Genome-wide mRNA expression correlates of viral control in CD4+ T-cells from HIV-1-infected individuals.

There is great interindividual variability in HIV-1 viral setpoint after seroconversion, some of which is known to be due to genetic differences among infected individuals. Here, our focus is on determining, genome-wide, the contribution of variable gene expression to viral control, and to relate it to genomic DNA polymorphism. RNA was extracted from purified CD4+ T-cells from 137 HIV-1 seroconverters, 16 elite controllers, and 3 healthy blood donors. Expression levels of more than 48,000 mRNA transcripts were assessed by the Human-6 v3 Expression BeadChips (Illumina). Genome-wide SNP data was generated from genomic DNA using the HumanHap550 Genotyping BeadChip (Illumina). We observed two distinct profiles with 260 genes differentially expressed depending on HIV-1 viral load. There was significant upregulation of expression of interferon stimulated genes with increasing viral load, including genes of the intrinsic antiretroviral defense. Upon successful antiretroviral treatment, the transcriptome profile of previously viremic individuals reverted to a pattern comparable to that of elite controllers and of uninfected individuals. Genome-wide evaluation of cis-acting SNPs identified genetic variants modulating expression of 190 genes. Those were compared to the genes whose expression was found associated with viral load: expression of one interferon stimulated gene, OAS1, was found to be regulated by a SNP (rs3177979, p = 4.9E-12); however, we could not detect an independent association of the SNP with viral setpoint. Thus, this study represents an attempt to integrate genome-wide SNP signals with genome-wide expression profiles in the search for biological correlates of HIV-1 control. It underscores the paradox of the association between increasing levels of viral load and greater expression of antiviral defense pathways. It also shows that elite controllers do not have a fully distinctive mRNA expression pattern in CD4+ T cells. Overall, changes in global RNA expression reflect responses to viral replication rather than a mechanism that might explain viral control.

Inhibition of adaptive immune responses leads to a fatal clinical outcome in SIV-infected pigtailed macaques but not vervet African green monkeys.

African green monkeys (AGM) and other natural hosts for simian immunodeficiency virus (SIV) do not develop an AIDS-like disease following SIV infection. To evaluate differences in the role of SIV-specific adaptive immune responses between natural and nonnatural hosts, we used SIV(agmVer90) to infect vervet AGM and pigtailed macaques (PTM). This infection results in robust viral replication in both vervet AGM and pigtailed macaques (PTM) but only induces AIDS in the latter species. We delayed the development of adaptive immune responses through combined administration of anti-CD8 and anti-CD20 lymphocyte-depleting antibodies during primary infection of PTM (n = 4) and AGM (n = 4), and compared these animals to historical controls infected with the same virus. Lymphocyte depletion resulted in a 1-log increase in primary viremia and a 4-log increase in post-acute viremia in PTM. Three of the four PTM had to be euthanized within 6 weeks of inoculation due to massive CMV reactivation and disease. In contrast, all four lymphocyte-depleted AGM remained healthy. The lymphocyte-depleted AGM showed only a trend toward a prolongation in peak viremia but the groups were indistinguishable during chronic infection. These data show that adaptive immune responses are critical for controlling disease progression in pathogenic SIV infection in PTM. However, the maintenance of a disease-free course of SIV infection in AGM likely depends on a number of mechanisms including non-adaptive immune mechanisms.

MicroRNA antagonism of the picornaviral life cycle: alternative mechanisms of interference.

In addition to modulating the function and stability of cellular mRNAs, microRNAs can profoundly affect the life cycles of viruses bearing sequence complementary targets, a finding recently exploited to ameliorate toxicities of vaccines and oncolytic viruses. To elucidate the mechanisms underlying microRNA-mediated antiviral activity, we modified the 3' untranslated region (3'UTR) of Coxsackievirus A21 to incorporate targets with varying degrees of homology to endogenous microRNAs. We show that microRNAs can interrupt the picornavirus life-cycle at multiple levels, including catalytic degradation of the viral RNA genome, suppression of cap-independent mRNA translation, and interference with genome encapsidation. In addition, we have examined the extent to which endogenous microRNAs can suppress viral replication in vivo and how viruses can overcome this inhibition by microRNA saturation in mouse cancer models.

