VGTI 5 Pillars of Growth - Research Summary
I. AIDS PATHOGENESIS
AIDS Pathogenesis and Immunity Program
L. Picker. M. Axthelm, S. Hansen, J. Sacha, H. Park, A. Okoye
One of the most advanced and mature research programs at the VGTI is the AIDS pathogenesis and vaccine program to focus on the development of novel immunotherapeutic approaches that provide for viral control (and perhaps even virus elimination) and/or immune restoration, in untreated infections or full normalization of immune function with optimally suppressive ART (anti-retroviral therapy).
Modeling and Evaluating HIV Cure Strategies in Nonhuman Primate Models
L. Picker. M. Axthelm, S. Hansen, J. Sacha, H. Park, A. Okoye
Current understanding of the residual virus remaining in HIV+ subjects on optimally effective cART suggests that HIV cure, either complete or functional, will likely require the combination of 2 mechanistically distinct interventions: 1) induction of viral gene expression in the transcriptionally “quiescent”, latent HIV reservoir (required for immune targeting of these cells) and 2) targeted immune destruction of all cells expressing HIV gene products. Given the known pathobiology of HIV infection, these two mechanisms will necessarily occur in diverse, complex and interacting microenvironments throughout the body, necessitating in vivo analysis for the rational development of effective interventions. It is difficult, if not impossible, in humans to access all of the tissue sites needed to understand the therapeutic activity being studied. Thus, the development of effective cure interventions will require in vivo pre-clinical studies in a relevant, manipulable animal model. In this regard, we have developed nonhuman primate (NHP) models of lentiviral latency in SIV-infected macaques given cART that faithfully recapitulate the known characteristics of HIV latency in humans, yet allow both aggressive, exploratory interventions and systemic, tissue-based analysis using the unparalleled cutting edge capabilities of our collaborative group for virologic and immunologic analyses in NHP models.
II. CYTOMEGALOVIRUS (CMV) VECTOR VACCINE AND DISEASE PROGRAMS
The CMV vaccine is on the NIH list of the “most promising medical advance of 2013”
L. Picker, S. Hansen, M. Axthelm, K. Früh, J. Nelson, J. Sacha , P. Caposio, D. Streblow, V. DeFilippis, H. Park, A. Okoye
A central program of the VGTI is the development of Cytomegalovirus (CMV) as a novel vector platform. The spectacular success of CMV vectors in protecting monkeys against highly virulent SIV has put CMV-vectored vaccines at the forefront of AIDS vaccine development. These results have received world-wide attention and the VGTI has been able to secure extensive funding from private foundations and the NIH to further the clinical development of this platform. We expect this program to continue to flourish and increase. We also anticipate the clinical development of these vectors will require increased investment in GMP manufacturing infrastructure.
This success is a direct result of multiple research groups at the VGTI contributing their strengths and expertise to this program:
- L. Picker is the director of this program and responsible for the overall scientific direction.
- S. Hansen is responsible for the non-human primate immunological analysis of CMV-vectors
- M. Axthelm is in charge of non-human primate veterinary care
- K. Früh is the co-director of the CMV vector program particularly with respect to vector design and manufacturing. Dr. Früh also heads the VGTI spinoff company TomegaVax and part of this work is funded via a phase II SBIR to TomegaVax with Eric Bruening PhD (COO) as Principal Investigator.
- J. Nelson is responsible for the improved vector design and testing of 2nd generation vectors
- P. Caposio is in charge of manufacturing and testing vectors in humanized mouse models
- D. Streblow directs research towards understanding the role of viral chemokines in tropism and T cell priming
- V. DeFilippis is responsible for optimizing vector manufacturing by developing production cell lines
- J. Sacha directs research towards understanding immunological principles that govern CMV-vector induced T cell priming
L. Picker, S. Hansen, M. Axthelm, K. Früh, J. Nelson, P. Caposio
The Bacille Calmette-Guérin (BCG) vaccine, created in 1921, is the only existing vaccine against TB. Unfortunately, it only provides some protection against severe forms of pediatric TB, but is unreliable against adult pulmonary TB which accounts for most of the disease burden worldwide. The world-wide reemergence of TB, including multi-drug resistant TB renders the development of novel TB vaccines a high priority for global health. We have demonstrated in rhesus macaques that CMV-vectored TB vaccines are far superior to BCG in limiting disease, dissemination and ultimately death, of immunized animals. Based on these highly encouraging results, we are advancing these studies in rhesus macaques including pre-clinical work in preparation for clinical development.
