Exceptional molecular and coreceptor-requirement properties of molecular clones isolated from an Human Immunodeficiency Virus Type-1 subtype C infection

<p>Abstract</p> <p>Background</p> <p>The pathogenic significance of coreceptor switch in the viral infection of HIV-1 is not completely understood. This situation is more complex in subtype C infection where coreceptor switch is either absent or extremely rare. To gain insights into the mechanisms that underlie coreceptor requirement of subtype C, we screened several primary viral isolates and identified a clinical sample that demonstrated a potential to grow on standard T-cell lines with no detectable CCR5 expression. The subject was diagnosed with HIV-1 associated dementia in the absence of opportunistic infections of the brain. To isolate molecular clones from this virus, we devised a novel strategy based on anchor primers that target a sequence in the reverse transcriptase, highly conserved among diverse subtypes of HIV-1.</p> <p>Results</p> <p>Using this strategy, we isolated 8 full-length molecular clones from the donor. Two of the eight molecular clones, 03In94_D17 and 03In94_D24, (D17 and D24) generated replication-competent viruses. Phylogenetic analysis of the full-length viral sequences revealed that both clones were non-recombinant subtype C viruses. They contain intact open reading frames in all the viral proteins. Both the viral clones are endowed with several unique molecular and biological properties. The viral promoter of the clones is characterized by the presence of four NF-kB binding elements, a feature rarely seen in the subtype C HIV-1 LTR. Interestingly, we identified the coexistence of two different forms of Rev, a truncated form common to subtype C and a full-length form less common for this subtype, in both proviral and plasma virus compartments. An exceptional property of the viruses, atypical of subtype C, is their ability to use a wide range of coreceptors including CCR5, CXCR4, and several others tested. Sequence analysis of Env of D17 and D24 clones identified differences within the variable loops providing important clues for the expanded coreceptor use. The V1, V2 and V4 loops in both of the molecular clones are longer due to the insertion of several amino acid residues that generated potential N-linked glycosylation sites.</p> <p>Conclusion</p> <p>The exceptional biological and molecular properties of these clones make them invaluable tools to understand the unique pathogenic characteristics of subtype C.</p

Evidence-based national vaccine policy

India has over a century old tradition of development and production of vaccines. The Government rightly adopted self-sufficiency in vaccine production and self-reliance in vaccine technology as its policy objectives in 1986. However, in the absence of a full-fledged vaccine policy, there have been concerns related to demand and supply, manufacture vs. import, role of public and private sectors, choice of vaccines, new and combination vaccines, universal vs. selective vaccination, routine immunization vs. special drives, cost-benefit aspects, regulatory issues, logistics etc. The need for a comprehensive and evidence based vaccine policy that enables informed decisions on all these aspects from the public health point of view brought together doctors, scientists, policy analysts, lawyers and civil society representatives to formulate this policy paper for the consideration of the Government. This paper evolved out of the first ever ICMR-NISTADS national brainstorming workshop on vaccine policy held during 4-5 June, 2009 in New Delhi, and subsequent discussions over email for several weeks, before being adopted unanimously in the present form

Molecular Epidemiology of HIV-1 Subtypes in India: Origin and Evolutionary History of the Predominant Subtype C

This thesis describes the translational genomics of HIV-1subtype C in India from its origin to therapeutic response with the aim to improve our knowledge for better therapeutic and preventive strategies to combat HIV/AIDS. In a systemic approach, we identified the molecular phylogeny of HIV-1 subtypes circulating in India and the time to most recent common ancestors (tMRCA) of predominant HIV-1 subtype C strains. Additionally, this thesis also studied drug resistance mutations in children, adolescents and adults, the role of host factors in evolution of drug resistance, and population dynamics of viremia and viral co-receptor tropism in perinatal transmission. Finally, the long term therapeutic responses on Indian national first-line antiretroviral therapy were also studied. In Paper I, we reported an increase in the HIV-1 recombinant forms in the HIV-1 epidemiology using a robust subtyping methodology. While the study confirmed HIV- 1 subtype C as a dominant subtype, its origin was dated back to the early 1970s from a single or few genetically related strains from South Africa, whereafter, it has evolved independently. In Paper II, the lethal hypermutations due to the activity of human apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3G (hA3G) was significantly associated with antiretroviral therapy (ART) failure in Indian HIV-1 subtype C patients. The presence of M184I and M230I mutations were observed due to the editing of hA3G in the proviral compartment but stop codons were also found in the open reading frames and the same drug resistance mutations were absent in plasma virus. Therefore, it is unlikely that the viral variants which exhibit hypermutated sequences and M184I and/or M230I will mature and expand in vivo and hence are unlikely to have any clinical significance. The high concordance of drug resistance genotyping in the plasma and proviral compartments in therapy-naïve patients, gives weight to the idea of using whole blood for surveillance of drug resistance mutations which precludes logistic challenges of cold chain transport. In Papers III and IV, we identified a substantial proportion of HIV-1 subtype C perinatally-infected older children who had a high burden of plasma viremia but also had high CD4+ T-cell counts. In addition, older children with HIV-1 subtype C infection presented a high prevalence of predicted X4 and R5/X4 tropic strains which indicates that HIV-1 subtype C strains required longer duration of infection and greater disease progression to co-receptor transition from R5- to X4-tropic strains (IV). Our studies also indicate that transmitted drug resistance is low among Indian HIV-1 infected children, adolescents (III) and adults (II). In Paper V, in a longitudinal cohort study, a good long-term response to the Indian national first-line therapy for a median of nearly four years with 2.8% viral failure, indicating the overall success of the Indian ART program. Our study also showed that three immunologically well patients with virological rebound and major viral drug resistance mutations (M184V, K103N and Y181C) during one study visit had undetectable viral load at their next visit. These findings suggest that use of multiple parameters like patients’ immunological (CD4+ T-cell count), virological (viral load) and drug resistance data should all be used to optimize the treatment switch to second line therapy. In conclusion, this translational genomics study enhances our knowledge about the HIV-1 subtype C strains circulating in India which are genetically distinct from prototype African subtype C strains. Considerably more research using appropriate models need to be performed to understand the phenotypic and biological characteristics of these strains to guide efficient disease intervention and management strategies

