Ignite Creation Date:
2024-05-06 @ 8:24 PM
Last Modification Date:
2024-10-26 @ 3:26 PM
Study NCT ID:
NCT06363500
Status:
COMPLETED
Last Update Posted:
2024-04-12
First Post:
2024-04-09
Brief Title:
Characterization of HIV-1 Reservoirs in HIV-1 Non-B Infected Adolescents on ART in Cameroon
Sponsor:
Chantal Biya International Reference Centre for Research on Prevention and Management of HIVAIDS
Organization:
Chantal Biya International Reference Centre for Research on Prevention and Management of HIVAIDS
Study Overview
Official Title:
Characterization of HIV-1 Reservoirs in HIV Adolescents
Status:
COMPLETED
Status Verified Date:
2024-04
Last Known Status:
None
Delayed Posting:
No
If Stopped, Why?:
Not Stopped
Has Expanded Access:
False
If Expanded Access, NCT#:
N/A
Has Expanded Access, NCT# Status:
N/A
Acronym:
AVIR
Brief Summary:
Background Combination antiretroviral therapy cART can bring HIV-1 in blood plasma to level undetectable by standard tests and allow a near-normal life expectancy for HIV-infected individuals Unfortunately cART is not curative as within a few weeks of treatment cessation HIV viremia in most patients rebounds except for rare elite or post-treatment controllers of viremia The primary source of this rebound is the highly stable reservoir of latent yet replication-competent HIV-1 proviruses integrated into the genomic DNA of resting memory CD4 T cells To achieve a cure for HIV understanding the cell reservoir environment is of paramount importance The size and nature of viral reservoir might vary per timing of therapy therapeutic response ART duration and immune response Mechanisms of reservoir maintenance generally depend on levelstype of immune recognition dynamics of viral persistence are different between pediatric and adult populations owing to but not limited to typesnumbers of target cells efficiency in clearing HIV-infected cells plasma viremia and HIV drug resistance patterns This difference could become more evident as these children grow toward adolescence increasing population due to ART benefits a stage during which suboptimal adherence is frequent leading to viral rebound and archiving of resistant patterns
Objectives We plan to conduct a cross sectional study with the aim to characterize HIV reservoirs and their variability according to virological and immunological profiles of non-B HIV-1 vertically infected adolescents receiving antiretroviral therapy Specifically we shall 1 evaluate the size of HIV reservoir 2 Determine HIV-1 genetic variability and drug resistance in cellular reservoirs 3 Characterize immune activationinflammation of HIV infected adolescents
Methods We plan to conduct an observational and comparative study involving 90 HIV-1 non-B infected adolescents aged 10-19 years vertically infected have been on ART for at least 12 months selected from a cohort of the ongoing EDCTP-READY study intravenous blood will be collected for CD4CD8 count plasmatic viral load PBMCs isolation immune activationinflammatory markers genotyping and Viral reservoir quantification We will as well recruit a group of 30 HIV-negative adolescents as control for immunological profiling
Overall impact Our findings will help in advancing knowledge on HIV reservoir in terms of size and genetic variability in adolescents living with HIV ADLHIV Such evidence will also help in understanding the effects of ART timing and duration on the size of reservoirs among ADLHIV a unique population from whom findings generated will largely contribute in designing functional cure strategies in this vulnerable population
Objectives We plan to conduct a cross sectional study with the aim to characterize HIV reservoirs and their variability according to virological and immunological profiles of non-B HIV-1 vertically infected adolescents receiving antiretroviral therapy Specifically we shall 1 evaluate the size of HIV reservoir 2 Determine HIV-1 genetic variability and drug resistance in cellular reservoirs 3 Characterize immune activationinflammation of HIV infected adolescents
Methods We plan to conduct an observational and comparative study involving 90 HIV-1 non-B infected adolescents aged 10-19 years vertically infected have been on ART for at least 12 months selected from a cohort of the ongoing EDCTP-READY study intravenous blood will be collected for CD4CD8 count plasmatic viral load PBMCs isolation immune activationinflammatory markers genotyping and Viral reservoir quantification We will as well recruit a group of 30 HIV-negative adolescents as control for immunological profiling
Overall impact Our findings will help in advancing knowledge on HIV reservoir in terms of size and genetic variability in adolescents living with HIV ADLHIV Such evidence will also help in understanding the effects of ART timing and duration on the size of reservoirs among ADLHIV a unique population from whom findings generated will largely contribute in designing functional cure strategies in this vulnerable population
Detailed Description:
Title Characterization of Viral Reservoirs among HIV-1 non-B Vertically Infected Adolescents receiving Antiretroviral Therapy in Cameroon
Acronym AVIR-study Adolescent viral reservoir
