Elite control of HIV and SIV infection
Small percentages of humans infected with HIV-1 and macaques infected with SIV spontaneously control the virus without the aid of antiretroviral drugs. By far the best predictor of an individual's ability to control the virus lies in which major histocompatibility complex class I (MHC-I) genes they express. MHC-I molecules present virus-derived peptides to CD8 T cells. We are studying rhesus macaques that express the MHC-I allele Mamu-B*17, the expression of which is associated with enhanced viral control, including in individuals that control the virus to undetectable levels. We are investigating the CD8 T cell responses these animals make against the virus as well as the specific manner in which the virus evolves to evade the response. We have found that, in macaques that express Mamu-B*17, CD8 T cells predominantly target the viral Nef protein and the virus evolves in a manner that compromises specific Nef functions, rendering the virus unfit and possibly enabling greater viral control. Thus, we are using immunological, virological and genetic techniques to understand effective immunity.
Identification of latently infected cells
One of the greatest obstacles to a cure for HIV is its propensity to integrate into the host genome and lie latent in certain cell types, including resting CD4 T cells and macrophages. A critical step towards a cure would be to identify and kill these latently infected cells before the virus begins to replicate. We are using high throughput RNA sequencing (RNA-seq) to identify novel markers of these cells, without the need to induce replication. In particular, we have found that the SIV genome remains transcriptionally active during a latency-like state where minimal viral replication is detected. Interestingly, we have found that the antisense strand of the viral genome also is transcribed but only in a small number of genomic locations to produce novel, presumably non-coding RNAs. Together, these data suggest that therapies designed to target particular viral products might be effective at eliminating the viral reservoir.
Mechanisms of protection from pathogenic SIV provided by novel SIV vaccines
Though not viable for clinical translation live attenuated SIV vaccines can serve as ideal models to discover the types of immune responses an effective vaccine needs to induce. Working with colleagues at the University of Pennsylvania, we are investigating a novel attenuated SIV vaccine that contains a two amino acid deletion in the SIV Envelope cytoplasmic tail. This virus replicates well initially but is rapidly controlled by the host (Pigtail macaques). These animals are then protected from a variety of pathogenic SIV challenges. This protection occurs in the absence of neutralizing antibodies and appears to be T cell mediated. We are exploring the mechanisms by which T cells induced by this vaccine can control pathogenic SIV challenges. Specifically, we are focused on how this particular deletion alters the distribution of molecular components of the virologic and immunologic synapses, leading to reduced viral spread and enhanced antiviral T cell function.
Zika infection of nonhuman primates
We are collaborating with a large team of scientists at Tulane and beyond to develop and optimize a nonhuman primate model of Zika virus infection in macaques, including pregnant females as well as adult and infant non-pregnant animals. Our goals are to comprehensively assess the pathogenicity of Zika in these animals and to assess immunological responses to the virus that might be responsible for preventing both viral dissemination into neurological and reproductive tissues and preventing maternal to fetal viral infection.
Evaluate potential novel vaccines for their safety and efficacy
Infection with Mycobacterium tuberculosis (Mtb) causes tuberculosis (TB) disease. As many as ~ 9 million new cases and ~ 1.5 million deaths globally, every year are reported. This global pandemic is made worse by widespread incidences of drug-resistance. The problem is compounded by the spectacular failure of BCG, the vaccine in use for a century to protect against TB. The development of one or more efficacious vaccines against TB, including one that can prevent the transmission of MDR-TB is of the utmost priority. We use a highly faithful nonhuman primate (NHP) model of TB to study the protective efficacy of a series of mycobacterial strains based on the sigH deletion. Preliminary data shows that the Mtb:D-sigH mutant, an avirulent, BSL2 strain, is able to protect against lethal Mtb challenge in NHPs, especially when delivered directly to the lungs. We are pursuing the development of an anti-TB vaccine candidate based on sigH deletion.
Study the role of host negative regulators of T cell function
IDO (IDO1), which encodes the tryptophan-degrading enzyme Indoleamine 2,3 dioxygenase, is a powerful immunosuppressant and may play a key role in modulating immune responses to TB. Its expression is highly induced in the lungs of NHPs and Kramnik mice with acute but not latent infection. It is expression is highly induced in cultured macrophages. Its presence is limited to the macrophage rich ring structure, adjoining the caseum. Thus, Mtb infection-mediated IDO induction may suppress the function of activated, interferon gamma producing CD4+ T cells and prevent them from accessing the center of the lesion. This could potentially facilitate the persistent survival of Mtb. We propose experiments to conclusively test the hypothesis that IDO expression in the inner ring of a TB granuloma modulates T cell function, thus aiding Mtb in its persistence.
