Theses and Dissertations

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Open Access
HIV prophylaxis: an essential role for T cells and adjuvants in recombinant mucosal Lactobacillus acidophilus vaccines
(Colorado State University. Libraries, 2016) LeCureux, Jonathan Spicer, author; Dean, Gregg, advisor; Aboellail, Tawfik, committee member; Chen, Chaoping, committee member; Zabel, Mark, committee member
Current HIV vaccines have poor efficacy, with inconsistent levels of protection following mucosal HIV exposure. Lactic acid bacteria offer an alternative vaccine vector targeting the primary site of HIV infection, the mucosa. In these studies we evaluated the immunogenicity of several strains of Lactobacillus acidophilus expressing HIV membrane proximal external region (MPER), a portion of HIV envelope that contains broadly neutralizing antibody binding sites. We evaluated MPER-only expressing strains along with strains expressing adjuvants (interleukin-1β or flagellin) to improve immunogenicity against the HIV MPER. We compared the adjuvant strains to the MPER-only strain by oral administration in BALB/c mice to observe these improvements, and in CD40L-/- mice to observe if T cell help was necessary. Some BALB/c animals were also placed on a modified diet supplemented with prebiotic rice bran to observe any influence on vaccine immunogenicity. Resulting antibody responses and interleukin-17 levels were measured by ELISA, and T and B cell levels were measured by flow cytometry. Here we show that the addition of adjuvants, including dietary rice bran, to L. acidophilus vaccine strains improves their immunogenicity against HIV MPER. Our results indicate that anti-MPER IgG and IgA levels, as well as the number of anti-MPER antibody secreting cells, are improved with adjuvants, and that T cell help is required for an effective immune response. These results, combined with the many advantages offered by this lactic acid bacteria vaccine system make L. acidophilus an attractive vaccine vector for primate and human trials.
Open Access
Modeling human immunodeficiency virus-1 (HIV-1) infection in the male reproductive tract (MRT) using humanized mice
(Colorado State University. Libraries, 2016) Fisher, Suhrim, author; Akkina, Ramesh, advisor; Aboellail, Tawfik, committee member; Dean, Gregg, committee member; Kendall, Lon, committee member; Weil, Michael, committee member
Thirty-five million people are currently living with human immunodeficiency virus (HIV) globally. While 9.7 million infected people are receiving antiretroviral therapy, 2.3 million people are newly infected every year. Transmission via semen is one of the most prevalent methods of HIV-1 transmission, accounting for up to 80% of new infections every year. However, the source of infected leukocytes and the method of virus replication in semen and/or in the male reproductive tract (MRT) is not well described. It has been shown that infected germinal cells such as spermatogonia and spermatocytes as well as infected leukocytes are present in the MRT of HIV infected human patients and simian immunodeficiency virus (SIV) infected macaque models. Studies have also shown that the seminal viral strain differs from the serum viral strains in HIV infected human patients. Furthermore, HIV-1 continues to persist in the semen and in the MRT of infected male patients on highly active antiretroviral therapy (HAART) despite undetectable serum viral load. To investigate the spread and replication of HIV-1 in the MRT in human patients, development of an animal model is essential. Use of humanized mice allows researchers to study the transmission, pathogenesis and drug interactions of HIV-1 in the human immune system in vivo. The human-hematopoietic stem cell (hu-HSC) mouse model created by transplantation of HSC into Rag2−/−γc−/− mice, has shown excellent susceptibility to HIV-1 and ability to sustain high viremia for months. However, so far no studies have been conducted to demonstrate whether or not HIV-1 infiltrates and replicates in the semen or MRT of infected humanized mice. The presence of HIV-1 in the semen or MRT of infected humanized mice could play a vital role in using humanized mice for describing the pathogenesis mechanism of the virus in semen and drug development in reducing seminal HIV-1 viral load in human patients. The aim of this study is to model HIV-1 infection in the MRT using humanized mice to 1) evaluate the severity of disease using histologic scoring of MRT and morphometric analysis of the testis and epididymis in viremic and negative control mice, 2) characterize leukocytes in the testis and epididymis of viremic and negative control mice via immunohistochemistry, 3) assess the presence of HIV-1 viral RNA in the MRT of viremic mice via in situ hybridization (HIV-1 mRNA), and 4) assess seminal viral loads from vaginal plugs of viremic mice via qRT-PCR. We showed that both viremic (16/25; 64%) and negative control (9/28; 32%) mice developed histopathologic lesions in their testis and epididymis parallel to the lesions found in the testes and epididymis of chronically infected HIV-1 and AIDS patients. When broken down by two age groups, 5-7 month old and 8-12 month old, the viremic mice from both age groups showed significantly higher frequency of testicular oligospermia/azoospermia (p<0.05) compared to the age-matched negative control groups. Five to seven month old viremic mouse group showed significantly higher frequency of testicular germinal cell degeneration (p<0.05) compared to the age-matched negative control group. The histopathological lesion scores were significantly higher in both 5-7 month old and 8-12 month old viremic mouse group compared to that of age-matched negative control mouse groups (p<0.05), suggesting the lesions in viremic mice are related to HIV-1 infection. Morphometric analysis of the testicular and epididymal epithelium however, did not show any statistical differences on the levels of germinal cell degeneration between the viremic (n=8) and negative control (n=4) group. We were able to identify the presence of CD4 positive cells in viremic (4/22; 22%) and negative control (7/17; 41%) mice in the testicular and epididymal interstitium. The majority of the CD4 cells were associated with histopathologic lesions within primary reproductive organs. RNA in situ hybridization showed successful infiltration of HIV-1 infected cells in the MRT of viremic mice (9/11; 82%), most often found in penis or prepuce (6/11; 55%), and less often in seminal vesicles (4/11; 9%), testes (3/11; 27%), epididymides (1/11; 9%) and prostate glands (1/11; 9%), suggesting that HIV-1 indeed can infect and infiltrate the MRT via CD4 positive cells. Vaginal plugs failed to show detectable HIV-1 viral loads using qRT-PCR. Our study offers first glance at HIV-1 infection in the MRT of viremic humanized mice and provides further evidence that leukocytes play an important role in HIV-1 infection in the MRT.
