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Browsing Theses and Dissertations by Author "Akkina, Ramesh, advisor"

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    Evaluation of novel therapeutics for HIV prevention and treatment in a humanized mouse model
    (Colorado State University. Libraries, 2011) Neff, Charles Preston Tagg, author; Akkina, Ramesh, advisor; Aboellail, Tawfik, committee member; Callahan, Gerald, committee member; Chen, Chaoping, committee member; Garrity, Deborah, committee member
    In the absence of an effective HIV-1 vaccine finding alternative therapeutics and preventative methods has become essential. In this regard preventative approaches such as pre-exposure chemo-prophylaxis that employ either topical applied microbicides or systemically administered anti-retroviral drugs show great promise. In these studies, we evaluated two new classes of clinically approved drugs with different modes of action namely, an integrase inhibitor raltegravir and a CCR5 inhibitor maraviroc as potential systemically and topically applied pre-exposure chemo-prophylaxis. Additionally, therapeutic strategies designed to combat HIV/AIDS using siRNAs show considerable promise. However, targeted delivery of these synthetic molecules into infected cells in vivo has been a formidable challenge. In addressing this need we sought to evaluate the efficacy of a chimeric construct consisting of an HIV-1 gp120 specific aptamer with viral neutralization capacity fused to a siRNA with proven efficacy against tat/rev viral transcripts. We also sought to evaluate the efficacy of structurally flexible, cationic PAMAM dendrimers as a siRNA delivery system. For these novel therapeutic strategies to succeed it is important to evaluate them in both in vitro and in vivo. The rhesus macaque has been a valuable research tool for comparative HIV-1 studies. However, aspects of this model render its usefulness limited considering its expensive nature and not utilizing HIV-1 itself. In this regard the recently developed humanized mouse model that permits multi-lineage human hematopoiesis is an excellent alternative to the non human primate model. To generate humanized mice, neonatal Rag2-/-yc-/- or Rag1-/-yc-/- mice were xenografted with human CD34+ hematopoietic stem cells, resulting in a model that can permit HIV-1 infection. Upon infection by HIV-1 chronic viremia develops with a subsequent loss of CD4 T cells. These mice also successfully mimic the predominant mode of HIV-1 transmission via the sexual vaginal route which also results in chronic viremia and helper T cell loss. Thus this small animal model permits the rapid preclinical evaluation of potential candidates for pre-exposure prophylactic (PrEP) efficacy as well as novel RNA-based therapeutics. Here we utilize these humanized mouse models to evaluate the PrEP efficacies of the drugs named above as well as the in vivo efficacy of siRNAs delivered by utilizing a chimeric aptamer construct or a PAMAM dendrimer. Our results showed that both of these approaches using either a chimeric aptamer or a PAMAM dendrimer resulted in suppression of viral loads in vivo and most importantly also resulted in protection from T-cell depletion, making these compounds attractive therapeutic candidates for the treatment of HIV-1 infection. Lastly, using the same humanized mouse model we also successfully tested a gene therapy strategy employing lentiviral vectors having RNA-based anti-HIV-1 constructs convey intracellular immunization against HIV-1 in vivo.
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    HIV and Zika: modeling pathogenesis and therapies in humanized mice
    (Colorado State University. Libraries, 2017) Charlins, Paige, author; Akkina, Ramesh, advisor; Liber, Howard, committee member; Aboellail, Tawfik, committee member; Ryan, Elizabeth, committee member
    Investigation into pathogenesis of critical viral diseases that pose a global threat is important not only for pathogen modeling but also for treatment discovery. Use of humanized mice to study viral pathogens, including HIV-1 and Zika virus, provide a novel insight into the mechanisms of viral persistence and act as a platform for therapeutic intervention and observation. In the context of HIV-1 research, humanized mice are ideal for parroting the infection mechanics. Recapitulation of HIV-1 viral pathogenesis enables the study of various facets of the virus lifecycle, concerning infection establishment, detection, and treatment. In this respect, humanized mouse modeling of HIV-1 infection is paramount for continued studies of HIV-1 in terms of HIV-1 latency, with focus on viral reactivation and outgrowth, and for research with novel therapeutic approaches for treatment and modulation of the infection. The model has been paramount in development of an ultra-sensitive assay for detection of latent HIV-1 and validation of aptamer-siRNA conjugates and conditionally replicating vectors for treatment of HIV-1 infection. Humanized mice can further be expanded for the study of additional emerging pathogens, such as Zika virus, in terms of infection pathology and immune response. Infection of humanized mice with a Puerto Rican strain of Zika virus resulted in high plasma viral loads detectable up to four months post inoculation with wide spread dissemination detected in various tissues by IHC and CLARITY. Understanding these different elements of various viral infections with the ability to establish innovative perspective into mechanics and therapeutics, contribute a previously inaccessible view of human specific viral pathogenesis and treatment.