K. Früh, L. Picker, S. Hansen, M. Axthelm
Malaria is a global burden with > 600,000 deaths, most of them children, and with >200 million clinical cases annually. Malaria can be prevented through a combination of chemoprophylaxis and personal protection measures. However, both require strict compliance and chemoprophylaxis cannot be used over longer periods of time. The development of a vaccine against malaria is therefore one of the highest priorities for global health research. Since it is known that cellular immunity to the pre-erythrocytic stage of the malaria parasite can provide sterile immunity against Plasmodium parasites there is a very strong rationale for using CMV-vectors in malaria vaccine development. In collaboration with the Naval Medical Research Center (NMRC) in Bethesda we performed a pilot study using CMV vectors to express a mixture of pre-erythrocytic and erythrocytic antigens in rhesus macaques. Preliminary results suggest that CMV-vectored malaria vaccines were able to reduce the pre-erythrocytic parasite burden. Based on these encouraging results we now applied for Department of Defense funding for continuation of this program. We are also in discussion with the malaria vaccine initiative (MVI/PATH). We anticipate that this program will grow over the coming years depending on funding success.
S. Wong, K. Früh, L. Picker, S. Hansen, M. Axthelm
KS caused by a gamm-2 herpesvirus (KSHV) and is the leading cancer in sub-saharan Africa. Funded by an NIH R21 exploratory grant, Dr. Wong and his collaborators are currently exploring in the rhesus rhadinovirus model whether CMV-vectors can prevent RRV-induced B cell hyperplasia. Depending on the outcome of this pilot experiment, we are appling for additional grants to further develop this program.
Cervical Cancer Vaccine
K. Früh, L. Picker, S. Hansen, M. Axthelm
While there is a prophylactic vaccine against human papillomavirus (HPV) the causative agent of cervical cancer, this vaccine is unable to clear HPV once the infection has been established, leaving infected women at risk for the development of cervical cancer. With funding by TomegaVax (SBIR) and by the Oregeon Nanotechnology and Microtechnologies Institute (Onami), we explore whether CMV-vectored HPV vaccine can prevent tumor formation in murine models and induce T cells in the cervix of female macaques. If successful, we will seek industry funding to develop a CMV-based therapeutic HPV vaccine.
Prostate Cancer Vaccine
D. Streblow, K. Früh, L. Picker, S. Hansen, M. Axthelm
A therapeutic vaccine against prostate cancer developed by Dendreon was the first cancer vaccine ever to be approved. The vaccine is expensive, difficult to generate and it does not provide the durable protection needed in a post-op setting. With a pilot grant from the Knight Cancer Institute we are currently examining whether CMV-vectors carrying the rhesus equivalent of the Dendreon antigen are able to elicit T cell responses to this self-antigen in rhesus macaques. If successful, we will seek funding from the NCI and other sources to develop a CMV-based cancer vaccine
Genital Herpes Vaccine
D. Streblow, K. Früh, L. Picker, S. Hansen, M. Axthelm, P. Caposio, J. Nelson
An approved vaccine for genital herpes is currently not available. Funded by TomegaVax (STTR as well as industry funding) Dr Streblow determines whether CMV vectors can protect against HSV2 in murine models. Using a novel HCMV vector platform (VGTI-1) developed by P. Caposio and J. Nelson with funding from TomegVax (SBIR), Drs Früh and Hansen further determine whether VGTI-1-vectored HSV2 vaccines induce lasting immune responses in the female genital tract of rhesus macaques. Our plan is to develop a CMV-based, therapeutic genital herpes vaccine using industry funding.