Granuloma Mechanisms of Protection from Tuberculosis
We utilize the nonhuman primate model of inhalation TB, to study Mtb-specific mechanisms of granuloma persistence and modulation of immune responses. We propose to characterize mechanisms that allows granulomas to control Mtb infection and identify events, which cause this control to dissipate. Our studies have the potential to inform fundamental aspects of the latent control of Mtb within granulomatous lesions.
Role of MAIT cells and CD1-restricted T cells in control of TB and opportunistic infections in the absence or presence of SIV infection of macaques. Examining the role of innate type-17 mucosal immunity in the process of accelerated aging and gut mucosal dysfunction/dysbiosis in SIV-infected macaques on long-term ART.
Infectious Disease Aerobiology and the Communicability of Airborne Disease
Research in the Division has focused on the study of respiratory-transmitted disease agents, with the long term goal of gaining a better understanding of the constellation of variables that are important to airborne infection. Over the last several years, through targeted scientific inquiry, we have dedicated our efforts to deciphering the factors essential to the initiation, transport, and primary infection through the aerosol route of exposure. Utilizing the tools and scientific resources unique to this line of investigation, we have harnessed laboratory-based engineered configurations for determining microbial efficiencies of high consequence pathogens when in
aerosol. For example, studies have led to discoveries about the unexpected long term survival of Monkeypox virus, a known human respiratory pathogen, when in aerosol form. Using this unique aerosol generation and sampling system, collectively housed in our high biocontainment (BSL-3) laboratories, we have been able to perform aerosol survivability studies with bacterial agents as well. Building upon basic approaches to better understand the aerosol ecology of infectious agents relevant to human disease, we are also heavily invested in the in-depth study of the modeling disease using advanced animal species, including the nonhuman primate, using experimental aerosols as our modality of infection. The application of our work in aerosol biology of infectious agents, including our extensive efficiency studies performed with a number of infectious agents, is leveraged in ongoing animal trials. Using this comprehensive approach has provided significant traction towards reaching our long term research goal, and has richly contributed to the ongoing development of a better understanding of the complexities associated with the study of airborne infection in man.
Aerosol Ecology & Transmission of Infectious Disease
Very little is known about the temporal development of infectious bioaerosol generation in infectious hosts as well as the risk presented from aerosol particle transport from a contagious individual in the context of disease induction. Our research attempts to study these phenomena experimentally, using devices and laboratory apparatus within a high containment environment that supports these activities in a safe manner.
There is a known particle size distribution associated with impacting the most susceptible region of the respiratory system, and as such is of the most intensively studied. The difficulty with focusing only upon the ‘respirable’ fraction of any distribution containing infectious particles is that the human condition, in stark contrast, dictates that aerosols of a highly heterogeneous distribution are generated in infectious individuals considered contagious. The effect of particle size upon the relative viability of the pathogenic organism is highly variable and may be dependent upon the generator and subsequent transport. In addition, there are other variables, such as genetic composition, and strain-specific modification, that may also be determinative of survival in aerosol. Collectively, this is an exceedingly difficult problem to investigate in a laboratory setting.
As an exemplar of our research approach to defining microbial ecology of aerosolized pathogenic agents, we investigated influenza viruses to better understand factors important in to aerosol survival using many of the scientific tools available in the aerobiology laboratories. We determined that that strain-specific composition, and subsequent morphological differences between H1N1 (swine mutant) and H2N3 (seasonal variant) in part, is determinative of the efficiency of each of these viruses when in aerosol. The study of viral particles subsequent to aerosol generation and capture by aerosol sampling showed that a filamentous virion common to the H1N1 strain, in part, was much more susceptible to the rigors of the aerosol environment, and thus is less likely to maintain a form consistent with cellular entry, replication, and ultimately the induction of infection.