Characterization of human immunodeficiency virus (HIV)-infected cells in infiltrates associated with CNS opportunistic infections in patients with HIV clade C infection

Infection with human immunodeficiency virus (HIV) clade C is the most common HIV infection worldwide, yet its impact on the nervous system remains largely unknown. Autopsy studies from regions affected by this virus are scarce, and HIV dementia has only rarely been reported from these countries. Most patients who develop neurologic complications die of opportunistic infections. We thus conducted a neuropathologic study from a single institution in India to characterize the HIV-infected cells in the inflammatory infiltrates in a total of 15 cases (5 patients each who died of either CNS toxoplasmosis, tuberculosis, or cryptococcal meningitis). Nearly, all patients had HIV-infected cells in the brain, although these cells were most abundant in patients with toxoplasma encephalitis. Interestingly, none of the patients had any multinucleated giant cells. HIV-infected cells were found in the parenchyma, perivascular regions, and choroid plexus and found infiltrating the parenchyma from the meninges, suggesting multiple portals of entry into the brain. These findings suggest the possibility that patients, even if successfully treated for an opportunistic inflection, may be at high risk of developing HIV encephalitis and subsequent dementia

Annotated phylogenetic trees for the <i>gag</i>- (Panel A), <i>env</i> (Panel B) and <i>pol</i>- (Panel C) genes.

<p>Phylogenetic trees were constructed using the Indian HIV-1 subtype C from clinical samples taken from different parts of India (shown in green), including southern (Karnataka, Tamil Nadu and Andhra Pradesh), northern (Punjab and Haryana), north-eastern (Manipur) and central (Madhya Pradesh) regions of India plus data base retrieved reference sequences from western India and other countries, and two out-group subtype B strains. The Indian tMRCA included in this study were shown with an arrow. <i>Pol-</i>gene analysis was considered to provide the most reliable estimate of tMRCA (shown with a red arrow) for Indian HIV-1C, indicating around 1971 (95% CI: 1965–1976)]. <b>Posterior probability plots showing the tMRCA estimates of Indian HIV-1C (Panel D):</b> The posterior probability density plots for the three genes (<i>env</i>, <i>pol</i> and <i>gag</i>) point to a tMRCA for Indian HIV-1C from 36.9 (<i>env</i>), 40.5 (<i>pol</i>) and 44.3 (<i>gag</i>) years ago, with an overall mean for the combined graph at 40.6 years ago before 2011, that is in 1970.4 (95% CI: 1960.3–1978.1).</p

Distribution of HIV-1 subtypes and recombinants in the clinical cohorts based on two genes.

<p>The regions [southern (Karnataka, Tamil Nadu and Andhra Pradesh), northern (Punjab and Haryana), north-eastern (Manipur) and central (Madhya Pradesh)] from where the samples were collected are shown in colors. The pie chart depicts the percentage of subtypes and recombinant strains in respective regions.</p

Bayesian skyline plot representing estimates of the effective number of infections through time.

<p>The demographic history from the pol BSP identified three epidemic growth phases divided by the vertical lines; an initial slow growth followed by an exponential phase which slowed down to a stationary phase around 1990. The identification of first HIV case in India is indicated by an arrow.</p

Genetic make-up of recombinant clinical strains.

a<p>Samples from northern India, ^bNorth-eastern India and ^cSouthern India. The recombinants were identified based on atleast two of the three viral genes, <i>gag</i>, <i>pol</i> and <i>env</i>. Recombination Identification Program ver 3.0 (RIP) and REGA subtyping tool ver 2 (REGA) were used. Recombinant strains were further confirmed by bootscan analysis in Simplot version 3.5.1 using 100 nucleotide window size and 20 nucleotide step size to map the precise breakpoint and region-specific phylogenetic analysis using ML tree in MEGA 5. NA: Not Assigned.</p