J. Sacha, K. Früh, L. Picker, S. Hansen, M. Axthelm
The reason why flu vaccines need to be given every year is that antibody responses are highly strain specific and short-lived. In contrast, the long-lived T cell responses induced by CMV vectors might overcome this problem. To determine whether T cells induced by CMV can prevent highly pathogenic flu in non-human primates, we will test CMV vectors against 1918 flu, a highly virulent virus that sickened and killed millions and is highly pathogenic in monkeys.
Other Exploratory CMV Vaccines
We are in the process of generating preliminary data for grant submissions using CMV vectors against the following diseases; each of these diseases has a strong rationale for testing CMV vectors and, except for HBV (note the HBV vaccine only works to prevent infection but cannot eradicate established infections), there are currently no approved vaccines:
Chikungunya Virus (CHIKV) D. Streblow, V. DeFilippis
Chagas disease D. Streblow
Dengue Virus J. Nelson
Hepatitis B K. Früh
Hepatitis C J. Nelson
CMV Disease Program
The CMV disease program encompasses basic science questions concerning mechanisms of viral persistence, replication and latency as well as animal model development to understand viral pathogenesis as describe in the following programs:
HCMV miRNA Regulation of Latency, Secretion, and Virion Maturation
J. Nelson. P. Caposio, R. Skalsky
Regulation of how the HCMV (Human cytomegalovirus) miRNAs target several members of the IL-1 and TNFa signaling pathways leading to NFkB activation, is a critical determinant of whether HCMV reactivates or remains latent. This project characterizes the role of these miRNAs in targeting NFkB activation in invitro and humanized mouse models (see below). We have initially observed that deletion of two of these miRNAs results in a virus that can establish latency but cannot reactivate. These results may be important for the generation of an HCMV vaccine vector that is safe. In addition, the HCMV encoded miRNAs target cellular genes in the secretory pathway that affect three important processes during viral infection. Using this information we are designing an HCMV vaccine vector that lacks the HCMV miRNAs that mediate these processes with the expectation that we can increase immunity to antigens inserted into these vectors by increasing cell surface MHC I, as well as, inflammatory cytokines besides attenuating the virus.
Generation of a Humanized Mouse Model to Study HCMV Latency, Reactivation, and Immune Response
P. Caposio, D. Streblow, William Flemming (Hematology), Morgan Hakki (Infectious Disease) and J. Nelson
HCMV remains a serious complication in patients receiving allogeneic hematopoietic stem-cell transplantation (HSCT). Myeloid lineage cells are one of the cellular reservoirs of latent HCMV and many groups have established in vitro cell systems to examine mechanisms of viral latency. Although these in vitro myeloid systems have proved useful to explore HCMV latency, an animal model is needed to assess the relevance of these findings. We have reported the first humanized mouse model in which human CD34+ hematopoietic progenitor cells (HPCs) engrafted NOD-scidIL2Rc null (NSG) mice infected with HCMV can support a latent viral infection. Moreover, we have shown that HCMV reactivates in human macrophages following G-CSF-induced mobilization of bone-marrow hematopoietic cells. These data recapitulate observations made in bone marrow transplant patients receiving G-CSF mobilized cells from HCMV seropositive donors. These observations also provide definitive evidence that CD34+ HPCs and monocytes harbor latent HCMV that results in dissemination and increased expression of virus in macrophages upon differentiation. The model is being used to analyze mechanisms of reactivation in HSCT patients as well as potential cellular sources of latent virus in collaboration with Harv Flemming (Knight Cancer Institute) and Morgan Hakki (Infectious Disease) and to study characteristics of growth persistence and immune response of the HCMV/HIV vaccine.
