of the current HBV vaccine. Despite its success in preventing HBV infection, the current HBV vaccine (recombinant HBsAg adsorbed to alum) has a number of characteristics that are suboptimal. First, it does not induce a protective antibody response in all immunized individuals. Second, between two and four doses of the vaccine are recommended to induce long-lasting immunity. This need for repeated immunization makes the vaccine somewhat challenging to administer in many regions of the world, especially those lacking the appropriate medical infrastructure. Third, the protective antibody response wancs after immunization, and declines to below protective levels (>10 IU/L) in up to 60% of vaccinated individuals. Fourth, escape mutations in the surface protein gene can produce virus that is resistant to the antibody response generated by the vaccine Finally, as discussed above, although it clicits a protective antibody response that prevents infection, the current vaccine does not generate a strong CD8 T cell response, and it has not been effective in clinical trials to control virus replication in those who are already infected with HBV.
Current therapies for CHB. Current standard of care for CHB includes anti-viral and immune-enhancing drugs, such as tenofovir, entecavir and PEGylated IFN which are very effective at slowing down disease, are curative only 8-12% of the patients treated. Cessation of antiviral therapy is often accompanied by a rebound in the viral load; therefore, lifelong treatment is required. Although available antiviral drugs can lead to suppression of serum HBV viral load to undetectable levels efficiently, however, they usually fail to achieve sero-clearance of hepatitis B surface antigen (HBsAg), which indicates eradication of HBV infection, the ultimate goal of antiviral treatment for CHB. The failure to achieve HBsAg sero-clearance may be duc to emerging drug-resistant HBV variants and the covalently closed circular DNA (cccDNA) in remaining infected hepatocytes. As none of these clinical therapies achieve long-term virological control in majority of patients with CHB, therefore, there is an urgent need to develop new therapies to improve HBsAg clearance and virological curc.
New Modalities for HBV Treatment. The failure of HBsAg sero-clearance requires the development of novel therapeutic strategies for achieving durable viral remission. One strategy is to target virus directly, by targeting viral entry, viral assembly/encapsidation, preS1 or hepatitis B surface antigen (HBsAg) secretion, envelopment and cccDNA. Another strategy is to interfere with the host mechanisms, by using Toll-like receptor (TLR) agonists, cytokines and the blocking of PD-1/PD-L1. In addition, therapeutic vaccines based on recombinant HBV proteins or HBV-envelope subviral particles, DNA and T-cell peptide epitope resent another promising strategy for HBV eradication. Therapeutic vaccination is aimed at eliminating persistent viral infection by augmenting the patient's immune responses. Individuals who become acutely infected but ultimately clear the virus have a relatively strong, multi-specific T-cell response to HBV. However, in those who become chronically infected, the T-cell response is much weaker in magnitude and is directed toward fewer viral antigens. This suboptimal immune response persists in chronically infected individuals despite the continual presence of viral antigens in the liver and blood. Although the current HBV vaccines induce potent antibody responses that prevent infection, they do not elicit the virus-specific T cells needed to control an established infection. New technologies that generate an effective T cell-dependent immune response to HBV are urgently needed. One promising approach for treating CHB is a therapeutic vaccine capable of inducing virus-specific CD8 T cells to clear HBV infection.
Functional cure of HBV. The ultimate goal of HBV treatment is ‘functional cure’. According to the meeting of AASLD and EASL, functional cure is defined as a sustained loss of HBsAg in scrum. In this scenario, although HBV cccDNA remains at low levels, a functional adaptive immune response ensures suppression of viral replication without treatment, analogous to that which occurs in clearance of acute HBV. A strong HBV-specific CD8 T cell response is required for HBV clearance in acute infection, but in CHB the T cell response is dysfunctional and is not fully restored by NUCs. As functional cure is rarely achieved with current therapy, alternative treatments that can be given in shorter and finite courses are urgently required. CHB infection is the result of complex interactions between HBV and the host, and an impaired immune response to viral antigens is believed to be a key factor associated with the CHB carrier state. If this state of immune tolerance could be overcome, the loss of HBeAg or HBsAg from the serum (seroclearance) and sustained control of the HBV infection would be achieved.