Importance and relevance Sub-Saharan Africa SSA is disproportionally affected with HIVAIDS with close to 70 of the global epidemics With the highest burden of paediatric HIV infections about nine out of every 10 children living with HIV found in the SSA region1 2 Adolescents and young people represent a growing share of people living with HIV worldwide In 2019 about 17 million 11 million-24 million adolescents between the ages of 10 and 19 were living with HIV worldwide representing about 5 of all people living with HIV about 15 million 10 - 21 million or 88 of HIV infected adolescent live in SSA In 2019 alone 460000 260000-680000 young people between the ages of 10 to 24 were newly infected with HIV of whom 170000 53000-340000 were adolescents between the ages of 10 and 19 To compound this most recent data indicate that only 27 of adolescent girls and 16 of adolescent boys aged 15-19 in Eastern and Southern Africa - the region most affected by HIV - have been tested for HIV in the past 12 months and received the result of the last test The testing rates in West and Central Africa and South Asia are even lower If current trends continue hundreds of thousands more will become HIV-positive in the coming years and without knowing their status adolescents will miss out on life-saving treatment Additionally a large population of children infected with HIV perinatally over the last decade are growing into adolescence 3 Of the estimated 690000 people who died of AIDS-related illnesses in 2019 110000 or approximately 16 of them were children under 20 years of age UNICEF global and regional trends July 2020
With the advent and scalability of Antiretroviral therapy ART there is a global decrease in AIDS-related deaths As of the end of 2019 254 million people were accessing antiretroviral therapy representing 67 of all people living with HIV However only 53 of children living with HIV were receiving ART Interestingly about 94 of all children receiving ART are from SSA 4 In this context of continuous new HIV paediatric infections and increasing coverage in paediatric ART the number of children living with HIV will increase suggesting a higher likelihood of reaching adolescent age and even adulthood if treatment regimens remain fully effective in controlling HIV infection 3 4 ADLHIV therefore constitutes an HIV population with growing health concerns and with very limited findings for generalizable best practices specific to this target population especially in SSA
Like several West and Central African countries Cameroon is still faced with a generalized HIV epidemiology 27 prevalence 5 with higher prevalence among pregnant women 57 and HIV-exposed infantschildren 58 95816638 positivity at first PCR and 15 at the end of the PMTCT cascade 6 As of June 2018 the national coverage of ART was 515 281083 which includes 12362 children below 15 years of age 6 Regarding response to ART in Cameroon we reported an overall rate of 794 viral suppression with significant disparities across age ranges 811 in adults 756 in children and only 533 in adolescent aged 10-19 years 7 Similar to UNAIDS reports adolescents living with HIV ADLHIV represent the most vulnerable and underserved population in response to the epidemics1 5 For a safer growth toward adulthood there is need to prioritize this population for the quest of innovative treatment strategies that ensure their well-being and their contribution for the development of SSA
In spite of the unquestioned benefits of ART there are limitations with current treatment strategies Of note the lifelong nature of current ART goes with challenges related adherence for most patients ART-attributed toxicities and persisting immune dysfunction have significant health impairments and HIV drug resistance HIVDR is increasing mostly in SSA countries where most ART-experienced patients are living 8 There is a threat of an emerging new HIV epidemic driven by HIVDR to existing antiretroviral These challenges are particularly true for paediatric populations due to limited ART options poor drug formulations and increasing events of non-adherence as they grow toward adolescence These challenges call for approaches toward HIV functional cure or remission especially for the most vulnerable populations ie ADLHIV 7 9
Concepts underpinning the project including ideas and models or assumptions HIV-1 remission or eradication strategies aim to achieve viral remission in the absence of antiretroviral therapy ART The development of an HIV-1 cure remains challenging due to the latent reservoirs The HIV-1 Latent Reservoir LR can be defined as the fraction of cells harbouring transcriptionally silent proviral DNA that are capable of producing infectious virions following activation10 Resting memory CD4T cells are the primary host of the LR but HIV-1 infection in these cells is inefficient due their low co-receptor expression and inherent restrictions to reverse transcription 11 12 Nevertheless there is evidence that HIV-1 can infect resting CD4T cells directly or via cell-to-cell transmission though infection in these cells is associated with slower replication kinetics 13 Alternatively latency is established when a subset of infected activated CD4T cells revert to a resting memory phenotype effectively silencing viral gene expression whilst sustaining the proviral DNA long-term14 The provirus is maintained in a quiescent state in these cells via host factors such as epigenetic