Animal Models of Aerosol Infection
Many of the high consequence respiratory human pathogens cannot be studied clinically nor are susceptible populations available to monitor because of the present-day episodic (or absence) incidence of disease. Research in our Division is focused on using animal species, with emphasis on the nonhuman primate, to model these diseases of interest. We leverage our knowledge about the aerosol characteristics of infectious agents as a cornerstone for experimental infection in many of these studies. Many of the agents that are of interest, such as ricin toxin, have no human clinical data from which to reference because there has never been a poisoning via inhalation with this biotoxin. Yet ricin is considered a biological threat agent of concern and thus there is a need for preventive or palliative measures in the event of a human exposure, ultimately necessitating the development of an animal model of (aerosol) intoxication. As such, research in our Division included an extensive investigation of the pathophysiology of aerosolized ricin exposure in the rhesus macaque, using our specifically engineered aerosol exposure delivery system within high containment. We showed that intoxication by this modality is exceedingly acute, per weight dose is similar to rodent species, and effects such as vascular leak induction occurs much earlier subsequent to exposure than originally envisioned. Also of interest in these studies was the previously unidentified cell migrations, specifically interstitial macrophages, into the lung parenchyma, and the associated architectural changes including extensive fibrosis noted in animals receiving sublethal aerosol doses of the toxin. This disease model subsequently was of intense interest to a number of collaborative groups seeking to evaluate candidate vaccines and therapeutics for prevention of the effects of ricin toxin.
Vaccine and Therapeutics Evaluation using Advanced Aerosol Infection Models
Development of advanced animal models that capture critical aspects of the human syndrome of disease has been a major focus of the Division for a number of years. The nonhuman primate represents the essential link between understanding human disease and development of measures to combat effects from infection. This relationship is most important for the study of high consequence human pathogens were little to no annual incidence of human disease exists nor does the epidemiology of these diseases support advanced evaluation and development of medical products. Accordingly, many of the animal models that incorporate aerosol as a modality of exposure have been used in the ongoing Divisional research for the evaluation of medical products.
A major effort involving the development of viable vaccines for the pathogenic alphaviruses – Eastern equine encephalitis, Western equine encephalitis, and Venezuelan equine encephalitis, and Chikungunya virus – was undertaken over the last several years. Individualized disease models for each virus were developed specifically to support each vaccine product due to differences in disease outcome and lack of cross protective capacity of the vaccines under development. Vaccine products with competing platforms (virally-vectored v. VLP) were evaluated using the nonhuman primate models that were developed as a consequence of this research effort. The disease models proved robust and durable for these purposes, and paired with immunogenicity, efficacy was achieved against all of the pathogenic viruses listed within this family.
The development and utilization of nonhuman primate models of alphaviral disease are one of a number of ongoing efforts that comprise the research in this area within our Division. We have leveraged many aspects of infectious disease aerobiology that although initially appearing disparate in focus, complements programmatically many of the research applications.
Non-Human Primate Models of Viral and Autoimmune Diseases of the Gastrointestinal Tract
Over past 12 years, Dr. Sestak’s research focus at Tulane was predominantly directed towards infectious and autoimmune diseases of the gastrointestinal tract such as enteric calicivirus, rotavirus infections and gluten-sensitive enteropathy. The main objective is the development and translational use of non-human primate models to address those issues of immunity, pathogenesis and prevention that cannot be readily dealt with in clinical studies. Dr. Sestak’s studies and collaborations led to isolation and in vitro adaptation of two groups of novel enteric viruses (rhesus rota- and caliciviruses), first ever description and recognition of rhesus gluten-sensitive enteropathy as the model for human celiac disease plus other models of human diseases. These models and agents are now available for intra- and inter-institution collaborative studies.
Gluten Sensitivity, Celiac Disease and Non-Celiac Gluten Sensitivity
It is estimated that there are at least 3 million people in the United States affected with celiac disease. Overall population prevalence ranges between 0.5-2% whereas certain predisposition groups show much higher numbers. The classical celiac disease is characterized by an autoimmune reaction against tissue transglutaminase in the small intestine of genetically predisposed individuals although other organs and tissues including the liver, skin, bones, reproductive and CNS can also be affected. Recent epidemiological surveys indicate that geographical distribution of CD is associated with genetic background and consumption of cereal grains.
