The biological significance of the C-?B motif: The stronger subtype-C viral promoter contains an additional NF-?B binding site (which we call the C-?B motif, (5’-GGGGCGTTCC-3’) that is genetically different from the two upstream canonical H-?B motifs (5’-GGGACTTTCC-3’). The C-?B motif is not only unique for subtype-C (absent from all other HIV-1, HIV-2 and SIV promoters), but is proximal to the Sp1 sites in the promoter. Moreover, the C-?B motif appears to be indispensable for subtype-C since none of the promoter sequences of subtype-C available in the extant databases lack this motif. Current research in our laboratory shows that the acquisition of the C-?B motif also catalyzed associated genetic variations in the proximal Sp1 site. The combination of the C-?B motif and the subtype-specific Sp1 site function with the highest degree of efficiency and cannot be substituted with the other functional equivalent motifs. Our work is suggestive of a coevolutionary selection of these two important transcription factor binding sites in subtype-C.
If acquiring a stronger promoter is important, subtype-C could have simply duplicated the upstream H-?B site to have three genetically identical NF-?B binding sites (HH to HHH). Subtype-C rather acquires a genetically different NF-?B site (HH to HHC). The acquisition of the variant ?B-site must confer not only a quantitative gain-of-function, as previously suggested by others, but, importantly, also a qualitative gain-of function. Work in progress in our laboratory confirms this proposition.
Acquisition of the C-?B motif in C-LTR also accentuated genetic variations in the proximal Sp1 site. What is the biological significance of these variations for the subtype-C promoter? We have several important leads in this direction.
Are the emerging 4-?B subtype-C viral strains likely to change the epidemic? Work from our laboratory demonstrated the rapid emergence of 4-?B viral strains in India and South Africa over the past decade. This observation is subsequently confirmed from Brazil and Mozambique by our collaborators in Brazil. Furthermore, we also found the expansion of a different promoter variant viral strain containing the RBEIII motif duplication, in all these geographical locations. Thus, unlike other viral subtypes of HIV-1, subtype-C alone appears to be undergoing important genetic variations in the promoter at the present time. Our study raises many important questions.
Why subtype-C alone, not any other viral subtype, acquires a stronger promoter by duplicating important transcription factor binding sites repeatedly? Subtype-C made a promoter of 3 NF-?B sites previously when all other subtypes had only one or two such elements. Presently, subtype-C appears to repeat that trick by producing a promoter of 4 NF-?B sites.
How can subtype-C contain a stronger promoter characterized with 4 NF-?Bmotifs yet capable of establishing and maintaining viral latency?
A stronger viral promoter causing higher magnitude of viral antigen expression should attract a stronger immune attack? Is it not counterintuitive that subtype-C should acquire a stronger promoter despite the immune response?
Is there a limit to the gain of strength of the viral promoter? Will there be strains evolving in the future with more NF-?B binding sites? In contrast, if there is a limit, where is that limit?
Why the acquisition of the NF-?B or RBEIII sites is mutually exclusive?
How is the sequence duplication in subtype-C regulated at the molecular level?
To address some of the above questions, we generated infectious HIV-1 promoter variant viruses that contain a variable number of NF-?B motifs (4, 3, 2, 1 or 0) in the natural context. In a cell-based latency model, the data are suggestive of the important role NF-?B plays in establishing and maintaining viral latency. Work is presently in progress.
Are the 4-?B viral strains of subtype-C more pathogenic? The pathigenic properties of HIV-1 can be studied only in the natural host. We plan to study this question in a multicentric, non-interventional, open-label study at 4 different sites (YRGCARE, Chennai; St. John’s Hospital, Bangalore; National AIDS Research Center, Pune and All India Institute of Medical Sciences, New Delhi). At each of the sites, 50 each HIV-infected subjects will be recruited into two arms that contain 3- or 4-?B viral infection. The CD4 cell count, plasma viral load and the activation markers on the T-cells will be monitored every 6 months in a follow up study of 2 years. At the end of the study, the CD4 cell slopes will be compared between the two groups to see if the 4-?B viral infection can cause a faster reduction of the CD4 cell count. The work is to begin soon.
|2. The host immune response to HIV-1 antiqgens.