Scientific Premise. Current standard-of-care therapies only rarely lead to a functional cure, characterized by sustained loss of HBsAg (with or without HBsAg antibody seroconversion). The goal for the next generation CHB therapies is to achieve a higher rate of functional cure with finite treatment duration. To address this urgent need, we developed targeted shRNA therapeutics for CHB based on VLV delivery platform. The shRNA can be developed as a stand-alone treatment or in combination with therapeutic vaccine to achieve a functional cure. Since the human immune system can control HBV but often fails to do so, immunotherapies including therapeutic vaccine represent a promising approach to cure CHB. However, the therapeutic immune responses generated in the persistent HBV infection are often weak due to CD8 T cell exhaustion. Exhaustion of virus-specific T cells may play an important role in HBV persistence. The interaction between programmed death-1 (PD-1) receptor on lymphocytes and its ligand PD-L1 plays a critical role in T-cell exhaustion by inducing T-cell inactivation indicating that the PD-1/PD-L1 pathway is a good therapeutic candidate for chronic HBV infection. Woodchucks infected with woodchuck hepatitis virus (WHV) can have increased hepatic expression of PD-1-ligand-1 (PD-L1), increased PD-1 on CD8+ T cells, and a limited number of virus-specific T cells. Others have shown that in these animals, combination therapy with aPD-L1 and entecavir (ETV) improved control of viremia and antigenemia compared to ETV treatment alone. In addition, others have shown that PD-L1 blockade synergistically augments HBV-specific CD4 T cells. Furthermore, there is accumulating evidence that immune checkpoint inhibitors can enhance ex vivo effector T-cell responses from patients with other chronic viral, bacterial, or parasitic infection, including HIV, tuberculosis, and malaria. We have found that therapeutic shRNA intervention targeting exhausted T cells by blocking these suppressive pathways can restore the function of these impaired T cells and lead to a functional curc.
We have found that alphavirus replicons are excellent vaccine vectors because they are highly immunogenic and target dendritic cells. The virus-like vesicles (VLV) vaccine platform is a capsid-free, self-replicating, antigen expression system that represents an attractive alternative to other virus-based vectors. VLV encodes a Semliki Forest virus (SFV) replicon and an additional structural protein, the vesicular stomatitis virus glycoprotein (VSV-G). Following in vitro evolution by 50 passages in BHK-21 cells that led to 10 amino acid changes in SFV nsP1-4, the evolved SFV nonstructural proteins promote high-titer VLV replication in cell culture through increased efficiency of VLV release. VSV-G expression allows for robust and pantropic infectivity, as infectious vesicles composed of SFV replication spherules derived from bulb-shaped plasma membrane invaginations are coated with VSV-G protein and bud from infected cells, spread to uninfected cells, and undergo multiple rounds of infection. VLV are nonpathogenic in mice and rhesus macaques, have little risk of genome integration or reversion to pathogenesis, and are immunogenic in the absence of adjuvant. Recent improvements to the system allow the generation of high-titers of VLV particles as well as high gene expression until multiple subgenomic promoters. Although VLVs mimic the immune stimulating properties of viral vectors, they are safe and non-pathogenic when administered to mice or rhesus macaques, nor do they display neurovirulence when injected directly into mouse brain. These vectors are significant because of their potency, case of high-titer particle production, and predicted safety due to the lack of viral structural proteins. Furthermore, VLVs have a demonstrated large capacity to deliver nucleic acids for the expression of several antigens resulting in induction of T cell and antibody responses against multiple epitopes of multiple antigens and thus help to maximize the potential efficacy of the proposed immunotherapy in patients.