A chronic diarrheal disease named “Gluten-Sensitive Enteropathy” (GSE) was described first in 2008 by Dr. Sestak’s group in a subset of captive rhesus monkeys fed gluten-containing chow. The presence of intestinal tissue transglutaminase autoantibodies, anti-gliadin serum antibodies, decreased resorption of nutrients, decreased xenobiotic metabolism, villous atrophy, lowered diversity of gut microbiome, chronic diarrhea, weight loss, cancer predisposition and immunogenetic (MHC II-linked) association were all reported in GS rhesus macaques. In GS macaques and in celiac patients, GSE can be induced by dietary gluten. In reverse, administration of gluten-free diet typically results in both species in disease remission. In addition to treating the selected captive GS rhesus macaques with gluten-free diet, there is a high interest from the commercial and academic sectors to utilize these animals in biomedical research, with novel celiac therapy approaches. Our group currently collaborates with Arcadia Biosciences on evaluation of proprietary therapies involving reduced gluten cereals. In addition, we currently work with other companies on preparation of project(s) for evaluation of novel immunotherapies for treatment of celiac disease. Another, tissue transglutaminase-independent and more prevalent form of gluten sensitivity e.g. non-celiac gluten sensitivity, was recently identified in humans and macaques.
Rotaviruses are the primary cause of severe diarrhea in young children and are responsible for ca. 500,000 deaths in the world each year - despite that efficacious rotavirus vaccines are already available. In 2004-2005, Dr. Sestak’s group isolated and characterized the new rotavirus strain of rhesus monkey origin named TUCH. Over past 10 years, TUCH rotavirus was used extensively by several groups in studies that focused on recombinant vaccine development, molecular evolution of naturally emerging, animal-human rotaviruses, VP6 rotavirus protein T-cell epitope mapping, and pathogenesis of pediatric biliary atresia. Recent collaborative studies with Cincinnati’s Children’s Hospital focused on function of rhesus rotavirus VP4 structural protein. It was determined that specificity of rhesus VP4 governs induction of biliary atresia in a mouse model. Because neonatal obstructive cholangiopathy e.g. biliary atresia remains the most common indication for pediatric liver transplantation in the United States, it is anticipated that rhesus-derived rotaviruses including TUCH will remain valuable research tool in studies that aim at the development of novel therapy approaches for biliary atresia.
Rhesus Enteric Caliciviruses
According to CDC, human noroviruses (NoVs) are annually worldwide responsible for more than one million hospitalizations and over 200,000 deaths in children less than 5 years of age. In the U.S. alone, an estimated 23 million cases of acute gastroenteritis, including 70,000 hospitalizations and 800 deaths are attributed to NoV infections each year. No robust in vivo or in vitro model exists to study human NoVs. Dr. Sestak’s group discovered and characterized in 2008 a new group of enteric caliciviruses of rhesus monkey host origin with the name Recovirus. Prototype of this new group of enteric caliciviruses is the Tulane Virus. Rhesus enteric caliciviruses are closely related to human NoVs and in contrast to human NoVs, can be grown in vitro. Since 2008, numerous studies were conducted with Tulane Virus to study enteric calicivirus biology and pathogenesis. Some of these investigations (with non-human primates as well as with human patients) revealed that rhesus enteric caliciviruses can infect not only macaques but also humans where they can produce an acute, diarrhea-like illness. Because of their biological features e.g. capability to grow in vitro and to cause gastroenteritis and fever, we plan to continue utilizing rhesus enteric caliciviruses as human NoV model.
Gut Microbiome Studies
Composition of the gut microflora affects health and metabolism of its host. Due to biological similarities and evolutionary closeness with humans, captive non-human primates represent valuable resource in biomedical research. Controlled studies that evaluate impact of diet, age, gender, genetics, environment, disease, cohabitation, exercise and other factors on the composition of gut microbiome are currently being performed. Ongoing work focuses on the differences in gut microbiome composition of captive rhesus macaques with chronic bacterial colitis, gluten-sensitive enteropathy and healthy controls. For the first time, the evidence was generated suggesting that above chronic inflammatory conditions of rhesus macaques significantly differ from each other – by composition of gut microflora. New collaborative studies utilizing these models, with emphasis on pathogenesis and therapy of gut dysbiosis, are planned for near future.