New tools provide a second look at HDV ribozyme structure, dynamics and cleavage.

The hepatitis delta virus (HDV) ribozyme is a self-cleaving RNA enzyme essential for processing viral transcripts during rolling circle viral replication. The first crystal structure of the cleaved ribozyme was solved in 1998, followed by structures of uncleaved, mutant-inhibited and ion-complexed forms. Recently, methods have been developed that make the task of modeling RNA structure and dynamics significantly easier and more reliable. We have used ERRASER and PHENIX to rebuild and re-refine the cleaved and cis-acting C75U-inhibited structures of the HDV ribozyme. The results correct local conformations and identify alternates for RNA residues, many in functionally important regions, leading to improved R values and model validation statistics for both structures. We compare the rebuilt structures to a higher resolution, trans-acting deoxy-inhibited structure of the ribozyme, and conclude that although both inhibited structures are consistent with the currently accepted hammerhead-like mechanism of cleavage, they do not add direct structural evidence to the biochemical and modeling data. However, the rebuilt structures (PDBs: 4PR6, 4PRF) provide a more robust starting point for research on the dynamics and catalytic mechanism of the HDV ribozyme and demonstrate the power of new techniques to make significant improvements in RNA structures that impact biologically relevant conclusions.

Co-orientation of replication and transcription preserves genome integrity.

In many bacteria, there is a genome-wide bias towards co-orientation of replication and transcription, with essential and/or highly-expressed genes further enriched co-directionally. We previously found that reversing this bias in the bacterium Bacillus subtilis slows replication elongation, and we proposed that this effect contributes to the evolutionary pressure selecting the transcription-replication co-orientation bias. This selection might have been based purely on selection for speedy replication; alternatively, the slowed replication might actually represent an average of individual replication-disruption events, each of which is counter-selected independently because genome integrity is selected. To differentiate these possibilities and define the precise forces driving this aspect of genome organization, we generated new strains with inversions either over approximately 1/4 of the chromosome or at ribosomal RNA (rRNA) operons. Applying mathematical analysis to genomic microarray snapshots, we found that replication rates vary dramatically within the inverted genome. Replication is moderately impeded throughout the inverted region, which results in a small but significant competitive disadvantage in minimal medium. Importantly, replication is strongly obstructed at inverted rRNA loci in rich medium. This obstruction results in disruption of DNA replication, activation of DNA damage responses, loss of genome integrity, and cell death. Our results strongly suggest that preservation of genome integrity drives the evolution of co-orientation of replication and transcription, a conserved feature of genome organization.

Safety and immunogenicity of a replication-defective adenovirus type 5 HIV vaccine in Ad5-seronegative persons: a randomized clinical trial (HVTN 054).

BACKGROUND: Individuals without prior immunity to a vaccine vector may be more sensitive to reactions following injection, but may also show optimal immune responses to vaccine antigens. To assess safety and maximal tolerated dose of an adenoviral vaccine vector in volunteers without prior immunity, we evaluated a recombinant replication-defective adenovirus type 5 (rAd5) vaccine expressing HIV-1 Gag, Pol, and multiclade Env proteins, VRC-HIVADV014-00-VP, in a randomized, double-blind, dose-escalation, multicenter trial (HVTN study 054) in HIV-1-seronegative participants without detectable neutralizing antibodies (nAb) to the vector. As secondary outcomes, we also assessed T-cell and antibody responses. METHODOLOGY/PRINCIPAL FINDINGS: Volunteers received one dose of vaccine at either 10(10) or 10(11) adenovector particle units, or placebo. T-cell responses were measured against pools of global potential T-cell epitope peptides. HIV-1 binding and neutralizing antibodies were assessed. Systemic reactogenicity was greater at the higher dose, but the vaccine was well tolerated at both doses. Although no HIV infections occurred, commercial diagnostic assays were positive in 87% of vaccinees one year after vaccination. More than 85% of vaccinees developed HIV-1-specific T-cell responses detected by IFN-γ ELISpot and ICS assays at day 28. T-cell responses were: CD8-biased; evenly distributed across the three HIV-1 antigens; not substantially increased at the higher dose; and detected at similar frequencies one year following injection. The vaccine induced binding antibodies against at least one HIV-1 Env antigen in all recipients. CONCLUSIONS/SIGNIFICANCE: This vaccine appeared safe and was highly immunogenic following a single dose in human volunteers without prior nAb against the vector. TRIAL REGISTRATION: ClinicalTrials.gov NCT00119873.