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    Modeling the evolution of SIV into HIV using humanized mice
    (Colorado State University. Libraries, 2016) Boddeda, Srinivasa Rao, author; Akkina, Ramesh, advisor; Laybourn, Paul, committee member; Juarrero, Mercedes Gonzalez, committee member; Ryan, Elizabeth, committee member
    Acquired Immunodeficiency syndrome (AIDS) is caused by two lentiviruses belonging to the family Retroviridae, namely HIV-1 and HIV-2, which originated from SIVcpz and SIVsmm respectively. Multiple independent cross-species transmission events of SIV from chimpanzees (SIVcpzPtt ) and gorillas (SIVgor) have given rise to four groups of HIV-1 (M, N, O, P), while transmission from sooty mangabeys (SIVsmm) is responsible for at least 8 groups of HIV-2 (A-H) (Ayouba et al., 2013; Gao et al., 1999; Hirsch et al., 1989). Some of these groups are extremely rare. However, four have established themselves in human populations as pandemic (HIV-1 group M) or epidemic (HIV-1 group O, HIV-2 groups A and B) outbreaks (Faria et al., 2014). While these data suggest that SIV transmission to a human host is by itself not sufficient to establish a new epidemic outbreak, it also implies that viral adaption is necessary for efficient spread of the virus within humans (Marx et al., 2004). However, exact role of factors such as immune selective pressure and genomic changes, contributed to successful SIV adaptation in humans remains unclear. Study of SIV transmission to humans has been limited by lack of an appropriate model. Ethical constraints prevent experimental challenge of human subjects with SIV and transmission studies have thus far been limited to non-human primates (NHPs) (Chahroudi et al., 2012). While NHP studies have been instrumental to identify key differences in innate and adaptive immune responses that influence transmissibility and virulence of SIV between species, until now experimental in vivo challenge of a functional human immune system with SIV has not been possible. Recently however, a new generation of humanized Rag2−/−γc−/− mice has been established that supports systemic engraftment of a functional human immune system (Akkina, 2013; Denton et al., 2012). These animals are susceptible to HIV-1 infection by mucosal routes and display key features of pathogenic HIV-1 infection in humans, including sustained plasma viremia, CD4+ T cell depletion and increased levels of immune exhaustion markers (Berges et al., 2006; Palmer et al., 2013). Additionally, humanized mice are able to mount adaptive immune responses to HIV-1 infection, producing antibodies and HIV-1 specific T cell responses. In the current study, we used humanized Rag2−/−γc−/− mice to study SIV transmission to humans. We hypothesized that humanized mice could support SIV infection, and that the presence of in vivo selective pressures, either innate or adaptive, would drive viral evolution. That is, we expected the majority consensus virus emerging in the plasma of productively infected humanized mice different from the stock virus used to infect the animals. Viral adaptation within infected humanized mice could produce a variant virus more fit for growth and transmission in humans. We infected humanized mice with both SIVsmmE041 and SIVcpzLB715 isolates and measured plasma viral loads and determined complete viral genome sequences. Both SIVsmm and SIVcpz showed stable viral loads over time for up to 7 months. SIVsmm virus from the infected mice was successfully passaged to three successive generations of humanized Rag2−/−γc−/− mice via intraperitoneal route. We were also able to show that human cell-adapted SIVsmm can infect humanized mice through the mucosal route. Sequence analyses of SIVsmm and SIVcpz output virus from these mice showed many functional mutations in various genes such as Gag, Pol, Env, Vif, Vpx, Vpr and Nef regions. In both viruses, Env regions showed the highest number of mutations suggesting that the envelope region of this virus might be under selective pressure for this virus to be able to replicate in human cells. We observed that the number of mutations in all genes increased over time, suggesting that the SIVsmm virus is continuously evolving and adapting for successful replication and transmission in these humanized mice. These studies will provide a flexible in vivo model for elucidating the mechanisms underlying SIV transmission and gain of function to replicate in humans.