Varicella Virus Infection, Latency, and Reactivation
The Varicella-zoster virus (VZV) causes clinically mild chickenpox in children and then becomes latent in ganglionic neurons. However, in older or immunocompromised individuals showing increasing loss of T-cell-mediated immunity with age results in virus reactivation, manifesting as zoster or shingles, primarily in the elderly. Zoster is a huge health issue, affecting approximately one million Americans each year. Neurological complications of zoster include postherpetic neuralgia, myelitis, meningoencephalitis, retinitis, vasculopathy and multiple ocular disorders. Zostavax immunization is highly successful, reducing zoster by 51% and neurological complication by 66%. Yet even if every person >60 yr old were vaccinated, there will still be at least 500,000 zoster patients, nearly half of whom will experience neurological complication. VZV only causes disease in humans and testing is limited by overall inability to obtain tissues.
Our laboratory has shown the nonhuman primate counterpart virus, simian varicella virus (SVV) infects monkeys, shows clinical, virological and pathological features of acute disease, latency and reactivation that parallel VZV infection in humans, thus providing an exceptional model to study the molecular pathogenesis of varicella infection. In the past five years, we showed that SVV becomes latent in ganglionic neurons and can be experimentally reactivated by immunosuppression. Yet how SVV spreads during primary infection and reactivation is unknown. Our work has shown that after primary exposure, virus infects alveolar macrophages and/or dendritic cells in lungs and memory T cells in blood. Later SVV-infected T cells were found in ganglia, lymph nodes and in skin lesions close to blood vessels. Early after zoster, we also found that ganglia contained few SVV antigen-positive neurons, and CD8 T-cell infiltration correlated with CXCL10 transcript levels but not with SVV antigen expression, whereas lymph nodes contained SVV DNA and abundant viral antigen. Because lung, lymph node and ganglia are infected before skin rash appears, and because the most pronounced histopathological changes have been found in lungs and lymph nodes, our current studies are focusing on the delineation of virus trafficking of the virus through these organs and the cells, molecular signals, and routes, whether hematogenous or trans-axonal, involved in virus dissemination. We are also testing immunomodulatory therapeutics for controls of infection are also underway. In additional collaborations, chimeric varicella viruses are being tested to delineate genes responsible for varicella latency, and ultimately, control of reactivation.
Varicella based AIDS Vaccine
A safe and effective vaccine is needed to control the worldwide AIDS epidemic caused by the human immunodeficiency virus (HIV). The Varicella-zoster virus (VZV) causes clinically mild chickenpox in children and then becomes latent in ganglionic neurons. In 1995, the live attenuated VZVOka vaccine, VARIVAX®, was FDA approved and licensed for routine childhood vaccination, in infants as early as 12months of age. VARIVAX® was shown to be highly immunogenic, very successful, with an efficacy rate of 90-95%, and extremely safe, even in some immunosuppressed patients, including HIV infected children with depressed CD4+ T cells. As such, this chickenpox virus is an attractive vaccine vector to express foreign antigens of other pathogens.
The simian varicella virus (SVV), closely related to VZV, causes a natural, varicella-like disease in nonhuman primates. We have developed a unique recombinant, experimental live-attenuated rSVV-SIV vaccine based on the SVV vector by inserting expressing SIV genes into the vector. Rhesus macaques immunized with this recombinant rSVV-SIV vaccine induced immune responses to SIV and reduced plasma SIV loads by as much as 100-fold following challenge with SIV by intravenous inoculation. The vaccine was shown to increase CD4+ proliferation, and SIV-specific polyfunctional CD4 and CD8 responses that were significantly associated with viral load reduction. Effector memory cells were suggested to be involved, similar to findings with another herpesvirus, CMV vector vaccine. These findings are significant as other studies have shown that similar reductions in SIV or HIV blood load may result in reduced disease transmission and progression.
