With improved survival cardiovascular disease (CVD) has become an increasingly important cause of morbidity and mortality among people with treated HIV-1 infection. HIV-1 infection is characterized by a chronic state of immune activation that is an independent predictor of disease progression. Residual immune activation in treated HIV-1 may be contributing to atherosclerosis but there is limited evidence supporting this hypothesis. The mechanisms that connect inflammation, CVD and activation of the immune system in HIV infection remain to be elucidated. Oxidized lipids may be a significant mediator in the interplay between inflammation, immune activation and atherosclerosis. While normal High Density Lipoprotein (HDL) binds oxidized lipids and protects against CVD, HDL can become oxidized and dysfunctional in the setting of chronic inflammatory diseases. The primary goal of this dissertation is to better define the role of dysfunctional HDL in immune activation and atherosclerosis associated with HIV-1 infection.

This was addressed using a variety of approaches. First we showed that immune activation may be a contributing factor to progression of atherosclerosis in HIV-1 infection. Using a cell based assay of HDL function we found that HIV-1 infected subjects have dysfunctional HDL. We then developed novel cell free biochemical assays that measure redox properties of HDL as tools to demonstrate that HIV-1 infected subjects have dysfunctional HDL that is independently associated with biomarkers of macrophage and T cell activation, and predictors of disease progression. We then investigated the direct in vitro effects of dysfunctional (oxidized) HDL on T cells, which are important for immune activation, HIV-1 immunopathogenesis and atherogenesis. We found that modified dysfunctional HDL directly upregulated markers of T cell activation in vitro, increased T cell proliferation and production of cytokines, specific killing of HIV-1 specific cytotoxic CD8+ T cells (CTLs) and expression of chemokine receptors (CXCR4, CCR5) on CD4+ T cells important for HIV-1 infectivity, whereas it impaired the ability of HIV-1 specific CTLs to suppress HIV-1 replication. Together these results suggest that dysfunctional HDL has a major role in immune activation and atherosclerosis associated with HIV-1 infection.

CD8+ Cytotoxic T-Lymphocytes (CTLs) are critical for control of viremia during Human Immunodeficiency Virus-1 (HIV-1) infection. While CTLs ultimately fail to fully suppress viral replication in most individuals, a small subset of infected persons can effectively control viremia for several decades. Understanding the factors that allow CTLs to mediate this control of viremia can provide a mechanistic basis for the failure of the CTL response in most individuals and help in the development of future HIV-1 vaccines.

Certain Major Histocompatibility-I (MHC-I) types are associated with better containment of viremia, but the mechanisms mediating control have been difficult to elucidate. In this dissertation, I construct HIV-1 libraries with saturation mutagenesis at several commonly targeted epitopes and utilize these libraries to demonstrate that epitopes presented by protective MHC-I types are highly constrained in their fitness landscapes and abilities to escape CTL clones targeting the epitope. I then demonstrate that an epitope presented by a non-protective HLA type has multiple fit variants, allowing for mutational escape from CTL recognition with little or no loss in replicative capacity.

Next, I examine the CTL response induced by the Mrk/Ad5 HIV vaccine to understand how mutational escape may have contributed to the vaccine’s failure. I demonstrate that the vaccine may have generated CTLs that were less efficient at cross recognizing fit epitope variants, and thus allowed for rapid mutational escape from CTL recognition, likely contributing to the failure of this vaccine trial.

In the final chapter of this dissertation, I focus on the HIV-1 Nef protein. Nef downregulates the expression of MHC-I on the surface of infected cells and represents another mechanism through which the virus can escape CTL recognition. I conduct a high throughput screen of an HIV-1 library containing thousands of point mutations throughout nef to identify amino acid residues essential for Nef’s ability to downregulate MHC-I and demonstrate that these residues are highly conserved in circulating virus isolates.

These the results support a mechanism wherein protective MHC-I types elicit superior CTL responses through immunodominance of highly fitness-constrained epitopes and underscore the importance of escape pathways for successful vaccines and immunotherapies based on CTLs.