suppression depletion of transcription factors such as NF-κB and transcriptional interference due to integration into expressed genes reviewed in more detail 15 Viral rebound from the LR following ART cessation is rapid leading to detectable viremia within weeks of therapy interruption 16 Additionally initiating ART early in infection is not sufficient to stop the formation of the LR suggesting the LR is established and disseminated early Chun et al 1998 Whitney et al 2014 Colby et al 2018 even in vertically infected children that started ART soon after birth 17 The latent reservoir is stable despite years of suppressive ART and is the source of rebound viremia following therapy interruption Latently infected cells therefore represent the principle barrier to an HIV- 1 cure and should be specifically targeted by novel treatment and eradication strategies
The progress toward the development of a functional or sterilizing cure for HIV-1 has been significantly hindered by the presence of the LR Currently two people have been cured of HIV-1 infection the so-called Berlin and London patients who since receiving allogenic stem cell transplantations from CCR53232 donors have consistently tested negative for viral rebound for over 10 and 2 years respectively without ART 18 In these cases the infected cell pool was significantly depleted during pre- transplant conditioning and replaced with donor cells that are resistant to infection with R5-tropic virus due a large deletion in the CCR5 co-receptor 19 Due to the relative paucity of CCR5 3232 donors and the unique circumstances predetermining these cases this type of cure is not feasible for widespread use it does however emphasize the basic principle of HIV-1 cure silence or eradicate the HIV-1 LR 20 Over the past decade understanding of where and how HIV persists in individuals on ART has transformed substantially with evidence that virus persists in multiple cell types and tissue sites and in both quiescent and proliferating long-lived latently infected cells Thus in the frame of a suppressive treatment accurate estimates of the viral reservoir would help in a better mastering of viral persistence which in turn might overcome existing barriers for achieving a cure21
Total HIV DNA is a reference biomarker that includes both integrated and unintegrated HIV DNA and reflects the global level of the viral reservoir Importantly Buzon et al reported a statistical correlation between the time from HIV infection to treatment initiation and the total HIV DNA level after 10 years of continuous treatment in a cohort of adults first treated with early infection 22 In children the HIV DNA level was markedly lower when viral control was achieved before the age of 1 year 23 By comparison with other markers total HIV DNA has the advantage of easy quantification by standardized sensitive real- time PCR including digital droplet PCR ddPCR 24
Generalized immune activation is a hallmark of HIV-1 infection In this state a variety of immune cells show an increase in expression of activation proliferation and apoptotic markers cellular turnover with aberrant cell cycle regulation production of pro-inflammatory cytokines and increased lymphoid tissue fibrosis 25 Immune activation is strongly associated with HIV-1 disease progression for instance T cell activation as measured by expression of CD38 and HLA-DR is more predictive of CD4T cell depletion and shorter survival than is the plasma viral load 26 27 Furthermore the level of immune activation early in HIV-1 infection as measured by CD8T cell activation predicts CD4T cell loss independently of plasma HIV-1 RNA levels 28 Suppression of viral replication with effective antiretroviral treatment ART reduces immune activation but even effective ART regimens are unable to reduce the levels of immune activation in HIV-infected individuals to levels seen in healthy individuals 25 HIV-infected children even if successfully treated with ART face a lifetime of elevated immune activation Thus evaluating the potential impact of chronic immune activation and inflammation on the developing immune system and on disease outcome in paediatric HIV infection is of particular importance Recent studies showed that immune activation HLA-DR CD38 and exhaustion markers Tim-3 PD-1 Lag-3 are strongly associated with reservoir size in ART- treated adults thus it might be anticipated that minimizing the viral reservoir with early ART might equally minimize the level of immune activation and the non-AIDS ageing diseases associated with persistent immune activation
There is limited evidence in characterizing HIV reservoirs in West and Central African region a geographical setting having a highest variability in circulating HIV-1 and HIV-2 strains 29 30 For example Cameroon a zoonotic epicentre of HIV-1 is host to an extensively diverse landscape of HIV driven by the CRF02_AG recombinant including most group M sub- subtypes a vast array of URFs and CRFs and group N O P and HIV-2 viruses31-33 Thus generating baseline data on the genotypic and quantitative profile of the viral reservoir across several HIV clades in setting like Cameroon would inform the design of optimal strategies for HIV cure Considering the aforementioned vulnerability of adolescents with vertical infection and the limited knowledge on viral reservoirs and immune activationinflammatory reaction in this population evidence generated from this target will be highly complementary to current global efforts Such evidence generated in a context of high burden of co-infections 34 35 might depict differential mechanisms of HIV persistence far from those reported in other parts of the World