In a bilateral international collaborative effort, several laboratories from India and South Africa have been working together to study cell-mediated host immune responses in defined clinical cohorts in both the countries. Under the aegis of the Department of Science and Technology, India, JNCASR, Bangalore; AIIMS, New Delhi; NARI, Pune and YRG CARE, Chennai have joined hands in this endeavor from India. At JNCASR, we made an important observation that subtype-C strains are capable of inserting long stretches of amino acids, as long as 14 residues, in p6 gag, unlike other subtypes that can duplicate only short stretches. The ability to duplicate long stretches of amino acids appears to enhance the replication fitness of the variant viral strains. We found that the 14 amino acid sequence insertion in gag duplicated the PTAP domain that is instrumental in recruiting host sorting machinery critical for viral budding. Using next-generation sequence analysis, we found that in subjects coinfected with the wild type (a single PTAP domain) and the variant type (PTAP duplication) viral strains, the variant type exerts absolute domination over the wild type in all the 7 or 8 subjects examined. We are presently comparing various biological properties of these two viral strains to understand how the replication fitness is regulated as a consequence of the PTAP motif duplication.
|3. Can the reverse transcriptase be different in different subtypes?
Subtype-C appears to be endowed with a special ability to duplicate longer stretches of nucleotides that permit the virus acquire enhanced replication fitness. The duplicated residues constitute a motif of biological importance, an important transcription factor binding site (NF-?B binding domain in the enhancer) or a domain that regulates a biological function such as viral budding (the PTAP duplication). Other viral subtypes appear to be devoid of this capability as they cannot duplicate the NF-?B site or can duplicate only short stretches of amino acids in gag that doesn’t seem to provide a functional advantage. There could be additional locations in subtype-C where long sequence stretches are duplicated for replication advantage. In addition to examining the replication advantage conferred on the variant viral strains by the sequence duplication, understanding how the process of the sequence duplication is regulated at the molecular level is important. An added twist is the flavor the viral subtypes add to this interesting phenomenon. Consider the following,
1. All the viral subtypes possess the capability to duplicate sequences, not randomly, but at certain hot spots. All HIV subtypes can duplicate the RBEIII site in the viral promoter, however, only subtype-C can duplicate the NF-?B site. What makes the difference?
2. All the viral subtypes can insert sequences of shorter length in p6 of gag, but only subtype-C appears to be capable of duplicating longer stretches and at a higher frequency, even in the absence of drug pressure. What makes the difference?
We believe that the differential sequence insertion properties could be a function of the collective activity of the cis (RNA folding differences) and trans (the reverse transcriptase) factors. We are isolating the molecular clones of primary viruses that possess the defined sequence duplications. Using these viral clones we will study the replication properties of the RT.
|4. The significance of the signature amino acid residues
Signature amino acids are those residues that are unique for or prevalent in one or a few viral subtypes but absent or less common in other subtypes. Serine 31 of Tat is a signature amino acid residue (SAR) for subtype-C since all the other subtypes contain a cysteine at this position. We demonstrated that the C31S variation disrupted the dicysteine motif in Tat (CC to CS) and the chemokine activity as a consequence. We, however, do not know why subtype-C galvanized this change when the presence of the dicysteine motif is indispensable for all the other HIV-1, HIV-2 and SIV subtypes. We believe that subtype-C is trading off the chemokine function of CC-Tat for some yet unexplored gain-of-function, through the CS-Tat. Using controlled expression of Tat proteins variant for C or S at position 31, we are comparing the modified host gene expression in Jurkat T-cells using the NGS platform. Consider the following facts.
1. HIV-1 is believed to have originated from a single zoonotic transmission approximately 100 years ago in Africa and undergone diversified evolution into multiple subtypes as it migrated to different geographical regions.
2. The divergent viral evolution must have been the direct consequence of the differences in the hostile host landscape that varies between different geographical regions. In an attempt to fine tune itself to the variable landscape of the host, the virus must undergo divergent evolution thus generating groups, subtypes, and strains. These variations gained under the selection pressure acquire subtype-specific nature, thus becoming SAR.
3. All the viral antigens have signature amino acid residues (likewise RNA nucleotide differences) specific to different viral subtypes. For instance, in subtype-C Tat we identified SAR at 7 of the 101 positions. The location and numbers of SAR in Tat are variable in different viral subtypes. We mapped the SAR in Tat of all the major HIV subtypes.
