Vaccines can be used to provide immune protection against pathogens, such as viruses, bacteria, fungi, or protozoans, as well as cancers.
Infectious diseases are the second leading cause of death worldwide after cardiovascular disease but are the leading cause of death in infants and children (Lee and Nguyen, 2015, Immune Network, 15(2):51-7). Vaccination is the most efficient tool for preventing a variety of infectious diseases. The goal of vaccination is to generate a pathogen-specific immune response providing long-lasting protection against infection. Despite the significant success of vaccines, development of safe and strong vaccines is still required due to the emergence of new pathogens, re-emergence of old pathogens and suboptimal protection conferred by existing vaccines. Recent important emerging or re-emerging diseases include: severe acute respiratory syndrome (SARS) in 2003, the H1N1 influenza pandemic in 2009, and Ebola virus in 2014. As a result, there is a need for the development of new and effective vaccines against emerging diseases.
Cancer is one of the major killers in the Western world, with lung, breast, prostate, and colorectal cancers being the most common (Butterfield, 2015, BMJ, 350:h988). Several clinical approaches to cancer treatment are available, including surgery, chemotherapy, radiotherapy, and treatment with small molecule signaling pathway inhibitors. Each of these standard approaches has been shown to modulate antitumor immunity by increasing the expression of tumor antigens within the tumor or causing the release of antigens from dying tumor cells and by promoting anti-tumor immunity for therapeutic benefit. Immunotherapy is a promising field that offers alternative methods for treatment of cancer. Cancer vaccines are designed to promote tumor-specific immune responses, particularly cytotoxic CD8+ T cells that are specific to tumor antigens. Clinical efficacy must be improved in order for cancer vaccines to become a valid alternative or complement to traditional cancer treatments. Considerable efforts have been undertaken so far to better understand the fundamental requirements for clinically-effective cancer vaccines. Recent data emphasize that important requirements, among others, are (1) the use of multi-epitope immunogens, possibly deriving from different tumor antigens; (2) the selection of effective adjuvants; (3) the association of cancer vaccines with agents able to counteract the regulatory milieu present in the tumor microenvironment; and (4) the need to choose the definitive formulation and regimen of a vaccine after accurate preliminary tests comparing different antigen formulations (Fenoglio et al., 2013, Hum Vaccin Immunother, (12):2543-7). A new generation of cancer vaccines, provided with both immunological and clinical efficacy, is needed to address these requirements.
Ebolaviruses, such as Zaire ebolavirus (EBOV) and Sudan ebolavirus (SUDV), and the closely related Marburg virus (MARV), are associated with outbreaks of highly lethal Ebola Hemorrhagic Fever (EHF) in humans and primates in North America, Europe, and Africa. These viruses are filoviruses that are known to infect humans and non-human primates with severe health consequences, including death. Filovirus infections have resulted in case fatality rates of up to 90% in humans. EBOV, SUDV, and MARV infections cause EHF with death often occurring within 7 to 10 days post-infection. EHF presents as an acute febrile syndrome manifested by an abrupt fever, nausea, vomiting, diarrhea, maculopapular rash, malaise, prostration, generalized signs of increased vascular permeability, coagulation abnormalities, and dysregulation of the innate immune response. Much of the disease appears to be caused by dysregulation of innate immune responses to the infection and by replication of virus in vascular endothelial cells, which induces death of host cells and destruction of the endothelial barrier. Filoviruses can be spread by small particle aerosol or by direct contact with infected blood, organs, and body fluids of human or NHP origin. Infection with a single virion is reported to be sufficient to cause Ebola hemorrhagic fever (EHF) in humans. Presently, there is no therapeutic or vaccine approved for treatment or prevention of EHF. Supportive care remains the only approved medical intervention for individuals who become infected with filoviruses.
As the cause of severe human disease, filoviruses continue to be of concern as both a source of natural infections, and also as possible agents of bioterrorism. The reservoir for filoviruses in the wild has not yet been definitively identified. Four subtypes of Ebolaviruses have been described to cause EHF, i.e., those in the Zaire, Sudan, Bundibugyo and Ivory Coast episodes (Sanchez A. et al., 1996, PNAS USA, 93:3602-3607). These subtypes of Ebolaviruses have similar genetic organizations, e.g., negative-stranded RNA viruses containing seven linearly arrayed genes. The structural gene products include, for example, the envelope glycoprotein that exists in two alternative forms, a secreted soluble glycoprotein (ssGP) and a transmembrane glycoprotein (GP) generated by RNA editing that mediates viral entry (Sanchez A. et al., 1996, PNAS USA, 93:3602-3607).