Preliminary work
Within the frame of the ongoing EDCTP READY-Study we have set up a cohort of 292 vertically infected adolescents 10-19 years receiving ART in Cameroon In this cohort we reported a rate of 40 undetectable viral load 40 copiesmL after a median of 8 years of ART about 20 immunological failure CD4250 cellsmm3 rate and less than 10 clinical failure ie WHO stages IIIIV 36 This population offers a unique opportunity for understanding the size and nature of the reservoir the variability of immune responsecytokine profiling and the effect of viral subtype treatment history ART regimen and duration gender disparities and adherence level on the control of viral reservoir
AIM
The aim of this study is to characterize HIV reservoirs and their variability according to virological and immunological profiles of vertically infected adolescents receiving antiretroviral therapy in Cameroon and therefore improve understanding of viral reservoirs and provide accurate and reliable data for HIV cure research
Specific Objectives
In this study we shall 1- Determine HIV-1 genetic variability and drug resistance in cellular reservoirs 2- Characterize immune activationinflammation in adolescents vertically infected with HIV-1 3- Evaluate the size of HIV reservoir 4- Evaluate the effect of antiretroviral therapy and immune response on the viral reservoir profile
Materials and Methods Study design We plan to conduct an observational and comparative study among adolescent living with HIV and receiving ART in Cameroon Participants will be selected and enrolled from an existing cohort of close to 300 vertically infected adolescents recruited for the EDCTP-READY study
The EDCTP-READY-Study main objective was to evaluate treatment response drug resistance and HIV-1 variability among adolescents on first- and second-line antiretroviral therapy in Cameroon 7 In this study we recruited 296 HIV adolescents and compared the HIV-1 genotypic profile in circulating-RNA Out of 30 89296 APHI experiencing virological failure VL1000 copiesmL
Sampling method
Blood samples will be collected from participants of EDCTP-READY study according to the eligibility criteria
Eligibility criteria
Inclusion criteria Will be enrolled in the study HIV adolescents aged 10-19 years old vertically infected have been on ART for at least 12 months who provide their written assent and whose legal guardian provide written proxy-consent
Non-inclusion criteria Will not be considered for enrolment HIV adolescent with incomplete ART history HBVHCV and malaria coinfections
Exclusion criteria Will be considered excluded from the study any participant who decides freely to withdraw after had given consent or unable to give blood sample
Sample size The minimum sample size N of the study is 90 participants calculated based on the following formula Np 1-p Zα2α2 N 0021-002 19620052 30 per arm arm-A VL40 copiesmL arm-B VL 40-999 copiesmL arm-C viral load 1000 copiesmL P is the prevalence of adolescents living with HIV in Cameroon with P2 3 α being the risk agrees with α 5 Zα being the small gap with Zα 196
NB A group of 30 HIV-negative adolescents will serve as control for immunological profiling
All participants will be assigned a new identification number and data will be centralized in a password-protected computer at the International Reference Centre Chantal Biya CIRCB for research on HIVAIDS prevention and management
Study procedures and variables Procedures and timelines The present study requires 18 months to be completed 3 months month 1-3 for administration and ethics approval 12 months month 4 to 15 for enrolment of participants sampling and laboratory analyses 3 months month 16 to 18 for data processing and reporting see Gantt chart for more details
Based on inclusion criteria HIV adolescents case report forms will be selected from EDCTP-READY study and their legal guardian will be contacted Assent and proxy-informed consent will be obtained from adolescents and legal guardian respectively Socio-demographic clinical data and ART history will be assessed again for the period during enrolment in EDCTP-READY study Then 90 or more HIV adolescents 30 per arm arm-A VL40 copiesmL arm-B VL 40-999 copiesmL arm-C viral load 1000 copiesmL will be enrolled A group of 30 HIV-negative adolescents will be enrolled to serve as control for immunological profiling Intravenous blood 5ml x 2 will be collected by a trained phlebotomist for lab analyses including PBMCs isolation plasma and buffy coat isolation CD4CD8 counts plasmatic viral load PVL immune activation inflammatory markers assessment HIV-RNA and HIV-DNA genotyping quantification of viral reservoirs
HIV negative participants will be recruited at the CIRCB this institution is a reference Centre for management of HIV infected individuals since 2006 and as such is renown reference centre for HIV testing Base on this advantage CIRCB organizes free HIVHBVHCV screening tests for several institutions and communities per year therefore a similar free testing campaign will be organized in a nearby secondary school and the present study will be presented to those fulfilling inclusion criteria Potential participants will be invited at CIRCB with their legal guardian for enrolment