Direct TLR2 signaling is critical for NK cell activation and function in response to vaccinia viral infection.

Natural killer (NK) cells play an essential role in innate immune control of poxviral infections in vivo. However, the mechanism(s) underlying NK cell activation and function in response to poxviruses remains poorly understood. In a mouse model of infection with vaccinia virus (VV), the most studied member of the poxvirus family, we identified that the Toll-like receptor (TLR) 2-myeloid differentiating factor 88 (MyD88) pathway was critical for the activation of NK cells and the control of VV infection in vivo. We further showed that TLR2 signaling on NK cells, but not on accessory cells such as dendritic cells (DCs), was necessary for NK cell activation and that this intrinsic TLR2-MyD88 signaling pathway was required for NK cell activation and played a critical role in the control of VV infection in vivo. In addition, we showed that the activating receptor NKG2D was also important for efficient NK activation and function, as well as recognition of VV-infected targets. We further demonstrated that VV could directly activate NK cells via TLR2 in the presence of cytokines in vitro and TLR2-MyD88-dependent activation of NK cells by VV was mediated through the phosphatidylinositol 3-kinase (PI3K)-extracellular signal-regulated kinase (ERK) pathway. Taken together, these results represent the first evidence that intrinsic TLR signaling is critical for NK cell activation and function in the control of a viral infection in vivo, indicate that multiple pathways are required for efficient NK cell activation and function in response to VV infection, and may provide important insights into the design of effective strategies to combat poxviral infections.

The increasing impact of human immunodeficiency virus infections, sexually transmitted diseases, and viral hepatitis in Durham County, North Carolina: a call for coordinated and integrated services.

BACKGROUND: Durham County, North Carolina, faces high rates of human immunodeficiency virus (HIV) infection (with or without progression to AIDS) and sexually transmitted diseases (STDs). We explored the use of health care services and the prevalence of coinfections, among HIV-infected residents, and we recorded community perspectives on HIV-related issues. METHODS: We evaluated data on diagnostic codes, outpatient visits, and hospitalizations for individuals with HIV infection, STDs, and/or hepatitis B or C who visited Duke University Hospital System (DUHS). Viral loads for HIV-infected patients receiving care were estimated for 2009. We conducted geospatial mapping to determine disease trends and used focus groups and key informant interviews to identify barriers and solutions to improving testing and care. RESULTS: We identified substantial increases in HIV/STDs in the southern regions of the county. During the 5-year period, 1,291 adults with HIV infection, 4,245 with STDs, and 2,182 with hepatitis B or C were evaluated at DUHS. Among HIV-infected persons, 13.9% and 21.8% were coinfected with an STD or hepatitis B or C, respectively. In 2009, 65.7% of HIV-infected persons receiving care had undetectable viral loads. Barriers to testing included stigma, fear, and denial of risk, while treatment barriers included costs, transportation, and low medical literacy. LIMITATIONS: Data for health care utilization and HIV load were available from different periods. Focus groups were conducted among a convenience sample, but they represented a diverse population. CONCLUSIONS: Durham County has experienced an increase in the number of HIV-infected persons in the county, and coinfections with STDs and hepatitis B or C are common. Multiple barriers to testing/treatment exist in the community. Coordinated care models are needed to improve access to HIV care and to reduce testing and treatment barriers.