It has been suggested that immunization can be useful in protecting against Ebola infection because there appears to be less nucleotide polymorphism within Ebola subtypes than among other RNA viruses (Sanchez A. et al., 1996, PNAS USA, 93:3602-3607). Until recently, developments of preventive vaccines against filoviruses have had variable results, partly because the requirements for protective immune responses against filovirus infections are poorly understood. Additionally, the large number of filoviruses circulating within natural reservoirs complicates efforts to design a vaccine that protects against all species of filoviruses.
Currently, there are several vaccine antigen delivery platforms that demonstrated various levels of protection in non-human primates (NHPs) exposed with high infectious doses of filoviruses. Vaccine candidates are in development based on a variety of platform technologies including replication competent vectors (e.g. Vesicular Stomatitis Virus; Rabies virus; Parainfluenza Virus); replication incompetent vectors (Adenovirus, Modified Vaccinia Ankara Virus); protein subunits inclusive of Virus Like Particles expressed in bacterial cells, insect cells, mammalian cells, plant cells; DNA vaccines; and/or live and killed attenuated filovirus (Friedrich et al., 2012, Viruses, 4(9):1619-50). The EBOV glycoprotein GP is an essential component of a vaccine that protects against exposures with the same species of EBOV. Furthermore, inclusion of the GP from EBOV and SUDV, the two most virulent species of ebolaviruses, can protect monkeys against EBOV and SUDV intramuscular exposures, as well as exposures with the distantly related Bundibugyo (BDBV), Taï Forest ebolavirus (TAFV; formerly known as Ivory Coast or Cote d'Ivoire) species. Likewise, inclusion of the GP from MARV can protect monkeys against intramuscular and aerosol MARV exposures. The development of medical countermeasures for these viruses is a high priority, in particular the development of a PAN-filovirus vaccine—that is one vaccine that protects against all pathogenic filoviruses.
Replication-defective adenovirus vectors (rAd) are powerful inducers of cellular immune responses and have therefore come to serve as useful vectors for gene-based vaccines particularly for lentiviruses and filoviruses, as well as other nonviral pathogens (Shiver et al., 2002, Nature, 415(6869): 331-5; Hill et al., 2010, Hum Vaccin 6(1): 78-83; Sullivan et al., 2000, Nature, 408(6812): 605-9; Sullivan et al., 2003, Nature, 424(6949): 681-4; Sullivan et al., 2006, PLoS Med, 3(6): e177; Radosevic et al., 2007, Infect Immun, 75(8):4105-15; Santra et al., 2009, Vaccine, 27(42): 5837-45).
Adenovirus-based vaccines have several advantages as human vaccines since they can be produced to high titers under GMP conditions and have proven to be safe and immunogenic in humans (Asmuth et al., 2010, J Infect Dis 201(1): 132-41; Kibuuka et al., 2010, J Infect Dis 201(4): 600-7; Koup et al., 2010, PLoS One 5(2): e9015; Catanzaro et al., 2006, J Infect Dis, 194(12): 1638-49; Harro et al., 2009, Clin Vaccine Immunol, 16(9): 1285-92). While most of the initial vaccine work was conducted using rAd5 due to its significant potency in eliciting broad antibody and CD8+ T cell responses, pre-existing immunity to rAd5 in humans may limit efficacy (Catanzaro et al., 2006, J Infect Dis, 194(12): 1638-49; Cheng et al., 2007, PLoS Pathog, 3(2): e25; McCoy et al., 2007, J Virol, 81(12): 6594-604; Buchbinder et al., 2008, Lancet, 372(9653): 1881-93). This property might restrict the use of rAd5 in clinical applications for many vaccines that are currently in development including Ebolavirus (EBOV) and Marburg virus (MARV).