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    Modeling the evolution of SIV progenitor viruses towards HIV-1 and HIV-2 in a humanized mouse surrogate model
    (Colorado State University. Libraries, 2020) Curlin, James Zachary, author; Akkina, Ramesh, advisor; Aboellail, Tawfik, committee member; Stenglein, Mark, committee member; Wiese, Claudia, committee member
    Human Immunodeficiency Virus Type 1 (HIV-1) and Type 2 (HIV-2), the causative agents of Acquired Immunodeficiency Syndrome (AIDS) first emerged in humans over the past century. Despite significant advances in treatment options, the pandemics continue with millions of new infections every year. Both HIV-1 and HIV-2 likely emerged through the cross-species transmission of primate lentiviruses originating from nonhuman primates (NHPs) including chimpanzees (SIVcpz), gorillas (SIVgor), and sooty mangabeys (SIVsm). SIVsm shares a remarkable degree of homology with HIV-2, while SIVcpz and SIVgor are most closely related to HIV-1. Nonhuman primates infected with these lentiviruses frequently come into contact with humans due to the prevalence of bushmeat hunting practices in various African countries. Other lentiviruses such as SIVmac239 represent independent instances of primate lentiviruses crossing into novel host species. The repeated exposure of primate lentiviruses to a human immune environment allowed the accumulation of adaptive genetic changes uniquely suited to overcoming the evolutionary pressures of a new host. Host-restriction factors such as tetherin, SAMHD1, APOBEC3G and SERINC3/5 exert species-specific antiviral activity and must be overcome for a virus to adapt to a new host cell. These evolutionary pressures could be a guiding force in the direction that these viruses adapt. In order to recapitulate these genomic cross-species adaptations, we used humanized mice engrafted with human hematopoietic stem cells (hu-HSC mice). These mice produce a full spectrum of human immune cells such as B cells, T cells, macrophages, monocytes, and dendritic cells, and are susceptible to HIV infection. Representative progenitor viruses of both HIV-1 (SIVcpzEK505, SIVcpzMB897, and SIVcpzLB715) and HIV-2 (SIVsmE041) as well as other viruses of interest, namely, SIVmac239, SIVhu and SIVB670 lineages were intraperitoneally injected into hu-HSC mice. Following successful infections, the derivative viruses were subsequently passaged serially through multiple generations to simulate the repeated exposures that originally produced HIV-1 and HIV-2. Viral adaptation was assessed primarily through three different criteria. Plasma viral RNA levels were measured on a weekly basis using qRT-PCR to determine changes in viral replication kinetics over time. We found that the plasma viral loads of the viruses tested varied during serial passages, and mostly increased over time in many cases. Human CD4+ T cell engraftment decline as assessed by flow cytometry biweekly acts as a measure of AIDS progression in cases of human infection. CD4+ T cell levels declined over time with increasing rapidity upon further passaging in many cases. Additionally, viral RNA collected from the infected mice at multiple timepoints in each generation was used to generate overlapping amplicons spanning the length of the viral genome in order to be used with Illumina-based deep sequencing. Numerous nonsynonymous mutations arose in the first generation of passaging and were maintained across multiple sequential passages. While the mutations occurred throughout the viral genome, the bulk of the mutations were found in env and nef. Many of these mutations were present in known CD4+ binding sites, motifs involved in protein interactions, and other areas involved in host-restriction factor antagonism. While these results are revealing, further inquiry is needed to determine the true functionality of these genetic changes. These data showcase the value of using humanized mice to model lentiviral evolution and provide important insights into understanding the origin of HIVs.