Role of CMV in the Acceleration of Transplant Vascular Sclerosis in Solid Organ Transplant Patients
D. Streblow and Susan Orloff (Liver Transplantation)
Approximately 75% of the solid organ donor/recipient population is infected with HCMV. As such, the majority of transplant recipients are themselves HCMV positive and/or receive infected donor allografts, which makes it very difficult to avoid this important problem. However, the viral mechanisms involved in HCMV reactivation from latency and subsequent acceleration of vascular disease and rejection are unknown. To date no vaccine exists to prevent CMV-disease and resistance to antiviral therapeutics renders them less effective in the clinic. To address this, we are characterizing CMV immunity and identifying biomarkers of CMV-induced rejection in a human cardiac transplant cohort at OHSU. We have established a rat heart transplant model of CR that closely recapitulates this human transplant scenario. In the model, RCMV infection significantly accelerates the time to develop CR and increases the degree of transplant vascular sclerosis (TVS). This represents a unique and powerful animal model that allows us to define the critical host and viral mechanisms that drive the vascular disease associated with CR.
III. Herpes Family Viruses Prevention & Treatment
NHP Model in AIDS-related malignancies
S Wong, R. Skalsky
The focus of this project is to gain new insights into the pathogenesis of chronic herpesvirus infections in the immunocompromised host. The laboratory is focused on the rhesus macaque (RM) as an animal model to study Kaposi's sarcoma-associated herpesvirus (KSHV) pathogenesis. KSHV is the etiological agent of Kaposi's sarcoma (KS) and specific B cell lymphoproliferative disorders (LPD); primary effusion lymphoma (PEL); multicentric Castleman's disease (MCD); and some non-Hodgkin's lymphomas (NHL). Dr. Wong isolated the first KHSV-like virus in Rhesus macaques and demonstrated that this virus causes lymphproliferative disease. With this model viral genes necessary for causing were characterized, as well as, a potential preventative vaccine. Future work examines the role of virally encoded miRNAs in latency and disease.
Japanese macaque encephalomyelitis (JME) model development and the role of JMRV
S. Wong, A. Moses, J. Douglas, R. Skalsky, M. Axthelm, D. Bourdette
The focus of this program is to evaluate the potential role of a novel herpesvirus, referred to as Japanese macaque rhadinovirus (JMRV) in Japanese macaque encephalomyelitis (JME), a disease possesses clinical and pathological features that resemble multiple sclerosis (MS). The disease occurs in both progressive and relapsing-remitting forms and is characterized by brain and spinal cord demyelination. The virus grows well in cell culture and DNA sequence analysis revealed the virus is closely related to RRV, yet is distinct as it possesses unique ORFs that are not present in RRV. The results from our studies confirmed that JMRV is capable of inducing an inflammatory and demyelinating lesion in the central nervous system of animals with the appropriate genetic background, and that JME possesses signatures of an immune-mediated disease like MS. We are working to search for a human virus correlate in MS patients using genes from JMRV as a probe.
KSHV and Kaposi Sarcoma
A. Moses, J. Douglas, R. Skalsky
The primary goal of this program is to identify and study KSHV-induced host genes that contribute to KSHV persistence and development of KS, and to characterize the viral pathway(s) responsible for host modulation. Endothelial cells (EC) are the target cells of interest, given that KS is a tumor of EC origin. The intent is to understand how a particular host/virus interaction contributes to disease and to utilize this insight to identify new anti-viral or anti-tumor therapeutic strategies. Pathways/mechanisms identified may also have broader significance for cancer, angiogenesis and host immunity. Dr. Moses was the first to identify c-kit as a cellular gene induced by KSHV and showed that treatment of KSHV-infected EC with the TKI inhibitor Imatinib (Gleevec) prevented their characteristic progression to in vitro tumor phenotypes.