4. The presence of SAR at a specific location is likely to modulate the overall biological function of the protein in a significant manner that can be evaluated experimentally (eg. the loss of the chemokine activity in C-Tat due to C31S variation) or marginally that is difficult to quantitate experimentally.
5. The overall replication fitness and pathogenic properties of the HIV-1 subtypes must be a summation of the diverse SAR functions collectively. Functional evaluation of SAR therefore will enhance our understanding of the viral biological properties and how the variations are likely to modify these functions.
6. Since the origin of the SAR is expected to lie in the adaptation of the virus in response to the variable host factor differences, the study of SAR may be helpful to understand the viral evolution that has already taken place up to the present time. In addition, the study of SAR may also provide leads as to the direction of the viral evolution HIV-1 may undertake in the future.
7. Can the study of SARS teach us why only a small number of the large number of the recombinant viral strains survive and establish spreading epidemics? Can we predict which of the emerging variant viral strains is likely to survive and which ones are likely to perish?
Studies are in progress in our laboratory to address the significance of the signature amino acid residues for viral replication fitness and evolution. We use the viral antigen Tat as a model to build questions around this theme.
|5. Needle-less immunization.
The major viral epidemics happened because of the contaminated needle-sharing. If drug and vaccine administration is possible with devices that do not use needles, the safety profile of medical intervention will improve. Additionally, needle-less immunization offers means of targeting specific cell subsets in the skin, which would be technically difficult otherwise. Different dendritic cell subsets in the skin are specialists in different properties, like antigen cross-presentation for instance. Using a needle-less device, we are studying the mechanisms of antigen presentation in mice.
What We Have Reported Already
|The Indian HIV-1 epidemic is dominated by subtype-C: Using a subtype-C specific PCR, we demonstrated that in all the 4 South Indian states, subtype-C is responsible for nearly 99% of the viral infections (602/608 primary infections examined). The work represents not only the largest study to appear from India on HIV-1 molecular subtyping analysis, but also the first study to characterize the nature of the HIV-1 strains in the southern regions of the country.More Details|
Study of the molecular epidemiology of HIV-1 in India
a) Introduction: The major group of HIV-1 consists of a minimum of 10 primary viral subtypes (A, B, C, D, A/E, F, G, H, J and K), at least 66 circulating recombinant forms and countless number of unique recombinant forms circulating in the world (http://www.hiv.lanl.gov/content/sequence/HIV/CRFs/CRFs.html). Of these diverse variant forms of HIV-1, subtype-C viruses and their recombinants cause nearly half of the global infections (Hemelaar et al., 2011). Subtype-C is presently identified in the most populous nations, including India, China, sub-Saharan Africa, Brazil and others.
b) Why study viral molecular epidemiology? The first HIV-1 infection in India was detected in 1987. The epidemics of India are known to be dominated by subtype-C strains except in the North-eastern parts of the country where Thai B subtype is more common. In addition to subtype-C, several non-C subtypes, including –A, -B, –D and -E have also been reported in different parts of the country. Additionally, a few HIV-2 infections have also been reported from India. Unfortunately, the real prevalence of HIV-1 and HIV-2 infections in diverse populations of several states and Union territories of India has not been estimated in a nationwide study. Monitoring the distribution of viral subtypes in a geographical region is important as the viral subtype distribution is a dynamic process and could change as a function of time.
c) Development of a subtype-C-distinguishing PCR: The standard techniques used for viral molecular subtyping and sequencing of the envelope and/or the heteroduplex mobility assay (HMA) are not ideal for examining a large number of clinical infections. These techniques are too cumbersome, technically difficult and expensive to apply to hundreds of samples. To address this problem, we developed a multiplex-PCR in the nested format that can distinguish subtype-C from all the other genetic subtypes of HIV-1 (Siddappa et al., 2004b). The PCR amplifies two different viral gene segments.(1) The long-terminal repeat (LTR), this amplification is specific to subtype-C and (2) a gag fragment that is amplified from all the HIV-1 subtypes including subtype-C. In this PCR, all the subtype-C strains amplify two DNA fragments of diverse size and all other viral subtypes only the gag fragment. We call this amplification technique ‘the C-PCR’. All the three primer pairs target highly conserved regions of the viral genes, hence the failure rate is very low – typically less than 1%.