Vectors of the present invention may generally be a plasmid or other vector encoding VLVs. The term “vector” is therefore inclusive of plasmids. The plasmids can generally comprise any required elements for VLV production. The vectors or plasmids can be defined by one or more sequence domains or components, or by one or more sequences. Generally, unless clear from the context, plasmids may comprise additional sequence domains or components as necessary or desirable. Sequences may be defined as polynucleotide sequences or corresponding amino acid sequences. Some sequence components (such as shRNAs) may not have corresponding amino acid sequences. Exemplary sequence domains are provided in Table 1 below:
In various embodiments, vectors or plasmids may comprise and/or encode one or more of SEQ ID NO: 1-15. In various embodiments, vectors or plasmids may comprise and/or encode a sequence or sequence portion having more than 70%, or more than 75%, or more than 80%, or more than 85%, or more than 90%, or more than 95%, or more than 96%, or more than 97%, or more than 98%, or more than 99% homology to one or more of SEQ ID NO: 1-15. In some embodiments, vectors or plasmids may comprise a sequence consisting of one or more of SEQ ID NO: 1-15. Where a vector or plasmid comprises a sequence consisting of or encoding one or more of SEQ ID NO: 1-15, it is intended that the vector or plasmid may comprise additional sequence domains.
In exemplary embodiments, plasmids may have a polynucleotide sequence corresponding to SEQ ID NO: 16 or SEQ ID NO: 17. In exemplary embodiments, plasmids may have a polynucleotide sequence having more than about 70%, or more than 75%, or more than 80%, or more than 85%, or more than 90%, or more than 95%, or more than 96%, or more than 97%, or more than 98%, or more than 99% homology to SEQ ID NO: 16 or SEQ ID NO: 17. In some embodiments, a plasmid may have a polynucleotide sequence consisting of SEQ ID NO: 16 or SEQ ID NO: 17. In some embodiments, a plasmid may have a polynucleotide sequence consisting essentially of SEQ ID NO: 16 or SEQ ID NO: 17. Where a plasmid has a polynucleotide sequence consisting essentially of SEQ ID NO: 16 or SEQ ID NO: 17, it is intended that the plasmid, having the same general sequence domains, may contain one or more nucleotide and/or amino acid substitutions, additions, or deletions in or between those domains which do not significantly impact the function of the plasmid.
Where a sequence “homology” or “identity” is contemplated, for a DNA sequence or an amino acid sequence, the same percentage “similarity” is also contemplated for the amino acid sequence or amino acid sequence corresponding to the DNA sequence. The term “similarity” is different from the term identity because it allows conservative substitutions of amino acid residues having similar physicochemical properties over a defined length of a given alignment. Generally, any reasonable similarity-scoring matrix known may be used to determine similarity.
In determining the sequence homology or identity of a first sequence compared to a second sequence, various identity calculations may be performed such as those implemented in the National Institute of Health's Basic Local Alignment Search Tool (BLAST). In some embodiments, the standard BLAST settings may be utilized. For example, a BLAST identity may be defined as the number of matching bases over the number of alignment positions.
VLVs can generally be produced by transfecting any appropriate cell line with appropriate plasmids or vectors. In an embodiment, VLVs are produced by transfecting BHK-21 or HEK293 T cells with a vector or plasmid, incubating the transfected BHK-21 or HEK293 T cells in a buffer solution for a suitable time and at a suitable temperature to propagate VLVs; and isolating the VLVs from the BHK-21 or HEK293 T cells and buffer solution by a technique selected from the group consisting of ultrafiltration, centrifugation, tangential flow filtration, affinity purification, ion exchange chromatography, and combinations thereof. In various embodiments, VLVs can be produced by any appropriate transduction, incubation, and isolation methods.
The produced VLVs are generally useful for therapeutic methods. The produced VLVs may be formulated as vaccine compositions for treatment of HBV with one or more diluents, excipients, or other ingredients. The compositions may generally be administered by any appropriate route, such as by oral, parenteral, intravenous, or other routes.
The figures are described in more detail as follows, with reference to the Examples presented herein.
The invention is now described with reference to the following Examples. These Examples are provided for the purpose of illustration only and the invention should in no way be construed as being limited to these Examples, but rather should be construed to encompass any and all variations which become evident as a result of the teaching provided herein.
Without further description, it is believed that one of ordinary skill in the art can, using the preceding description and the following illustrative examples, make and utilize the compounds of the present invention and practice the claimed methods.