Open Access
The role of domestic ducks in the maintenance and spread of avian influenza viruses in Indonesia
(Colorado State University. Libraries, 2012) Pabilonia, Kristy L., author; Bowen, Richard, advisor; Aboellail, Tawfik, committee member; Hyatt, Doreene, committee member; Knight, Anthony, committee member
Wild waterfowl and aquatic birds serve as the natural reservoir host for influenza A viruses. As the reservoir, wild waterfowl play an important role in the persistence and transmission of influenza viruses among bird populations and to other mammalian species. In many Asian countries, domestic ducks are raised for meat and egg production. Some of these domestic ducks are ranged on rice paddies or post-harvest rice fields. The ducks provide service to the rice fields by fertilizing the field with feces and aerating the field by swimming and walking through the ground cover. Additionally, the ducks serve as a form of insect control through their natural grazing behaviors. The role that domestic ducks play in the ecology of influenza viruses is poorly understood. Highly pathogenic avian influenza H5N1 virus (HPAI H5N1) originated in Guangdong Province, China in 1996, which was followed by global dissemination of the virus that began in 2003. This virus is unprecedented in geographical spread, economic consequences and public health significance. At the present time, HPAI H5N1 virus is endemic six countries, including Indonesia. Indonesia has experienced the highest incidence of human infections with HPAI H5N1 virus and one of the highest case fatality rates. Control of the virus in Indonesia has proven extremely challenging, due to its diverse and complex poultry and domestic duck production systems. HPAI H5N1 virus is highly virulent in chickens and turkeys and causes severe systemic disease. Outbreaks of HPAI H5N1 in poultry populations are accompanied by high mortality. In contrast, HPAI H5N1 virus is typically nonpathogenic or mildly pathogenic in ducks and mortality in duck flocks during outbreaks of the virus is absent or limited. This allows ducks to serve as silent carriers of the virus, as they may shed large quantities of virus without displaying clinical signs of illness allowing infected ducks to evade detection by flock owners or government livestock officials. Domestic duck production is common in Southeast Asia. Indonesia has a large domestic duck population, estimated at more than 34 million ducks. Because HPAI H5N1 induces only mild disease in domestic ducks, outbreaks of the virus are difficult to detect and are rarely reported by domestic duck flock owners. Thus, domestic duck flocks have been left out of many government HPAI H5N1 surveillance and control programs. While a number of studies have demonstrated that the presence of domestic ducks in a country or at a specific location may be a risk factor for the presence of HPAI H5N1 virus, few studies have been conducted evaluating the role that domestic ducks play in the ecology of HPAI H5N1 virus. The objectives of the studies described in this dissertation were to elucidate the role of domestic ducks in the maintenance and spread of avian influenza viruses, particularly HPAI H5N1 virus, by evaluating domestic duck flock characteristics and behaviors, estimating the prevalence and incidence of avian influenza viruses in these flocks and characterizing HPAI H5N1 viruses detected in the field. To meet the objectives, two studies were conducted in West Java, Indonesia. The first study was a cross-sectional study aimed at characterizing domestic duck flocks and estimating the point prevalence and seroprevalence of avian influenza viruses, particularly HPAI H5N1 virus. This study was followed by a 7 month longitudinal study, aimed at estimating the incidence of avian influenza viruses, particularly HPAI H5N1 virus, in domestic duck flocks and evaluating flock illness and mortality during avian influenza virus outbreaks. A subset of samples from each of the studies was transported to the United States for virus characterization. The findings of the studies conducted demonstrate that domestic duck flocks are raised in complex production systems, are highly mobile, have significant contact with wild and domestic birds and mammals, are frequently ill and are provided with little formal veterinary care. The prevalence and incidence of avian influenza virus, including HPAI H5N1 virus, are high in domestic duck flocks in Indonesia. Clinical signs of illness and increased mortality did not correlate with the presence of avian influenza virus in the flock. Interestingly, there was also no correlation between increased flock mortality and the presence of HPAI H5N1 virus in the flock, demonstrating that domestic duck flocks can be asymptomatically infected with HPAI H5N1 virus while shedding high quantities of virus. Characterization of some of the viruses isolated from domestic duck flocks demonstrated that the flocks can be infected with more than one avian influenza virus at one time, as demonstrated by one flock that was positive for HPAI H5N1, as well as H3 and H7 avian influenza viruses. These situations are concerning, as domestic duck flocks may serve as mixing vessels for avian influenza viruses and co-infections in these flocks may result in the emergence of novel influenza viruses that may have capabilities for human-to-human transmission. It is likely that domestic ducks play an important role in the maintenance and spread of avian influenza viruses, including HPAI H5N1 virus. A number of domestic duck flock practices, including extensive flock movement, frequent introduction and sale of ducks, free-ranging of ducks in areas where they have contact with wild birds and animals and continual contact of duck flocks with other duck and poultry flocks, increasingly adds to the difficulty of control of HPAI H5N1 virus within this production system and makes eradication of the virus within a country extremely challenging.