Samples will be collected only once at enrolment as well as all relevant socio-demographic and clinical data Samples will be transported to CIRCB the same day for PBMCs isolation DNA plasma and buffy coat isolation CD4CD8 counts and activation inflammatory markers assessment using flow cytometry Plasma will be collected and stored at -80oC for plasmatic viral load PVL genotyping immune activation inflammatory markers assessment using ELISA tests or flow cytometry HIV-DNA will be subsequently extracted from PBMCs and stored at -80oC HIV-DNA will be shipped every three months to the department of experimental medicine University of Rome Tor Vergata Rome Italy for HIV viral reservoir quantification see letter of collaboration in annex section
Laboratory procedures CD4CD8 count CD4CD8 measurements will be determined using flow cytometry machine as previously described 37
Plasma viral load Plasma viral load will be determined using m 2000rt Abbott real time system as previously described 38
HIV-RNA extraction ARN will be extracted from plasma samples using Qiamp viral RNA Mini Kit Qiagen as described by the manufacturer
Pro-viral DNA extraction from buffy coat Pro-viral DNA will be extracted using DNeasy blood and tissue extraction kit Qiagen as described by the manufacturer
PBMCs isolation PBMCs will be isolated by density gradient centrifugation using Ficoll using an in-house protocol
HIV-DNA extraction from PBMCs HIV-DNA will be extracted using QIAmp DNA Mini Kit Qiagen as described by the manufacturer
HIV-1 Genes amplification and sequencing Sanger Sequencing for HIV-1 drug resistance testing will be performed for adolescent experiencing virological failure an in-house protocol as previously described by our research group39 Reverse transcriptase DNA sequences will be analysed for drug resistance mutations using the Stanford University database genotypic resistance interpretation algorithm wwwstanfordedu
Phylogenetic analysis Neighbour Joining phylogenetic trees will be created using MEGA software Kimura 2-parameter model 200 bootstrap replications and FigTree 40 41
Immune profiling Both immune activationinflammatory cytokines IL-1 IL2IL4 IL6 IL10 IL12 and TNF-alpha will be assessed using commercial ELISA kits and flow cytometry
Quantification of HIV viral reservoirs viral reservoirs will be quantified using droplet digital PCR ddPCR as previously described Of note this quantification will be performed in the department of experimental medicine University of Rome Tor Vergata Rome Italy as stated in the letter of collaboration found in annex of this proposal A material transfer and a data sharing agreement will be signed between CIRCB and UTV prior to sample shipment to UTV It is an in-house protocole for HIV-1 B subtype and will be adjusted for HIV-1 non-B subtypes expected from Cameroonian samples
Data analysis Data collected during the study will be analyzed using SPSS software Associated factors will be evaluated using multivariate logistic regression with an estimate approach for the unbiased effect of different parameters The data will be reported as median Non-parametric tests will be used for data not normally distributed comparisons of medians among different groups virological success vs virological failure will be performed by the U-Mann- Whitney test Correlations will be made with Spearman test and p values less than 005 will be considered to be statistically significant
Ethical considerations This study will be conducted according to the declaration of Helsinki on ethical principles for medical research involving human subjects Ethical clearance will be obtained from the Cameroon National Ethics committee for research on human health after an informed notice on the study a written proxy-informed will be obtained from legal guardian and a written assent will also be obtained from the participating HIV adolescent without any coercion Privacy and confidentiality will be ensured through the use of unique identifiers and a password protected database and will be accessed only by authorized staff Participants will be free to deliberately leave the study at any time without any effect on their routine monitoring at the study clinic Phlebotomy will be non-invasive venipuncture and will be performed by a trained nurse
Quality assurance
Our study team will include a quality assurance officer who will be responsible of all SOPs for the study protocol and will manage proficiency testing and data validation during the entire study
Management of potential risks The risks to the patients are minimal since the only procedure the volunteer is subjected to is venipuncture by a phlebotomist or physician The venipuncture may be slightly painful but is practically without risk of complication The potential risks to subjects none of which are likely to occur may include momentary pain bruising at the site or possible but extremely unlikely infection If such complications arise participant will be provided with emergency medical treatment
Acronym AVIR-study Adolescent viral reservoir