Replication-defective adenovirus vectors, rAd26 and rAd35, derived from adenovirus serotype 26 and serotype 35, respectively, have the ability to circumvent Ad5 pre-existing immunity. rAd26 can be grown to high titers in Ad5 E1-complementing cell lines suitable for manufacturing these vectors at a large scale and at clinical grade (Abbink, et al., 2007, J Virol, 81(9):4654-63), and this vector has been shown to induce humoral and cell-mediated immune responses in prime-boost vaccine strategies (Abbink, et al., 2007, J Virol, 81(9):4654-63; Liu et al., 2009, Nature, 457(7225): 87-91). rAd35 vectors grow to high titers on cell lines suitable for production of clinical-grade vaccines (Havenga et al., 2006, J Gen Virol, 87: 2135-43), and have been formulated for injection as well as stable inhalable powder (Jin et al., 2010, Vaccine 28(27): 4369-75). These vectors show efficient transduction of human dendritic cells (de Gruijl et al., 2006, J Immunol, 177(4): 2208-15; Lore et al., 2007, J Immunol, 179(3): 1721-9), and thus have the capability to mediate high level antigen delivery and presentation.
Modified Vaccinia Ankara (MVA) virus is related to Vaccinia virus, a member of the genera Orthopoxvirus in the family of Poxviridae. Poxviruses are known to be good inducers of CD8 T cell responses because of their intracytoplasmic expression. However, they may be poor at generating CD4 MHC class II restricted T cells (see for example Haslett et al., 2000, Journal of Infectious Diseases, 181: 1264-72, page 1268). MVA has been engineered for use as a viral vector for recombinant gene expression or as recombinant vaccine.
Strains of MVA having enhanced safety profiles for the development of safer products, such as vaccines or pharmaceuticals, have been developed by Bavarian Nordic. MVA was further passaged by Bavarian Nordic and is designated MVA-BN, a representative sample of which was deposited on Aug. 30, 2000 at the European Collection of Cell Cultures (ECACC) under Accession No. V00083008. MVA-BN is further described in WO 02/42480 (US 2003/0206926) and WO 03/048184 (US 2006/0159699), both of which are incorporated by reference herein in their entirety.
MVA-BN can attach to and enter human cells where virally-encoded genes are expressed very efficiently. MVA-BN is replication incompetent, meaning that the virus does not replicate in human cells. In human cells, viral genes are expressed, and no infectious virus is produced. MVA-BN is classified as Biosafety Level 1 organism according to the Centers for Disease Control and Prevention in the United States. Preparations of MVA-BN and derivatives have been administered to many types of animals, and to more than 2000 human subjects, including immune-deficient individuals. All vaccinations have proven to be generally safe and well tolerated. Despite its high attenuation and reduced virulence, in preclinical studies MVA-BN has been shown to elicit both humoral and cellular immune responses to vaccinia and to heterologous gene products encoded by genes cloned into the MVA genome [E. Harrer et al. (2005), Antivir. Ther. 10(2):285-300; A. Cosma et al. (2003), Vaccine 22(1):21-9; M. Di Nicola et al. (2003), Hum. Gene Ther. 14(14):1347-1360; M. Di Nicola et al. (2004), Clin. Cancer Res., 10(16):5381-5390].
Protective immunity to infection relies on both the innate and adaptive immune response. The adaptive immune response includes production of antibodies by B cells (humoral immune response) and the cytotoxic activity of CD8+ effector T cells (cellular immune response) and CD4+ T cells, also known as helper T cells, who play a key role in both the Immoral and the cellular immune response.
CD4+ T cells are stimulated by antigens to provide signals that promote immune responses. CD4+ T cells act through both cell-cell interactions and the release of cytokines to help trigger B cell activation and antibody production, activation and expansion of cytotoxic CD8+ T cells, and macrophage activity.
Antibody-mediated protection can be extraordinarily long-lived, and neutralizing antibodies present at the time of pathogen encounter can prevent rather than combat infection, thereby achieving ‘sterilizing’ immunity (Swain et al., 2012, Nat Rev Immunol, 12(2): 136-148). Following viral infection, CD4+ signaling is necessary to direct the formation of germinal centers, where CD4+ cells promote B cell isotype switching and affinity maturation of antibody responses as well as the generation of B cell memory and long-lived antibody-producing plasma cells. Thus, CD4+ cells are likely to be important for generating long-lived antibody responses and protective immunity to most, if not all, pathogens.
The role of CD4+ T cells in helping the priming, effector function, and memory of CD8+ T cells is especially important in the case of chronic infections, when CD8+ T cells rely on continued rounds of expansion, for which CD4+ T cell cytokine production is critical (Swain et al., 2012, Nat Rev Immunol, 12(2): 136-148).