CD8+ cytotoxic T lymphocytes (CTL) play an important role in HIV-1 infection. However, in most persons the HIV-1-specific CTL become dysfunctional. It has been suggested that anergy, a mechanism of T cell tolerance, may play a role in HIV-1-specific CTL dysfunction. Since HIV-1 is known to escape CTL recognition through mutation of epitopes to produce weak agonist altered peptide ligands (APL)—and anergy is thought to occur through suboptimal recognition—we hypothesized that weakly-recognized epitope variants may suboptimally stimulate the T-cell receptor (TCR) and trigger aberrant TCR signaling, leading to CTL anergy and enhanced viral persistence. We investigated weak agonist APL-mediated anergy as a potential mechanism of dysfunction in three HIV-1-specific CTL clones targeting three different epitopes. Clones were exposed to weakly-recognized epitope variants and screened for cytokine secretion, gene expression of markers of anergy and other forms of T cell dysfunction, and subsequent capacity to kill index peptide-presenting target cells in secondary challenge. Under those conditions, although suboptimal stimulation dampened cytokine secretion, we observed no patterns in gene expression profile consistent with anergy. Additionally, pre-exposure of CTL to weak agonist epitope variants had no discernable effect on their subsequent capacity to kill index epitope-bearing target cells. These data suggest that while weak agonist epitope variants evade CTL pressure through reduced recognition, their weak agonism does not appear to drive dysfunction of HIV-1-specific CTL, arguing against a role of APL-induced anergy in HIV-1-specific CTL dysfunction. However, under different experimental conditions, one HIV-1-specific CTL clone appeared to demonstrate varying degrees of reduced recognition of index epitope after pre-treatment with weak agonist APL stimulation. To try to reconcile these seemingly contradictory results, additional literature on the role of costimulation in both anergy and CD28- effector CTL is incorporated into the discussion. In Chapter 7, I propose a speculative model of how HIV-1-derived weak agonist APL, in conjunction with blockade of CD28-independent costimulatory pathways, may be able to cause HIV-1-specific CTL anergy in vivo. In Chapter 8, alternative approaches are suggested for possible future studies to resolve the confounding issues that still surround the field of APL-induced T cell anergy.

It is well established that CD8+ cytotoxic T lymphocytes (CTL) are key players in the protective immune response against Human Immunodeficiency Virus Type 1 (HIV-1).

However, there are clear differences in the effectiveness with which particular CTL

control viral infection. Understanding the factors that contribute to the individual

differences in CTL antiviral efficiency will have great implications for vaccine design, as

such information will provide important insights on the mechanism of efficacious antiviral

activity against HIV-1.

One key factor that affects the CTL-mediated antiviral efficiency is the cell-surface

downregulation of human leukocyte antigen class I (HLA-I) mediated by the HIV-1 Nef

protein. While the degree of Nef interference on CTL antiviral efficiency has been shown

to be epitope specific, the factors that determine the susceptibility of CTL to Nef are not

clear. Previous work has indicated that, the timing of epitope expression, functional

avidity, and protein targeting as well as HLA-I restriction can have varying degrees of

influence on how well a CTL can recognize infected cells and suppress HIV-1

replication. The primary goal of this dissertation is to better define the role these factors

play in influencing CTL antiviral activity.

This was addressed using a variety of viral constructs, and HIV-1 specific CTL clones.

In Chapter Three, I first examine CTL susceptibility to Nef-mediated HLA-I

downregulation using a previously described viral suppression assay, and identify

epitope properties that determine susceptibility to Nef. Then in Chapter Four, I focus on

the role of protein targeting in CTL antiviral activity. I specifically compare the role of

Gag versus Env targeting in viral suppression efficiency, using viral constructs where a

Gag epitope is translocated to the Env protein.

The data presented in this dissertation show that the kinetics of epitope presentation are

an important determinant of CTL antiviral activity. Individual epitope presentation

kinetics vary independently of protein, and the earlier the epitope is presented on the

cell surface before Nef-mediated HLA-I downregulation, the more effectively CTL can

eliminate virus-infected cells and suppress viral replication. Factors including protein

properties, HLA-I restriction of the epitope, and functional avidity, are poor predictive

properties of Nef susceptibility and CTL antiviral efficiency. Together these results

suggest that the properties of individual epitopes, such as epitope expression kinetics,

most strongly influence CTL antiviral activity. Lastly, future research could potentially

focus on the role of host factors, such as T cell receptor functions, in determining CTL

antiviral activity against HIV-1. A few proposed ideas are summarized in Chapter Five.