Importance and relevance Sub-Saharan Africa SSA is disproportionally affected with HIVAIDS with close to 70 of the global epidemics With the highest burden of paediatric HIV infections about nine out of every 10 children living with HIV found in the SSA region1 2 Adolescents and young people represent a growing share of people living with HIV worldwide In 2019 about 17 million 11 million-24 million adolescents between the ages of 10 and 19 were living with HIV worldwide representing about 5 of all people living with HIV about 15 million 10 - 21 million or 88 of HIV infected adolescent live in SSA In 2019 alone 460000 260000-680000 young people between the ages of 10 to 24 were newly infected with HIV of whom 170000 53000-340000 were adolescents between the ages of 10 and 19 To compound this most recent data indicate that only 27 of adolescent girls and 16 of adolescent boys aged 15-19 in Eastern and Southern Africa - the region most affected by HIV - have been tested for HIV in the past 12 months and received the result of the last test The testing rates in West and Central Africa and South Asia are even lower If current trends continue hundreds of thousands more will become HIV-positive in the coming years and without knowing their status adolescents will miss out on life-saving treatment Additionally a large population of children infected with HIV perinatally over the last decade are growing into adolescence 3 Of the estimated 690000 people who died of AIDS-related illnesses in 2019 110000 or approximately 16 of them were children under 20 years of age UNICEF global and regional trends July 2020
With the advent and scalability of Antiretroviral therapy ART there is a global decrease in AIDS-related deaths As of the end of 2019 254 million people were accessing antiretroviral therapy representing 67 of all people living with HIV However only 53 of children living with HIV were receiving ART Interestingly about 94 of all children receiving ART are from SSA 4 In this context of continuous new HIV paediatric infections and increasing coverage in paediatric ART the number of children living with HIV will increase suggesting a higher likelihood of reaching adolescent age and even adulthood if treatment regimens remain fully effective in controlling HIV infection 3 4 ADLHIV therefore constitutes an HIV population with growing health concerns and with very limited findings for generalizable best practices specific to this target population especially in SSA
Like several West and Central African countries Cameroon is still faced with a generalized HIV epidemiology 27 prevalence 5 with higher prevalence among pregnant women 57 and HIV-exposed infantschildren 58 95816638 positivity at first PCR and 15 at the end of the PMTCT cascade 6 As of June 2018 the national coverage of ART was 515 281083 which includes 12362 children below 15 years of age 6 Regarding response to ART in Cameroon we reported an overall rate of 794 viral suppression with significant disparities across age ranges 811 in adults 756 in children and only 533 in adolescent aged 10-19 years 7 Similar to UNAIDS reports adolescents living with HIV ADLHIV represent the most vulnerable and underserved population in response to the epidemics1 5 For a safer growth toward adulthood there is need to prioritize this population for the quest of innovative treatment strategies that ensure their well-being and their contribution for the development of SSA
In spite of the unquestioned benefits of ART there are limitations with current treatment strategies Of note the lifelong nature of current ART goes with challenges related adherence for most patients ART-attributed toxicities and persisting immune dysfunction have significant health impairments and HIV drug resistance HIVDR is increasing mostly in SSA countries where most ART-experienced patients are living 8 There is a threat of an emerging new HIV epidemic driven by HIVDR to existing antiretroviral These challenges are particularly true for paediatric populations due to limited ART options poor drug formulations and increasing events of non-adherence as they grow toward adolescence These challenges call for approaches toward HIV functional cure or remission especially for the most vulnerable populations ie ADLHIV 7 9
Concepts underpinning the project including ideas and models or assumptions HIV-1 remission or eradication strategies aim to achieve viral remission in the absence of antiretroviral therapy ART The development of an HIV-1 cure remains challenging due to the latent reservoirs The HIV-1 Latent Reservoir LR can be defined as the fraction of cells harbouring transcriptionally silent proviral DNA that are capable of producing infectious virions following activation10 Resting memory CD4T cells are the primary host of the LR but HIV-1 infection in these cells is inefficient due their low co-receptor expression and inherent restrictions to reverse transcription 11 12 Nevertheless there is evidence that HIV-1 can infect resting CD4T cells directly or via cell-to-cell transmission though infection in these cells is associated with slower replication kinetics 13 Alternatively latency is established when a subset of infected activated CD4T cells revert to a resting memory phenotype effectively silencing viral gene expression