The role of viral and cellular miRNAs in B-cell lymphomagenesis
One in six cancers can be linked to viral infection, and a portion of these can be attributed to infection with Epstein-Barr virus (EBV). EBV is a ubiquitous virus, infecting >90% of adults worldwide. Primary infection is commonly asymptomatic or presents as mononucleosis which resolves within a few weeks; however, in certain circumstances, life-long, latent infection can lead to epithelial and lymphoid malignancies, including lymphoma. Recent studies demonstrate roles for both EBV-encoded microRNAs (miRNAs) and EBV-induced cellular miRNAs in B cell transformation and survival in vitro. The goals of this project are to comprehensively identify and characterize critical miRNA-regulated genes during EBV infection of B cells in order to understand their contributions to viral persistence and oncogenesis. EBV infection of B cells in vitro provides us a valuable model system to investigate the molecular mechanisms driving the development and progression of uncontrolled B cell proliferation.
IV. Emerging Disease Program
Another one of the major research programs at the VGTI is centered on the development of vaccines and therapeutics for Emerging and Re-emerging diseases that pose a threat to the USA and the world. The VGTI emerging disease program encompasses three main areas of emphasis: development of vaccines for vulnerable populations such as the elderly, use of Systems Biology to develop antiviral therapeutics and adjuvants for existing vaccines, animal model development to test vaccines and antivirals, and novel vaccine approaches primarily using the CMV vector platform.
Development of CMV Vector based vaccines for TB, Malaria, Dengue Virus (DENV), and CHIKV (described in the CMV vaccine program Section II.)
Chikungunya virus (CHIKV) Animal Model Development
D. Streblow, M. Axthelm
CHIKV is a NIAID Category C Biodefense priority mosquito-transmitted Alphavirus that causes a febrile, sometimes-fatal disease that involves incapacitating myalgia and arthralgia that can persist for years. FDA approved anti-CHIKV therapeutics and vaccines are currently not available. A safe and effective vaccine that can protect vulnerable populations from CHIKV infection is clearly warranted. Using our NHP-CHIKV infection model, we have demonstrated that aged animals exhibit higher susceptibility to persistent CHIKV infection as a result of defects in the development of T cell responses directed against the virus. Traditional vaccines against acute viruses focus on eliciting neutralizing antibody responses, which is more difficult in immunologically vulnerable populations such as the aged. In addition, we have demonstrated that neutralizing antibody therapy does not reduce pre-existing CHIKV infection of NHP joint tissues, indicating that T cell responses are crucial for clearing the virus. In light of this, we are developing efficacious T cell-based vaccines against CHIKV to be tested in our CHIKV-NHP model. In addition, we are currently determining whether there exist differences in joint disease, viral load, distribution and/or immune infiltrates between CHIKV infected adult and aged animals at early times post infection in an effort to lay the foundation for the design of successful vaccine strategies to prevent CHIKV-associated morbidities in vulnerable populations.
Anti-viral Therapeutic Programs
CHIKV Therapeutic Program
D. Streblow, V. DeFilippis
This program aims to identify novel small molecules capable of inhibiting replication of diverse members of the Alphavirus genus. Alphaviruses are arthropod-transmitted RNA viruses comprising seven antigenic complexes that include multiple Biodefense Category B and C priority pathogens. Two distinctive virus-dependent pathologies are manifest during Alphavirus infection. Neurological disease including encephalitis is primarily associated with New World species (Eastern (EEEV), Venezuelan (VEEV), and Western Equine Encephalitis (WEEV) viruses) and can present high mortality rates especially in hosts with weakened or immature immune systems, the young and aged populations. Arthralgia and inflammatory syndromes are typically associated with Old World species (Chikungunya (CHIKV), Ross River (RRV), and Semliki Forest (SFV) viruses) and while these are uncommonly fatal they can elicit incapacitating effects that persist long after viral clearance. Currently no FDA approved vaccines or antiviral therapeutics are available to prevent Alphavirus infection or treat Alphavirus-associated disease. Our goal is the identification of lead molecules with appropriate activity and pharmacokinetic properties will be evaluated using CHIKV and VEEV mouse and NHP models of acute and persistent infection and disease. Parallel screening against multiple virus families using a Alphavirus screening platform library will dramatically increase the likelihood of identifying antiviral compounds that are efficacious against a broad spectrum of agents.