Key points include:
Remarkably, we have now attained a reduction in serum biomarker levels in >80% AAV-HBV mice (n=10) with high antigenemia, indicating that CARG-201 (HBcAg and MHBs: two HBV antigens) can break tolerance in highly tolerant models. To attain this reduction, we combined an enhanced gene expression strategy and a robust prime-boost regimen to achieve complete clearance of HBsAg in mice. We have therefore selected CARG-201 for advancement to the clinic based on the following results: (i) complete reduction of HBsAg in most of but not all treated animals in a mouse model of persistent HBV replication, (ii) reduction of HBV RNA in the liver to undetectable levels, and (iii) induction of multi-specific HBV T cells and antibodies. The reduction in intrahepatic HBV RNA may be the result of strong immune control under a high level of CD8+ and CD4+ T-cell responses, as observed in patients with resolution of acute HBV infection (
These data we have generated establish that (i) we can enhance the antigenic load with a concomitant increase in immunogenicity by modifying the VLV to harbor two or more subgenomic promoters and (ii) the VSV G serotype switch is an effective prime-boost strategy. An optimized single-antigen (MHBs) or double-antigen (MHBs and HBc) vector can drive complete clearance of HBsAg in mice (
As seen in
shRNA inhibits PD-L1 expression in stably transfected BHK21 cells in vitro (
CARG-201-mediated decrease of serum biomarker is correlated with decreasing population of PD-1+/CD8+ in high HBsAg AAV-HBV chronic model (
A CARG-2020 construct expressing both rIL-12 and PD-L1 shRNA down regulates the expression of multiple immune checkpoints (
As seen in Table 2, we have completed the generation of CARG-301 secreting all three antigens (secCARG-301). We have also engineered CARG-301 to incorporate shRNA alone (CARG-301.sh) or both secretion signals and shRNA (secCARG-301.shNA). We will now test the immunogenicity of these constructs and prioritize them for efficacy studies in a chronic mouse model of HBV infection. The availability of constructs in both serotypes will allow us to employ a prime boost regimen if necessary.
1.2 × 1010
9 × 109
The entire disclosure of each of the patent documents, including certificates of correction, patent application documents, scientific articles, governmental reports, websites, and other references referred to herein is incorporated by reference herein in its entirety for all purposes. In case of a conflict in terminology, the present specification controls.
The invention can be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The foregoing embodiments are to be considered in all respects illustrative rather than limiting on the invention described herein. In the various embodiments of the compositions and methods of the present invention, where the term comprises is used with respect to the compositions or recited steps of the methods, it is also contemplated that the compositions and methods consist essentially of, or consist of, the recited compositions or steps or components. Furthermore, it should be understood that the order of steps or order for performing certain actions is immaterial so long as the invention remains operable. Moreover, two or more steps or actions can be conducted simultaneously.
In the specification, the singular forms also include the plural forms, unless the context clearly dictates otherwise. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In the case of conflict, the present specification will control.
Furthermore, it should be recognized that in certain instances a composition can be described as being composed of the components prior to mixing, or prior to a further processing step such as drying, binder removal, heating, sintering, etc. It is recognized that certain components can further react or be transformed into new materials.
All percentages and ratios used herein are on a volume (volume/volume) or weight (weight/weight) basis as shown, or otherwise indicated.
This continuation application claims the benefit of and priority to International Patent Application No. PCT/US2023/060237, filed Jan. 6, 2023, which claims priority to U.S. Provisional Patent Application No. 63/297,728, filed Jan. 8, 2022, and to U.S. Provisional Patent Application No. 63/308,103, filed Feb. 9, 2022, each of which are incorporated by reference in their entireties herein.
This invention was made with government support under Grant No. 2R44 DK113858 awarded by the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK), an institute within the National Institutes of Health (NIH). The government has certain rights in the invention.
| Number | Date | Country | |
|---|---|---|---|
| 63297728 | Jan 2022 | US | |
| 63308103 | Feb 2022 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/US2023/060237 | Jan 2023 | WO |
| Child | 18755876 | US |