HIV-1 transmission is typically associated with the genetic bottleneck in which a single viral isolate, known as the transmitted/founder (T/F) virus, establishes successful infection in a new host. Current understanding of T/F virus properties is mostly limited to studies of envelope and very little is known about other post-entry viral properties. Here, T/F viruses from 3 successive generations of mother-to-child transmission (MTCT) were characterized. Nef mediated downregulation of human leukocyte antigen (HLA) class I and CD4, and Gag-Pol associated replication capacity appeared normal. Our results revealed no significant differences in these functions. The entry efficiency of T/F viruses and the maternal source were also similar however, the Envs of two maternal subsets varied in CD4 and CCR5 requirements for viral entry. Further understanding of whether there are unique functional characteristics of T/F viruses may perhaps provide an opportunity to design a vaccine or other intervention to prevent HIV-1 transmission.

Cytotoxic T cells (CTL) play a central role in providing protective immunity in humans by eliminating infected cells and establishing immunologic memory against re-infection. The study of modern immunobiology has made many significant discoveries into the mechanisms involved in the CTL response against foreign allogeneic cells (alloresponse) and infecting pathogens. Using technologies refined in our laboratory for the study of genetic diversity in T cell populations, we have investigated how to effectively implement therapies to control alloresponses that lead to immune mediated disease, as well as examined the factors that direct a more effective antiviral response.

The population of lymphocytes mediating acute cellular rejection after solid organ transplantation is restricted in the usage of specific genetic elements of the T cell receptor (TCR) gene. We have shown this genetic bias in the graft infiltrating lymphocyte (GIL) population mediating rejection persists throughout periods of undetectable to severe clinical rejection. In order to make use of this genetic restriction to implement more narrowly directed immunomodulatory therapies to treat acute rejection, we have developed an approach to categorize and predict these biases based on the alloreactive surface antigens present in donor and host. We have also devised a strategy to characterize CTL alloresponses within smaller populations of lymphocytes, allowing for the ability to detect and monitor for acute rejection through safer clinical sampling.

The immune response against HIV-1 infection is initially mediated by virus specific CTL, and this early response is likely key in determining the overall outcome of disease. We investigated how clonal breadth can influence the ability of the CTL response to avoid viral evasion in acute HIV-1 infection. We identified the HIV-1 specific CTL responses and target epitopes in six acutely infected individuals and tracked the evolution of viral variant species throughout early infection. CTL isolated ex vivo from these subjects demonstrated a limited ability to expand after activation in culture, constraining our ability to measure breadth based on cell expansion. We therefore developed a method to measure the clonal breadth of CTL responses based on cytokine secretion, allowing for breadth to be studied in acute infection without the limitations of poor ex vivo expansion of CTL. Our findings will give insight into the development of more effective cellular immunity based vaccines and gene therapies.

The CD8⁺ cytotoxic T lymphocyte (CTL) is a critical component in the antiviral immune response to Human Immunodeficiency Virus type-1 (HIV-1). However, the efficacy of immune containment is limited due the high viral turnover, rapid rate of mutation, and remarkable sequence plasticity of HIV-1. Studies have shown that immune selection pressure exerted by CTL can also readily produce epitope escape variants. Although these virions have the capacity to elude immune detection, escape events occur only if the benefit of immune evasion outweighs the fitness cost of the mutation. The dynamics of escape and the effect of such mutations on viral replication, however, have been generally poorly understood.

Here, we measured the fitness costs of HIV-1 mutations across different viral proteins and evaluated their overall impact on replication. We examined the effect of specific mutations in the Envelope gp41 membrane-proximal tyrosine-based sorting signal on the susceptibility to CTL and developed a potential model for in vivo attenuation of Simian Immunodeficiency Virus (SIV) containing analogous mutations that confer subsequent protection from wild type challenge. We investigated pairing-disruptive synonymous mutations within a novel RNA secondary structure identified in the pol gene and their impact on HIV-1 reproductive success while, in parallel, used chemically-based procedures to confirm RNA structure modification. Finally, we analyzed the differential fitness costs of immune-mediated escape mutations identified in gag-pol in persons with acute or chronic HIV-1 infection and assessed the breadth of escape mutations with associated fitness impairments across individuals expressing class I human leukocyte antigen (HLA-I) types not implicated with superior control of viral replication.