whilst sustaining the proviral DNA long-term14 The provirus is maintained in a quiescent state in these cells via host factors such as epigenetic suppression depletion of transcription factors such as NF-κB and transcriptional interference due to integration into expressed genes reviewed in more detail 15 Viral rebound from the LR following ART cessation is rapid leading to detectable viremia within weeks of therapy interruption 16 Additionally initiating ART early in infection is not sufficient to stop the formation of the LR suggesting the LR is established and disseminated early Chun et al 1998 Whitney et al 2014 Colby et al 2018 even in vertically infected children that started ART soon after birth 17 The latent reservoir is stable despite years of suppressive ART and is the source of rebound viremia following therapy interruption Latently infected cells therefore represent the principle barrier to an HIV- 1 cure and should be specifically targeted by novel treatment and eradication strategies
The progress toward the development of a functional or sterilizing cure for HIV-1 has been significantly hindered by the presence of the LR Currently two people have been cured of HIV-1 infection the so-called Berlin and London patients who since receiving allogenic stem cell transplantations from CCR53232 donors have consistently tested negative for viral rebound for over 10 and 2 years respectively without ART 18 In these cases the infected cell pool was significantly depleted during pre- transplant conditioning and replaced with donor cells that are resistant to infection with R5-tropic virus due a large deletion in the CCR5 co-receptor 19 Due to the relative paucity of CCR5 3232 donors and the unique circumstances predetermining these cases this type of cure is not feasible for widespread use it does however emphasize the basic principle of HIV-1 cure silence or eradicate the HIV-1 LR 20 Over the past decade understanding of where and how HIV persists in individuals on ART has transformed substantially with evidence that virus persists in multiple cell types and tissue sites and in both quiescent and proliferating long-lived latently infected cells Thus in the frame of a suppressive treatment accurate estimates of the viral reservoir would help in a better mastering of viral persistence which in turn might overcome existing barriers for achieving a cure21
Total HIV DNA is a reference biomarker that includes both integrated and unintegrated HIV DNA and reflects the global level of the viral reservoir Importantly Buzon et al reported a statistical correlation between the time from HIV infection to treatment initiation and the total HIV DNA level after 10 years of continuous treatment in a cohort of adults first treated with early infection 22 In children the HIV DNA level was markedly lower when viral control was achieved before the age of 1 year 23 By comparison with other markers total HIV DNA has the advantage of easy quantification by standardized sensitive real- time PCR including digital droplet PCR ddPCR 24
Generalized immune activation is a hallmark of HIV-1 infection In this state a variety of immune cells show an increase in expression of activation proliferation and apoptotic markers cellular turnover with aberrant cell cycle regulation production of pro-inflammatory cytokines and increased lymphoid tissue fibrosis 25 Immune activation is strongly associated with HIV-1 disease progression for instance T cell activation as measured by expression of CD38 and HLA-DR is more predictive of CD4T cell depletion and shorter survival than is the plasma viral load 26 27 Furthermore the level of immune activation early in HIV-1 infection as measured by CD8T cell activation predicts CD4T cell loss independently of plasma HIV-1 RNA levels 28 Suppression of viral replication with effective antiretroviral treatment ART reduces immune activation but even effective ART regimens are unable to reduce the levels of immune activation in HIV-infected individuals to levels seen in healthy individuals 25 HIV-infected children even if successfully treated with ART face a lifetime of elevated immune activation Thus evaluating the potential impact of chronic immune activation and inflammation on the developing immune system and on disease outcome in paediatric HIV infection is of particular importance Recent studies showed that immune activation HLA-DR CD38 and exhaustion markers Tim-3 PD-1 Lag-3 are strongly associated with reservoir size in ART- treated adults thus it might be anticipated that minimizing the viral reservoir with early ART might equally minimize the level of immune activation and the non-AIDS ageing diseases associated with persistent immune activation
There is limited evidence in characterizing HIV reservoirs in West and Central African region a geographical setting having a highest variability in circulating HIV-1 and HIV-2 strains 29 30 For example Cameroon a zoonotic epicentre of HIV-1 is host to an extensively diverse landscape of HIV driven by the CRF02_AG recombinant including most group M sub- subtypes a vast array of URFs and CRFs and group N O P and HIV-2 viruses31-33 Thus generating baseline data on the genotypic and quantitative profile of the viral reservoir across several HIV clades in setting like Cameroon would inform the design of optimal strategies for HIV cure Considering the aforementioned vulnerability of adolescents with vertical infection and the limited knowledge on viral reservoirs and immune activationinflammatory reaction in this population evidence generated from this target will be highly complementary to current global efforts Such evidence generated in a context of high burden of co-infections 34 35 might depict differential mechanisms of HIV persistence far from those reported in other parts of the World