Flavivirus Therapeutic Program
A. Hirsch, J. Nelson
The Dengue Viruses (DENV) antiviral program is part of a larger Antiviral Drug Discovery and Development Center that includes a group from multiple universities funded through NIAID’s “Centers for Excellence in Translational Research” program. The flaviviruses are mosquito-borne viruses that are associated with significant worldwide morbidity and mortality. The dengue viruses (DENV) are members of a group associated with dengue fever, dengue hemorrhagic fever and dengue shock syndrome. These viruses are endemic in most of the tropical and sub-tropical world, and roughly one-third of the Earth’s populace lives in areas at risk for dengue transmission. A second member of the flaviviruses, West Nile virus (WNV) is a neurotropic virus– infection can result in encephalitis/ meningitis and subsequent long term neurologic complications or death. For both viruses, there are no available anti-viral therapeutics, nor are there vaccines approved for use in humans. This collaborative project includes high-throughput screening of libraries of >200,000 small molecule compounds to identify inhibitors of DENV and WNV. The VGTI will characterize active compounds with regard to efficacy and mechanism of action, as well as extend analysis of promising lead compounds in murine models of virus infection.
V. Protective Immunity in Elderly Populations
Interface between Innate and Adaptive Immunity
A. Hirsch, V. DeFilippis, J. Nelson
The greatest and most cost effective deterrent to microbial pathogen-mediated morbidity and mortality in humans are vaccines. Although current vaccines have been generally effective in providing protective immunity in adults, infectious disease remains a significant cause of morbidity and mortality in the elderly, particularly in individuals over the age of 65. Older adults suffer disproportionately from certain types of infectious diseases that are readily controlled by the adult population, in large part due to the aging of the immune system termed immune senescence. This phenomenon remains one of the least well-understood areas of medicine. Age-related decline in immunity that results in a profound vulnerability of the elderly to infectious disease. One part of this program is designed to elucidate mechanisms of immune dysfunction in aged individuals (immune senescence). Using a murine model of West Nile virus (WNV) infection, which recapitulates the greater susceptibility of old humans to viral disease, we have observed functional defects in the innate and adaptive immune system of old animals. We have observed that there are functional defects in the innate immune response of old dendritic cells that allows their maturation and ability to present antigen to B and T cells as well as defects in miRNA expression that allow CD8 T cell maturation. We have observed that both replication competent vaccines as well as addition of adjuvants to protein-based vaccines can overcome these defects in old animals. These studies form the basis for the adjuvant vaccine program described below.
Vaccine Adjuvant Program
J. Nelson, V. DeFilippis, A. Hirsch, Dan Streblow
The major goal of this program is to rationally enhance protective immunity in the elderly by developing immune stimulating agents that will counteract the effects of immune senescence. While replication competent vaccines can overcome defects in the immune system that prevent development of a protective response, these vaccines are not safe for older adults and immune compromised individuals. Protein-based vaccines are safe but do not efficiently induce a protective response unless adjuvants are added to the inoculum. Currently, only two adjuvants (Alum and AS03) have FDA approval for use in the USA as well as another (MF59) that has been approved for use in Europe. These adjuvants elicit generalized effects in immunized patients but there is a need for novel targeted adjuvants. This program in collaboration with Michael Gale at the University of Washington and Kineta Inc. will identify small molecule leads that target members of PRR pathways activated in Dendritic cells (DC). Dentritic cells are potent myeloid-derived antigen presenting cells that function at the interface of innate and adaptive immunity. DCs primarily engender innate immune activities in response to microbes and pathogen-associated molecular patterns yet are critical to the initiation of adaptive immunity. These leads will be developed into formulated adjuvants that will be tested in our animal models.