d) The HIV-1 epidemic of India is dominated by subtype-C: We applied C-PCR to more than 600 clinical samples collected from dozens of rural villages, towns and urban centers spanning across all the four southern states of India. A subset of these samples was also analyzed using HMA and/or envelope sequencing to confirm the results. Our study identified that nearly 99% percent of these samples (602/608) were due to subtype-C strains thus confirming the previous reports. Additionally, we identified one B and two A viruses. This study represents the largest report on HIV-1 molecular epidemiology from India.
e) Identification of B/C recombinant viruses in India: Targeting two different regions (LTR with C-PCR and env with HMA and/or sequencing) of a subset of 115 samples of our cohort, we identified three strains that contained B envelops (Siddappa et al., 2005). One of the three envelops phylogenetically associated with Thai-B viruses that are common in China and Thailand. The two other viruses unexpectedly associated with subtype-B viruses of the USA and Europe. It is presently not known if there is an independent epidemic emerging in India due to B/C recombinants. The real incidence of such a possibility could be addressed only by a large scale epidemiological study. Interestingly, B/C recombinant viruses containing env parts of subtype-B viruses of the USA and Europe have not been identified before this.
Siddappa NB et al, Journal of Clinical Microbiology, 42, 2742-51, 2004
Siddappa NB et al, AIDS, 19, 1426-9, 2004
|Novel promoter variant strains of subtype-C are emerging in India and other places:During 2000-2003, we found that 2 or 3 different types of subtype-C strains were emerging in India (Siddappa et al., 2004a). The new viral strains contained an additional transcription factor binding site (NF-κB or RBEIII binding site, but usually not both) in the viral promoter (Bachu et al., 2012a). At that time, the promoter variant strains were only a small minority (1-2%). Ten years later in 2010-2013, however, we found that the new viruses have grown rapidly in numbers (20-35%) replacing the standard subtype-C strains (Bachu et al., 2012b). We did an extensive comparative study of the standard subtype-C virus that contains only 3 NF-κB binding sites with an engineered virus that contained 4 NF-κB binding sites in the promoter. In all the experiments, the 4-κB virus dominated the 3-κB virus, thus confirming replication fitness. The newly emerging HIV-1 subtype C viruses containing a stronger viral promoter produced more viral particles and a higher viral load, probably providing an enhanced transmission advantage. A virus like HIV-1 acquiring a stronger promoter is surprising and unexpected. See the explanation below why this finding is unexpected and surprising. The emergence of the novel promoter variant subtype-C viral strains was recently confirmed from Brazil and Africa (Boullosa et al., 2014). More Details.|
All the 10 plus proteins of HIV-1 are expressed from a single viral promoter called the long-terminal repeat (LTR). Any important change in the viral promoter therefore can have a significant impact on the viral biological properties and fitness. Unlike the other genetic subtypes of HIV-1 that have only two identical NF-?B binding sites in the viral enhancer, subtype-C contains 3 ?B-binding sites. The strength of the promoter is expected to be proportional to the number of the ?B-sites. Some scientists believe that the stronger promoter of subtype-C may be one reason why this viral family of HIV-1 is causing half of the global infections.
In this backdrop, during 2000-2003, we were quite surprised to find a small minority (1-2%) of viral strains of HIV-1 in India containing 4 NF-?B binding sites in the viral promoter. Why were we surprised? Consider the following facts.
1. How can a virus like HIV-1 that must establish ‘latency’ (remaining silent without gene expression, a strategy to trick the immune system) can have a much stronger promoter and still establish and maintain viral latency?
2. Additionally, stronger promoter means more viral antigens and stronger lash back by the immune system. Isn’t it, therefore, counter intuitive that subtype-C keeps making progressively stronger promoter?
3. Moreover, if gaining more NF-?B motifs is an advantage, why not other HIV-1 subtypes do the same?
4. Furthermore, where is the limit for the acquired strength of the viral promoter? Are we likely to see subtype-C with more and more number of NF-?B motifs in the future? Sure, there must be a limit, but, where is that limit and how to find it?