The Ontogeny of Mucosal and Systemic Antibody Responses to HIV-1 Infection

The humoral immune system plays a critical role in the clearance of numerous pathogens. In the setting of HIV-1 infection, the virus infects, integrates its genome into the host's cells, replicates, and establishes a reservoir of virus-infected cells. The initial antibody response to HIV-1 infection is targeted to non-neutralizing epitopes on HIV-1 Env gp41, and when a neutralizing response does develop months after transmission, it is specific for the autologous founder virus and the virus escapes rapidly. After continuous waves of antibody mediated neutralization and viral escape, a small subset of infected individuals eventually develop broad and potent heterologous neutralizing antibodies years after infection. In this dissertation, I have studied the ontogeny of mucosal and systemic antibody responses to HIV-1 infection by means of three distinct aims: 1. Determine the origin of the initial antibody response to HIV-1 infection. 2. Characterize the role of restricted VH and VL gene segment usage in shaping the antibody response to HIV-1 infection. 3. Determine the role of persistence of B cell clonal lineages in shaping the mutation frequencies of HIV-1 reactive antibodies.

After the introduction (Chapter 1) and methods (Chapter 2), Chapter 3 of this dissertation describes a study of the antibody response of terminal ileum B cells to HIV-1 envelope (Env) in early and chronic HIV-1 infection and provides evidence for the role of environmental antigens in shaping the repertoire of B cells that respond to HIV-1 infection. Previous work by Liao et al. demonstrated that the initial plasma cell response in the blood to acute HIV-1 infection is to gp41 and is derived from a polyreactive memory B cell pool. Many of these antibodies cross-reacted with commensal bacteria, Therefore, in Chapter 3, the relationship of intestinal B cell reactivity with commensal bacteria to HIV-1 infection-induced antibody response was probed using single B cell sorting, reverse transcription and nested polymerase chain reaction (RT- PCR) methods, and recombinant antibody technology. The dominant B cell response in the terminal ileum was to HIV-1 envelope (Env) gp41, and 82% of gp41- reactive antibodies cross-reacted with commensal bacteria whole cell lysates. Pyrosequencing of blood B cells revealed HIV-1 antibody clonal lineages shared between ileum and blood. Mutated IgG antibodies cross-reactive with both Env gp41 and commensal bacteria could also be isolated from the terminal ileum of HIV-1 uninfected individuals. Thus, the antibody response to HIV-1 can be shaped by intestinal B cells stimulated by commensal bacteria prior to HIV-1 infection to develop a pre-infection pool of memory B cells cross-reactive with HIV-1 gp41.

Chapter 4 details the study of restricted VH and VL gene segment usage for gp41 and gp120 antibody induction following acute HIV-1 infection; mutations in gp41 lead to virus enhanced neutralization sensitivity. The B cell repertoire of antibodies induced in a HIV-1 infected African individual, CAP206, who developed broadly neutralizing antibodies (bnAbs) directed to the HIV-1 envelope gp41 membrane proximal external region (MPER), is characterized. Understanding the selection of virus mutants by neutralizing antibodies is critical to understanding the role of antibodies in control of HIV-1 replication and prevention from HIV-1 infection. Previously, an MPER neutralizing antibody, CAP206-CH12, with the binding footprint identical to that of MPER broadly neutralizing antibody 4E10, that like 4E10 utilized the VH1-69 and VK3-20 variable gene segments was isolated from this individual (Morris et al., 2011). Using single B cell sorting, RT- PCR methods, and recombinant antibody technology, Chapter 4 describes the isolation of a VH1-69, Vk3-20 glycan-dependent clonal lineage from CAP206, targeted to gp120, that has the property of neutralizing a neutralization sensitive CAP206 transmitted/founder (T/F) and heterologous viruses with mutations at amino acids 680 or 681 in the MPER 4E10/CH12 binding site. These data demonstrate sites within the MPER bnAb epitope (aa 680-681) in which mutations can be selected that lead to viruses with enhanced sensitivity to autologous and heterologous neutralizing antibodies.