Open Access
Western equine encephalitis virus: neuroinvasion, pathogenesis, and immunomodulatory treatment strategies
(Colorado State University. Libraries, 2013) Phillips, Aaron Timothy, author; Olson, Kenneth, advisor; Brennan, Carol Blair, committee member; Aboellail, Tawfik, committee member; Tjalkens, Ronald, committee member
Western equine encephalitis virus (WEEV; Alphavirus) is a mosquito-borne virus that can cause severe encephalitis in humans and equids. WEEV is closely-related to eastern equine encephalitis virus (EEEV) and may model similar pathogenesis in a mouse model. Previous studies have shown that intranasal infection of outbred CD-1 mice with the WEEV McMillan (McM) strain result in high mortality within 4 days of infection, thus providing a model of exposure to airborne encephalitic alphavirus. In addition, WEEV McM causes high mortality within 5-7 days following peripheral inoculation of mice. Therefore, WEEV McM may be used to model infection following exposure to infected mosquitos. The route of WEEV entry into the central nervous system (CNS) is not well-understood. In the studies presented here, bioluminescence (BLM) imaging and recombinant reporter viruses based on WEEV McM were applied to detect and track virus in mice following intranasal or subcutaneous inoculation, and used to determine correlation between BLM and viral titer. Additionally, histopathology analysis was guided by corresponding BLM images and used to identify specific CNS regions affected during infection. The major findings from these studies indicate that WEEV McM uses a different route for entry into the CNS for each of the two inoculation methods (intranasal or footpad). Intranasal challenge resulted in neuroinvasion occurring primarily through cranial nerves, mainly in the olfactory tract. Olfactory bulb neurons were initially infected followed by spread of the infection into different regions of the brain. WEEV distribution was confirmed by immunohistochemistry as having marked neuronal infection but very few infected non-neuronal glial cells. Axons displayed infection patterns consistent with viral dissemination along the neuronal axis. The trigeminal nerve served as an additional route of neuroinvasion showing significant FLUC expression within the brainstem. Neuroinvasion from footpad inoculation demonstrated a consistent pattern in the spatiotemporal distribution of virus among the imaged brains, none of which involved the olfactory bulb. These studies support the hypothesis that neuroinvasion of WEEV likely occurs in areas of the CNS where the blood-brain barrier is naturally absent. These areas include the median eminence of hypothalamus (hypothalamic output), posterior pituitary, pineal body, and the area postrema. There are no antiviral therapies against alphaviral disease and current vaccine strategies target only a single alphavirus species. In an effort to develop new tools for a broader response to outbreaks, a novel alphavirus vaccine comprised of cationic-lipid-nucleic acid complexes (CLNCs) and the ectodomain of WEEV E1 protein (E1ecto) was designed and tested. Interestingly, the CLNC component alone had therapeutic efficacy as it increased survival of CD-1 mice following lethal WEEV infection. Immunization with the CLNC-WEEV E1ecto mixture (lipid-antigen-nucleic acid complexes; LANACs) using a prime/boost regimen provided strong protection in mice challenged with WEEV subcutaneously, intranasally, or via mosquito. In addition, the LANAC immunization protocol significantly increased survival of mice following intranasal or subcutaneous challenge with EEEV, indicating potential as a 'pan-alphavirus' vaccine candidate. Mice immunized with LANAC mounted a strong humoral immune response, but did not produce neutralizing antibodies.

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Browsing Theses and Dissertations by Author "Aboellail, Tawfik, committee member"

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