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    Pharmacokinetic and pharmacodynamic evaluation of HIV-1 pre-exposure prophylaxis candidates in humanized mice
    (Colorado State University. Libraries, 2014) Veselinovic, Milena, author; Akkina, Ramesh, advisor; Aboellail, Tawfik, committee member; Gonzalez-Juarrero, Mercedes, committee member; Peersen, Olve, committee member
    In the absence of a vaccine, alternative preventative approaches against HIV-1 are needed. In pre-exposure prophylaxis (PrEP) approach, antiretroviral drugs, broadly neutralizing antibodies (bnAb) or other biological molecules are administered orally or topically for the prevention of HIV-1 infection. For successful PrEP design pharmacokinetic (PK) and pharmacodynamic (PD) studies are needed to define protective levels of antiretrovirals in mucosal tissues. The RAG-hu mice used here represent a small animal model in which human immune system is reconstituted by haematopoietic stem cells (HSC) in the immunodefficient BALB/c- Rag1-/- γc-/- and BALB/c- Rag2-/- γc-/- mice. This model was previously shown to be suitable for HIV-1 mucosal transmission and protection studies. In the experiments presented here we evaluated the utility of RAG-hu mice for the study of PK-PD aspects of antiretroviral drugs in the context of PrEP. The PK studies focused on tissue distribution of the RT inhibitor Tenofovir (TFV), the integrase inhibitor Raltegravir (RAL) and the entry inhibitor Maraviroc (MVC) following single and combinatorial oral application. Drug kinetics were examined systemically in blood plasma, and in vaginal, rectal and colonic mucosal tissues, which are the sites of HIV-1 transmission and initial viral spread. Antiretrovirals were applied in human equivalent doses to achieve steady state kinetics. Data obtained from single oral applications verified favorable PrEP profile of TFV. While results showed that RAL and MVC represent promising PrEP candidates, the data suggest that the PrEP doses would need to be higher than therapeutic ones in order to allow for once a day dosing. In combinatorial TFV/RAL and TFV/MVC oral application studies, increase in the active form of TFV (Tenofovir diphosphate, TFV-DP) accompanied by agonistic effect for the second drug in combination was observed, which can be characterized as highly favorable for PrEP applications. This is the first report on combinatorial PK of TFV, RAL and MVC in mucosal tissues which informs further testing of TFV/MVC and TFV/RAL PrEP approaches in non-human primates (NHP) and in clinical settings. For topical PrEP potential, PK profiles of TFV, RAL and MVC were also evaluated in vaginal mucosa following topical application of gel formulations. With all three drugs, one to two log higher concentrations were achieved in vaginal mucosa compared to oral application reflecting previous findings in humans. Intracellular concentrations of TFV-DP in humanized mice corresponded to the levels observed previously in human vaginal mucosa. In PD studies, the protective effect of topical PrEP with single drug and combinatorial TFV, RAL and the RT inhibitor UC781 gels was evaluated against mucosal HIV-1 transmission. High level of protection was seen with combinatorial microbicide gels - 80% (4/5) protection by TFV/UC781 gel and 87.5% (7/8) protection by TFV/RAL gel, indicating their suitability for further testing in preclinical trials. In another PD study, protective efficacy of bnAb VRC01 was examined against mucosal transmission of HIV-1 in the form of topical PrEP. The VRC01 gel (1 mg/ml) conferred protection in 77.7% (7/9) animals, while the combination of b12, 4E10, 2F5 and 2G12 bnAb which target different epitopes on the HIV-1 envelope conferred complete protection (5/5 animals) against HIV-1 mucosal transmission. These data suggest that bnAb could be effective agents for topical PrEP against HIV-1. In summary, these proof of concept PK and PD studies validated RAG-hu mouse model for preclinical evaluation of new anti-HIV-1 drugs and bnAb for oral and topical PrEP, thus providing data for further NHP studies and human clinical trials.
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    Pseudotyping of lentiviral vector with novel vesiculovirus envelope glycoproteins derived from Chandipura and Piry viruses
    (Colorado State University. Libraries, 2016) Hu, Shuang, author; Akkina, Ramesh, advisor; Quackenbush, Sandra, committee member; Aboellail, Tawfik, committee member; Ryan, Elizabeth, committee member
    Lentiviral vector system is widely used in gene therapy. Although the envelope glycoprotein of vesicular stomatitis virus (VSV-G) has been mostly used to pseudotype lentiviral vectors, its disadvantages such as low transduction levels in certain cell types and sensitivity to inactivation by human complement hinders the usage of VSV-G pseudotyped lentiviral vectors in some cells or its direct in vivo clinical application. Aiming at overcoming some of these drawbacks of VSV-G, we evaluated two novel vesiculovirus envelope glycoproteins from Chandipura virus and Piry virus (CNV-G and PRV-G), as alternatives to VSV-G. Our results showed that pseudotyped lentiviral vectors could be generated with both these envelopes with high titers and stabilities similar to VSV-G. While displaying a more selective tropism than VSV-G, both CNV-G and PRV-G pseudotypes were found to be efficient in transducing a variety of cell types that include neuronal, fibroblastic and epithelial cells from across different species in addition to a number of human T-lymphocyte cell lines in vitro. Additionally, both the novel pseudotypes were found to be more resistant to human sera inactivation than the VSV-G pseudotype, thus providing better candidates for systemic administration. These data, taken together, establish that both Chandipura and Piry viral glycoproteins are suitable alternative candidates for lentiviral vector pseudotyping with an additional advantage for potential in vivo use in various gene therapy-based applications.