Further studies are underway in my laboratory to ultimately establish a pediatric, live, attenuated recombinant varicella-AIDS vaccine protocol to vaccinate and produce virus specific immunity in infants and young children, at a time when their immune systems are extremely active and well before sexual maturity to protect them against mucosal HIV infection. We are currently conducting additional preclinical trials to further test the ability of this unique rSVV-SIV vaccine to be boosted with either SIV DNA or SIV protein and if, with extended rest prior to SIV challenge will 1) be safe, 2) promote maximum virus specific immune responses 3) provide protection against mucosal SIV challenge without increasing SIV susceptibility and 4) preexisting SVV immunity will not diminish the vaccine induced immune responses. We are currently testing how these Boost immunization strategies and mucosal SIV challenge in SVV naïve and SVV seropositive infant rhesus are affected by immunization route, virus specific immune responses, targeted epitopes, effects of preexisting SVV immunity, and protection against an SIV mucosal challenge. Such outcomes would hasten development of a counterpart rVZV-HIV vaccine, streamlined approvals and subsequent clinical human trials. A safe and effective pediatric rVZV-HIV vaccine would be a monumental AIDS prevention strategy, especially for children in areas of the world with endemic HIV infection.
Papillomavirus Infection and Progression to Cervical Cancer
Human papillomavirus (HPV) is one of the most common sexually transmitted infections and a significant cause of cervical, anal, and other cancers worldwide. Human immunodeficiency virus (HIV) positive men and women have a higher prevalence of HPV infections and HPV-associated disease and cancers than HIV negative individuals. Rhesus papillomavirus virus type 1 (RhPV-1), isolated from a metastatic rhesus penile cancer shares many genetic and phenotypic similarities to the highly carcinogenic human isolate HPV type 16.
Our laboratory conducted an infection study to characterize inoculum dose response with inoculation of Rhesus macaques each with increasing doses of RhPV1 as well as a control group inoculated with non-replicating RhPV1. Monthly evaluations were performed. After ten months, all 12 animals were intravenously inoculated with SIVmac239 and monitored clinically for an additional three months and RhPV1 and SIV viral loads were monitored for effects of SIV co-infection on pathology. Cervical infection of female rhesus macaques with RhPV1 resulted in 100% infection in all dose groups confirming the susceptibility of the rhesus macaque and utility of RhPV1 and RhPV1-SIV rhesus models for pathogenesis studies. Clinical evidence of RhPV1 virus-specific progressive cellular changes were observed for 33% of the high-dose RhPV1 inoculated animals. Animals receiving the mid and low dose RhPV1 inoculum were neither consistently infected nor showed progression to disease. Following SIV inoculation, viral loads and kinetics of co-infection were similar in all RhPV1 dose groups and showed losses of CD4 cells and critical immune functions by SIV infection. Progression of cervical cytological changes in the high-dose RhPV1 infected animals following SIV co-infection suggests that SIV immunodeficiency may contribute to RhPV1 progressive disease.
Respiratory Syncytial Virus Infection and Pathogenesis
Respiratory Syncytial virus (RSV) is a major cause of serious lower respiratory tract disease in infants and young children accounting for over 120,000 hospitalizations annually in the U.S. alone. No effective vaccine is available, and the current antiviral drug available has limited use. Severe disease, characterized by bronchiolitis or pneumonia, may be life-threatening, especially in infants between 6 weeks and 6 months of age. Hospitalized children with underlying heart and lung conditions are at the most risk for severe complications. Virtually all children are exposed to RSV by age 2. Immunity to RSV is not long-lasting and symptomatic re-infection is common at all ages. RSV is also a major cause of severe respiratory disease in older adults with over 78% of all RSV related deaths occurring in persons age 65 or older. Annually in the U.S., RSV is responsible for an estimated 180,000 hospitalizations and 11,000 – 17,000 deaths of older adults. Individuals in nursing homes, those with underlying respiratory and cardiopulmonary disease, cancer patients, and immunocompromised patients are at greatest risk for severe, even fatal, RSV infection. Even otherwise healthy elderly individuals are susceptible to debilitating RSV respiratory disease.
An effective RSV vaccine is currently unavailable, but is urgently needed since there is no effective antiviral agent against RSV. A formalin inactivated RSV vaccine given to children in the 1960's failed to induce protective immunity and actually led to an enhanced severe disease following natural exposure to RSV. The enhanced disease was associated with cell-mediated pulmonary inflammation and eosinophilia. Live, attenuated RSV vaccines have had limited success in human clinical trials and are hampered by safety concerns. Subunit vaccines, including those composed of purified RSV F and/or G, do not stimulate long-lasting immunity and may induce enhanced pulmonary disease after RSV challenge. A nonhuman primate model of RSV infection and disease has been developed in my laboratory and is being utilized to test various RSV vaccine candidates and therapeutics.