In Chapter 5, I have completed a comparison of evolution of B cell clonal lineages in two HIV-1 infected individuals who have a predominant VH1-69 response to HIV-1 infection--one who produces broadly neutralizing MPER-reactive mAbs and one who does not. Autologous neutralization in the plasma takes ~12 weeks to develop (Gray et al., 2007; Tomaras et al., 2008b). Only a small subset of HIV-1 infected individuals develops high plasma levels of broad and potent heterologous neutralization, and when it does occur, it typically takes 3-4 years to develop (Euler et al., 2010; Gray et al., 2007; 2011; Tomaras et al., 2011). The HIV-1 bnAbs that have been isolated to date have a number of unusual characteristics including, autoreactivity and high levels of somatic hypermutations, which are typically tightly regulated by immune control mechanisms (Haynes et al., 2005; 2012b; Kwong and Mascola, 2012; Scheid et al., 2009a). The VH mutation frequencies of bnAbs average ~15% but have been shown to be as high as 32% (reviewed in Mascola and Haynes, 2013; Kwong and Mascola, 2012). The high frequency of somatic hypermutations suggests that the B cell clonal lineages that eventually produce bnAbs undergo high-levels of affinity maturation, implying prolonged germinal center (GC) reactions and high levels of T cell help. To study the duration of HIV-1- reactive B cell clonal persistence, HIV-1 reactive and non HIV-1- reactive B cell clonal lineages were isolated from an HIV-1 infected individual that produces bnAbs, CAP206, and an HIV-1 infected individual who does not produce bnAbs, 004-0. Single B cell sorting, RT-PCR and recombinant antibody technology was used to isolate and produce monoclonal antibodies from multiple time points from each individual. B cell sequences clonally related to mAbs isolated by single cell PCR were identified within pyrosequences of longitudinal samples of these two individuals. Both individuals produced long-lived B cell clones that persisted from 0-232 weeks in CAP206, and 0-238 weeks in 004-0. The average length of persistence of clones containing members isolated from two separate time points was 91.5 weeks both individuals. Examples of the continued evolution of clonal lineages were observed in both the bnAb and non-bnAb individual. These data indicated that the ability to generate persistent and evolving B cell clonal lineages occurs in both bnAb and non-bnAb individuals, suggesting that some alternative host or viral factor is critical for the generation of highly mutated broadly neutralizing antibodies.

Together the studies described in Chapter 3-5 show that multiple factors influence the antibody response to HIV-1 infection. The initial antibody response to HIV-1 Env gp41 can be shaped by a B cell response to intestinal commensal bacteria prior to HIV-1 infection. VH and VL gene segment restriction can impact the B cell response to multiple HIV-1 antigens, and virus escape mutations in the MPER can confer enhanced neutralization sensitivity to autologous and heterologous antibodies. Finally, the ability to generate long-lived HIV-1 clonal lineages in and of itself does not confer on the host the ability to produce bnAbs.

Preexisting compensatory amino acids compromise fitness costs of a HIV-1 T cell escape mutation.

BACKGROUND: Fitness costs and slower disease progression are associated with a cytolytic T lymphocyte (CTL) escape mutation T242N in Gag in HIV-1-infected individuals carrying HLA-B*57/5801 alleles. However, the impact of different context in diverse HIV-1 strains on the fitness costs due to the T242N mutation has not been well characterized. To better understand the extent of fitness costs of the T242N mutation and the repair of fitness loss through compensatory amino acids, we investigated its fitness impact in different transmitted/founder (T/F) viruses. RESULTS: The T242N mutation resulted in various levels of fitness loss in four different T/F viruses. However, the fitness costs were significantly compromised by preexisting compensatory amino acids in (Isoleucine at position 247) or outside (glutamine at position 219) the CTL epitope. Moreover, the transmitted T242N escape mutant in subject CH131 was as fit as the revertant N242T mutant and the elimination of the compensatory amino acid I247 in the T/F viral genome resulted in significant fitness cost, suggesting the fitness loss caused by the T242N mutation had been fully repaired in the donor at transmission. Analysis of the global circulating HIV-1 sequences in the Los Alamos HIV Sequence Database showed a high prevalence of compensatory amino acids for the T242N mutation and other T cell escape mutations. CONCLUSIONS: Our results show that the preexisting compensatory amino acids in the majority of circulating HIV-1 strains could significantly compromise the fitness loss due to CTL escape mutations and thus increase challenges for T cell based vaccines.