Some researchers believe that subtype-C virus has a relatively high degree of attenuation as compared to other subtypes and as a result is less pathogenic. Our current results propose that subtype-C virus exploits a small window of opportunity to make higher viral load without eliciting a higher magnitude of immune activation. Future studies remain needed to further validate this notion. (See below work in progress currently)
Bachu M et al, AIDS Research and Human Retroviruses, 28, 1362-8, 2012
Bachu M et al, Journal of Biological Chemistry, 287, 44714-35, 2012
|The Tat protein of subtype-C is a defective chemokine: We demonstrated that in subtype-C of HIV alone, but not in other subtypes of HIV-1, HIV-2 or SIV (the primate viruses), the dicysteine motif in the viral protein Tat is disrupted by the substitution of cysteine by serine at position 31 (Ranga et al., 2004). We proposed that the variant Tat is a compromised chemokine and cannot disrupt the blood-brain-barrier thereby failing to attract monocytes into the brain. This may result in a less magnitude of neuron killing and less prevalence of HIV associated dementia as reported from India (Satishchandra et al., 2000). Our work thus offered a molecular explanation for the low prevalence of HIV-associated dementia reported from India (only 1-6% in India as opposed to 20-35% in the advanced countries). This is the first demonstration that different HIV-1 subtypes could demonstrate different pathogenic properties. The subtype-specific differences of Tat were subsequently confirmed by many other publications including our own collaborators.More Details.|
a) Incidence of HIV-associated dementia (HAD) is low in India: Two prospective studies from India demonstrated that the prevalence of HAD is significantly low in India (1 to 6%) (Satishchandra et al., 2000; Wadia et al., 2001) as opposed to that seen in the USA and Europe (15-30%) (Heaton et al., 1995; White et al., 1995). In addition to demographic, diagnostic and other differences influencing these results, could the biological differences between the HIV-1 subtypes contribute to the differential prevalence of dementia? For instance, could subtype-C strains be not capable of accentuating dementia?
b) Tat protein of HIV-1 is a monocyte chemokine: Tat protein of HIV-1 is implicated in HAD (Nath et al., 1998). As a secreted protein, Tat is believed to recruit monocytes to brain either directly or indirectly through astrocyte-MCP1 pathway (Weiss et al., 1999). Under the influence of Tat, activated monocyte breach blood-brain-barrier and migrate to the brain in large numbers and trigger neuron death through several pathways (Nath, 2002). A dicysteine motif of Tat (amino acid residues 30 and 31) is critical for the molecular mimicry of the Tat chemokine activity (Albini et al., 1998). The dicysteine motif is highly conserved in Tat proteins of all the non-C HIV-1 subtypes. Additionally, this motif is conserved also in HIV-2 and SIV Tat proteins, suggestinga positive evolutionary selection pressure.
c) We identified a natural variation in the Tat protein of subtype-C viruses: The dicysteine motif is surprisingly subjected to a natural variation in the Tat protein of subtype-C viruses (C-Tat). In C-Tat, cysteine at position 31 is replaced by serine regardless of geographical origin. We hypothesized that the replacement of the dicysteine motif (CC) with CS must make C-Tat a defective monocyte chemokine since in all the natural chemokines the integrity of the CC-motif is critical for their biological function. Our experiments using recombinantly expressed C-Tat and its cysteine variants supported this hypothesis (Ranga et al., 2004). This variation, however, did not affect other properties of the viral protein such as the transactivation. Interestingly, we identified that the variant serine at position 31 in C-Tat is phosphorylated suggesting acquisition of novel signaling properties for C-Tat.
e) Are subtype-C viruses incapable of triggering HAD? We suggest that possibility. Although we do not rule out the contribution made by other factors, we emphasize the importance of taking viral subtype differences into consideration while examining HIV-associated dementia. The role of other viral proteins like env and nef must be evaluated to rule out the possibility of a functional compensation.
