Since its discovery 25 years ago, the HIV virus has infected more than 34 million people in the
world; the infection caused by this virus has led the world to its most terrible pandemic, and the
greatest global health crises of our times.
The host-virus interaction and natural history of the disease are influenced by the distinctive
interface the virus has with each infected individual. The infection with HIV-1 is characterized by the destruction of CD4+ T-cells during the typical course of the infection, but HIV has the arsenal to infect practically all the major blood leukocytes. Taken from these observations, it is apparent that HIV has the innate ability to subvert and manipulate the host gene machinery at the transcriptomic
The highly active anti-retroviral therapy (HAART) consists of a combination of powerful drugs,
which serve as potent defence mechanism against the ways in which the HIV virus attacks the
human bod y. Although these drugs are not able to rid the body of HIV virus, they can significantly
delay the onset of AIDS and reduce the incidence of opportunistic infections, morbidity and
mortality related to HIV infection. After the introduction of HAART treatment, it is observed that most patients with good adherence respond to HAART, which is defined by a decrease of plasma
viral load to undetectable levels and an immune reconstitution with a significant increase of CD4+ T cell levels. Around 30% of the patients fail to achieve this response and continue to express high plasma viral load and low CD4+ T cell numbers. In contrast, some rare HIV+ patients maintain below detectable levels of plasma viremia without the treatment. These are termed long-term nonprogressors(LTNPs), less commonly called elite controllers. These rare individuals are infected with HIV, but have the natural ability to control the infection with the strength of their immune system. Many of th ese patients have been HIV positive for 30 years or more and! off the rapy for the entire duration of their infection, showing high CD4+T cells counts and no progression to the disease. In this context, it is important to mention that the genomic basis of this natural effective immunological control of viremia in LTNPs, as opposed to drug-mediated control of HIV, remains
unknown. The development of high throughput microarray platforms and bioinformatic platforms to visualize and analyse the complex dataset has enabled considerable progress in the field of viral genomics, and also the visualization of host-virus interactions at the molecular level.
In chapter III, we carried out a comparative genome-wide (encompassing all 25,000 human genes)
pharmacogenomic study using whole primary peripheral blood mononuclear cells (PBMC) derived
from 14 HIV+ patients at two time points: pre-HAART (TP-1 with detectable viremia) and post
(TP2: below detectable level (BDL) of plasma HIV <40 copies of HIV RNA/mL plasma),
to ascertain how genomically distinct viremic phase is from the phase in which virus is fully
controlled with HAART. Another goal was to define the underlying pharmacogenomic basis of HIV
control during HAART. In the second study shown in Chapter IV, we compared the two time points
against the 9 LTNPs to unravel the genomic basis of natural control of viremia in therapy naïve
LTNPs showing below detectable levels of viremia (<20 copies of HIV RNA/mL plasma) and high
and stable CD4+T cell counts. Genomic RNA extracted from the PBMCs was used in genome-wide
microarray analysis, using HT-12v3 Illumina chips. Quantile normalization was performed to
normalize the data and inter-patient variability. Illumina®BeadStudio Data Analysis Software wa s
used to obtain differentially expressed (DE) genes. Only the significant genes with p value <0.01 and FDR of <5% (for the comparison between TP1 and TP2) and FDR <1% (for the comparison
between LTNP vers us TP1 and LTNP versus TP2) were considered appropriate for ! analysis .
Pathway analysis was performed in MetaCoreTM from GeneGo, Inc to derive functional annotations.
Functionally significant genes were validated by quantitative real time PCR. Between TP1 and TP2,
234 genes were differentially expressed. Within these genes, 212 were down-regulated and 22 upregulated. Between the comparison between LTNP vs TP1, 965 genes were differently expressed
(706 genes were up-regulated and 259 genes were down-regulated), and when LTNP was compared
to the TP2 group, we found 1181 DE genes (with 727 genes up-regulated and 454 genes downregulated). In the first part of this study, comparing the TP1 and TP2 only, we found that of the top 10 pathways, 8 belonged to the immune response system. This was the most significant pathway up-regulated in TP1 when compared to TP2. This comprised of genes that were involved in antiviral action of interferon (IFN) and their signalling function, antiviral response, dendritic cell maturation
a nd migration, and cell metabolism. Map folder and enrichment analysis corroborated with our
findings, thereby confirming an intrinsic role of the immune, inflammatory and interferon response
family-related genes during HIV viremia in the absence of treatment. But a closer examination of
this contrast also showed a mirror down-regulation of genes involved in innate and adaptive immunity, inflammation, apoptosis and antiviral functions. This directly implies a functional relevance of these pathways, through their modulation in TP1 and TP2 stages. Although these data are intuitive and expected, such analysis has never been performed before.
The second part of this study, on comparisons between LTNP and TP1 and LTNP against TP2, we
show the first evidence demonstrating that the natural control of HIV in LTNP is guided by the
genes enriched in the immune response, cytoskeleton remodelling, apoptosis and T cell signalling pathways. Another striking ob servation was that, even though the LTNP and TP2 groups
maintai ned BDL of plasma viremia (<40 copies), the LTNP group was genomically distinct from the TP2, which controlled viremia with HAART. This highlights the qualitative distinction and
critical role of enriched pathways in natural control of viremia in the LTNPs. Seventeen genes
encompassing all these pathways were validated by q-PCR, which showed consistent trends
between microarray and q-RT PCR. One gene in particular, the thrombospondin (THBS1) (R2=
0.942) was identified as a biomarker in our study, discriminating between viremic patients and
LTNPs at the genomic (R2= 0.942, p<2.654e.08) and proteomic levels (p<0.003761). The levels of
expression of THBS1 showed excellent correlation with plasma viremia (R2=0.81557;p<.0.0003761), the first description of such an important protein. This is the most unique finding of
this work, which has significance in HIV disease prognostics and diagnostics, in addition to
predicting the strength of the ho st immune system, as evident from its down-regulation and low expression in the natural controllers.
Overall, through these analyses, we have shown that, although there are common set of key genes
associated with HIV at all stages, each stage also showed unique molecular signatures. This was
demonstrated with the identification of molecular signatures for the control of virus with HAART
therapy, as well as for the natural control of HIV in LTNPs. Especially for the LTNPs, the downregulation of the apoptosis was the most significant feature, which may have important implications in therapy, especially in the context of using apoptosis as a target for future therapies.