Recent data has indicates that the role of CD4+ T cells extend further than that of cytokine production. For example, CD4+ T cells can recruit key lymphoid populations into secondary lymphoid tissue or sites of pathogen infection (Sant and McMichael, 2012, J Exp Med, 209(8):1391-5). Specifically, CD4+ T cells can promote engagement of CD8+ T cells with dendritic cells in secondary lymphoid tissue, cause influx of lymphoid cells into draining lymph nodes, and recruit effectors to the site of viral replication. In addition, CD4+ T cells can also protect against pathogens through direct cytolytic activity.
Following the resolution of primary immune responses, or after successful vaccination, most pathogen-specific effector CD4+ T cells die, leaving behind a small population of long-lived memory cells. Memory CD4+ T cells enhance early innate immune responses following infections in the tissues that contribute to pathogen control (Swain et al., 2012, Nat Rev Immunol, 12(2): 136-148). Importantly, CD4+ T cells provide more rapid help to B cells, and potentially to CD8+ T cells, thereby contributing to a faster and more robust immune response.
The range of functions of CD4+ T cells during an immune response highlights their key role in generating highly effective immune protection against pathogens. Recent studies have provided new evidence for CD4+ T cells as direct effectors in antiviral immunity (Sant et al., 2012, J. Exp. Med. 209: 1391-1395). Preexisting influenza-specific CD4+ T cells were reported to correlate with disease protection against influenza challenge in humans (Wilkinson et al., 2012, Nature Medicine, 18: 274-280).
Several assays are used to detect immune responses, including, e.g., ELISA (enzyme-linked immunosorbent assay), ELISPOT (enzyme-linked immunospot), and ICS (intracellular cytokine staining). ELISA assays analyze, e.g., levels of secreted antibodies or cytokines. When ELISA assays are used to determine levels of antibodies that bind to a particular antigen, an indicator of the humoral immune response, they may also reflect CD4+ T cell activity, as the production of high-affinity antibodies by B cells depends on the activity of CD4+ helper T cells. ELISPOT and ICS are single-cell assays that analyze, e.g., T cell responses to a particular antigen. ELISPOT assays measure the secretory activity of individual cells, and ICS assays analyze levels of intracellular cytokine. CD4+ specific and CD8+ specific T cell responses can be determined using ICS assays.
There are published papers testing methods for using MVA-Ad prime-boost regimens in animals, such as monkeys and mice. However, no MVA-Ad prime-boost regimen has been shown to be more effective at stimulating an immune response than the complementary Ad-MVA prime-boost regimen until now. For example, Barouch et al. (2012, Nature, 482(7383):89-93) found that, in monkeys, a heterologous regimen comprising MVA/M26 was “comparatively less efficacious than Ad26/MVA or Ad35/Ad26, which reduced viral load set-points by greater than 100-fold.” In particular, the cellular immune response to SIV Gag, Pol, and Env in rhesus monkeys was less-pronounced for the MVA/Ad26 prime-boost regimen administered on a 0-24 week schedule than for the opposite Ad26/MVA regimen, as measured by IFN-gamma ELISPOT and ICS assays. The antibody response was also less effective for the MVA/Ad26 regimen than for the Ad26/MVA regimen, as evidenced by an ELISA assay, though to a lesser extent. Roshorm et al. (2012, Eur J Immunol, 42(12):3243-55) found that an MVA/ChAdV68 prime-boost regimen administered in mice on a 0-4 week schedule was no more effective at inducing an immune response to HIV Gag than the opposite ChAdV68/MVA regimen, as measured by an ICS assay for CD8+ T cell activity. Gilbert et al. (2002, Vaccine, 20(7-8):1039-45) found that an MVA/Ad5 prime-boost regimen administered in mice on a 0-14 day schedule was slightly less effective in producing an immune response to Plasmodium CS than the opposite Ad5/MVA regimen, as measured by an ELISPOT assay. The MVA/Ad5 regimen was even less effective than the Ad5/MVA regimen when both were administered on a 0-10 day schedule. Additionally, the MVA/Ad5 regimen was less effective in protecting immunized mice against a challenge infection (80% vs. 100% protection). None of these reports indicate that an MVA/Ad regimen can result in a stronger humoral and/or cellular immune response in humans, than an Ad/MVA regimen.
There is an unmet need for improved vaccines that elicit broad and strong immune responses in humans against antigenic proteins, and particularly vaccines that provide protective immunity against the deadly Ebola and Marburg filoviruses.
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