Ranga U et al, Journal of Virology, 78, 2856-90, 2004
Rao VR et al, Journal of Neuroscience, 28, 10010-6, 2010 (Prasad’s Lab)
Rao VR et al, Retrovirology, 10, 61, 2013 (Prasad’s Lab)
|Controlling chronic immune activation using a poly-herbal formulation: HIV- 1differs remarkably from other infectious diseases in subjecting the host immune system to prolonged and incessant activation called ‘chronic immune activation’. The immune system trapped in this highly activated status has no escape from it and progressively deteriorates and finally collapses leading to AIDS (Ranga U and Arunachalam PS, NARI Bulletin, 2012). Thus, in HIV infection, the problem is immunological and the solution must also be immunological. Can an immune-modulator help the immune system against HIV by controlling the chronic immune activation? We addressed this question by using a poly-herbal formulation in a pilot clinical trial in 63 HIV-1 seropositive subjects. The clinical trial of 2 years suggested clinical stabilization in the arm (n=32) administered the polyherbal medicine as compared to the arm administered the anti-retroviral therapy (n-31) (Asokan et al., 2013). A subsequent immune analysis of the T-cells derived from the participants suggested attenuation of the immune activation in the poly-herbal arm providing an explanation for the clinical observation. We identified several biomarkers that can serve as potential surrogate markers for studying immune activation (Asokan M et al, VirusDisease, in press). Our study represents only a pilot clinical trial, not a randomized, multi-centric and placebo-controlled large-scale clinical trial. The experimental data are only indicative but not assertive of the beneficial effects of the poly-herbal medicine. In spite of the encouraging results, under no circumstances, the poly-herbal formulation evaluated in the present clinical trial, or any other such formulation, must be considered a ‘drug’ for HIV-AIDS.More Details.|
Introduction: The HIV-AIDS laboratory at the Jawaharlal Nehru Centre for Advanced Scientific Research (JNCASR), Bengaluru evaluated a polyherbal formulation (PHF) for its safety and efficacy against HIV-AIDS. The work consisted of an active collaboration with an NGO Samraksha, Bengaluru and several hospitals that specialize in HIV management, including the Seva Free Clinic, Bengaluru; Chest and Maternity Centre, Bengaluru; National Institute of Mental Health and NeuroSciences (NIMHANS), Bengaluru; Shanmugha Arts, Science, Technology and Research Academy (SASTRA), Thanjavur; All India Institute of Medical Sciences (AIIMS), New Delhi. The project was funded by the Department of Science and Technology (DST), Government of India. The work reported in a research article titled ‘Evident stabilization of the clinical profile in HIV/AIDS as evaluated in an open label clinical trial using a polyherbal formulation’ has appeared in The Indian Journal of Medical Research (MangaiarkarasiAsokan et al, the June issue, 2013) and is available at http://www.icmr.nic.in/ijmr/2013/June/0613.pdf.
The question addressed: Although complementary and alternative medicines are popular in HIV-AIDS management, they have not been systematically evaluated, especially in India. HIV infection differs remarkably from many other infectious diseases in its ability to cause chronic immune activation. In chronic immune activation, the body's defense mechanism is pushed to the top gear in an attempt to remove the viral infection. The immune system is trapped in this highly activated status and has no escape from it as the body once infected cannot remove the virus. Under the heavy workload, the immune system progressively deteriorates and finally collapses leading to AIDS. Thus, in HIV infection, the problem is immunological and the solution must also be immunological. Can an immunomodulator help the immune system against HIV by controlling the chronic immune activation? We attempted to address this question by using a polyherbal formulation in a pilot clinical trial.
The research methods: The herbal formulation, prepared by Vedic Drugs Pvt. Ltd., Bengaluru, consists of ingredients from 58 different plant species and lacks heavy metals. In a prospective, open-label, non-randomized, controlled, investigator-blinded clinical trial, 32 subjects under the PHF arm were administered the herbal formulation orally for only 4 months and followed for a period of 2 years. For comparison, 31 other subjects under the standard antiretroviral therapy (ART) were followed for the same period. All the subjects at the time of the study beginning were HIV-1 positive, did not take anti-HIV medicines previously, contained a CD4+ cell number in the blood between 200 and 250 cells/µl and lacked presenting opportunistic infections. In all the study participants, the plasma viral RNA load, CD4 cell count, blood chemistry and AIDS-related clinical symptoms were monitored at 3-month intervals using standard techniques. The trial was registered with the Clinical Trials Registry of India, CTRI/2008/091/000021.
The results and interpretation: The clinical trial found that the polyherbal formulation was safe for human administration. This finding was also confirmed in the toxicity studies using a small animal model. Importantly, the results suggested a stabilized immune profile in the polyherbal arm over the study period of 2 years. In the PHF subjects, the CD4 cell loss was remarkably low, on the average only 14 cells lost per year (as opposed to the expected loss of 60-100 cells or more per year). On the other hand, the viral load in the blood remained stable without a significant increase during the study period. In the ART arm, the results were as expected, with a significant improvement in the CD4 cell count (154.4 cells gained per year) and a significant control of the viral load in the blood within 3 to 6 months.Importantly, when the PHF and ART arms were compared for the AIDS-related illnesses (opportunistic infections, malignancies, or death etc), there were no significant differences between these two groups over the study period. This is an important observation. For instance, a previous study from India showed that in the absence of medical intervention, nearly half of the subjects with low CD4 cell numbers are likely to die within a span of 33 months (Kumarasamy N et al, Natural history of human immunodeficiency virus disease in southern India. Clinical Infectious Diseases, 36,79-85, 2003). Although all the participants in the present clinical trial contained a low CD4 cell count (200-250 cells) at the beginning of the study, the AIDS-related clinical manifestations in the PHF group were as low as in the ART group.
The significance of our work: Collectively, our results appear to suggest that the polyherbal formulation may have provided protection by slowing the loss of CD4 cells in the blood. In the absence of a potential therapeutic vaccine for HIV-AIDS and given the various limitations characteristic of the anti-retroviral therapies, the identification of a potential ‘immunomodulator’ to control HIV-AIDS is of paramount importance. Our study generated high quality data from meticulous study design and execution. Inferences drawn from this prospective study are supported by a large number of repeat observations from each participant, as many as 10 in a large number of subjects and at least 5 in others. Monitoring the AIDS-related illnesses added strength to the present study. Our study also used an arm consisting of the standard anti-retroviral therapy for comparison thus adding authenticity to the analyses. The progress of the study was monitored by the NGO and its Institutional Ethics Committee, but not by the study investigators, thus guarding against any possible vested interests.
An important caution: In spite of the encouraging results,under no circumstances, the polyherbal formulation evaluated in the present clinical trial must be considered a ‘drug’ for HIV-AIDS. The experimental data are only indicative but not assertive of the beneficial effects. This is because our study was only a pilot clinical trial, not a randomized, multi-centric and placebo-controlled large-scale clinical trial. The intrinsic limitations of the study design, that were unavoidable due to practical and ethical considerations, do not permit drawing an assertive inference from the data. Despite these limitations, the study generated high quality data and offered potential leads. The beneficial effects, however, must be confirmed in future clinical trials that are rigorous and uncompromising in study design.
A follow up of the clinical trial: The only way to confirm our results is to conduct a larger and a rigorous clinical trial which was an expensive proposition. As a way out, we did a follow up analysis using the blood cells stored in the laboratory from the above pilot clinical trial. Using flow cytometry, we tested the hypothesis that if the quality of life of the study participants is improved in the polyherbal arm, the function of their blood cells (the CD4 T-cells) must have also improved. The immune analysis of the follow up study identified five independent and potential biomarkers, including soluble CD14, all of them collectively suggesting the control of chronic immune activation in the polyherbal arm which should be clinically beneficial. The follow up study also suggested a strong association between the enhanced immune responses and the improved liver function. Our studies thus collectively offer several important leads towards novel ways of HIV disease management.
Ranga U and Arunachalam PS, HIV-AIDS is different: what goes up doesn't come down, NARI Bulletin, 3, 2-6, 2012
Asokan M et al, Evident stabilization of the clinical profile in HIV/AIDS as evaluated in an open label clinical trial using a polyherbal formulationIndian Journal of Medical Research, 137, 1128-44, 2013
Asokan M et al, Attenuation of Immune Activation in an Open-Label Clinical Trial for HIV-AIDS Using a Polyherbal Formulation,VirusDisease, 2014 (in press)
|Optimization of DNA vaccines by engineering molecular adjuvants (HIV-1 Tat as a modular antigen): DNA vaccines offer the advantage of targeting the encoded antigens to the MHC class I pathway. However, DNA vaccines are not efficient in eliciting strong immune responses in larger animals on one hand and augmenting humoral immune responses on the other hand. One way to improve the function of DNA vaccines is to engineer molecular adjuvants into the DNA expression vectors that non-specifically induce desirable immune responses. We evaluated a few such molecular approaches to improve antigenicity of DNA vaccines in the mouse model.|