Preliminary work
Within the frame of the ongoing EDCTP READY-Study we have set up a cohort of 292 vertically infected adolescents 10-19 years receiving ART in Cameroon In this cohort we reported a rate of 40 undetectable viral load 40 copiesmL after a median of 8 years of ART about 20 immunological failure CD4250 cellsmm3 rate and less than 10 clinical failure ie WHO stages IIIIV 36 This population offers a unique opportunity for understanding the size and nature of the reservoir the variability of immune responsecytokine profiling and the effect of viral subtype treatment history ART regimen and duration gender disparities and adherence level on the control of viral reservoir
AIM
The aim of this study is to characterize HIV reservoirs and their variability according to virological and immunological profiles of vertically infected adolescents receiving antiretroviral therapy in Cameroon and therefore improve understanding of viral reservoirs and provide accurate and reliable data for HIV cure research
Specific Objectives
In this study we shall 1- Determine HIV-1 genetic variability and drug resistance in cellular reservoirs 2- Characterize immune activationinflammation in adolescents vertically infected with HIV-1 3- Evaluate the size of HIV reservoir 4- Evaluate the effect of antiretroviral therapy and immune response on the viral reservoir profile
Materials and Methods Study design We plan to conduct an observational and comparative study among adolescent living with HIV and receiving ART in Cameroon Participants will be selected and enrolled from an existing cohort of close to 300 vertically infected adolescents recruited for the EDCTP-READY study
The EDCTP-READY-Study main objective was to evaluate treatment response drug resistance and HIV-1 variability among adolescents on first- and second-line antiretroviral therapy in Cameroon 7 In this study we recruited 296 HIV adolescents and compared the HIV-1 genotypic profile in circulating-RNA Out of 30 89296 APHI experiencing virological failure VL1000 copiesmL
Sampling method
Blood samples will be collected from participants of EDCTP-READY study according to the eligibility criteria
Eligibility criteria
Inclusion criteria Will be enrolled in the study HIV adolescents aged 10-19 years old vertically infected have been on ART for at least 12 months who provide their written assent and whose legal guardian provide written proxy-consent
Non-inclusion criteria Will not be considered for enrolment HIV adolescent with incomplete ART history HBVHCV and malaria coinfections
Exclusion criteria Will be considered excluded from the study any participant who decides freely to withdraw after had given consent or unable to give blood sample
Sample size The minimum sample size N of the study is 90 participants calculated based on the following formula Np 1-p Zα2α2 N 0021-002 19620052 30 per arm arm-A VL40 copiesmL arm-B VL 40-999 copiesmL arm-C viral load 1000 copiesmL P is the prevalence of adolescents living with HIV in Cameroon with P2 3 α being the risk agrees with α 5 Zα being the small gap with Zα 196
NB A group of 30 HIV-negative adolescents will serve as control for immunological profiling
All participants will be assigned a new identification number and data will be centralized in a password-protected computer at the International Reference Centre Chantal Biya CIRCB for research on HIVAIDS prevention and management
Study procedures and variables Procedures and timelines The present study requires 18 months to be completed 3 months month 1-3 for administration and ethics approval 12 months month 4 to 15 for enrolment of participants sampling and laboratory analyses 3 months month 16 to 18 for data processing and reporting see Gantt chart for more details
Based on inclusion criteria HIV adolescents case report forms will be selected from EDCTP-READY study and their legal guardian will be contacted Assent and proxy-informed consent will be obtained from adolescents and legal guardian respectively Socio-demographic clinical data and ART history will be assessed again for the period during enrolment in EDCTP-READY study Then 90 or more HIV adolescents 30 per arm arm-A VL40 copiesmL arm-B VL 40-999 copiesmL arm-C viral load 1000 copiesmL will be enrolled A group of 30 HIV-negative adolescents will be enrolled to serve as control for immunological profiling Intravenous blood 5ml x 2 will be collected by a trained phlebotomist for lab analyses including PBMCs isolation plasma and buffy coat isolation CD4CD8 counts plasmatic viral load PVL immune activation inflammatory markers assessment HIV-RNA and HIV-DNA genotyping quantification of viral reservoirs
HIV negative participants will be recruited at the CIRCB this institution is a reference Centre for management of HIV infected individuals since 2006 and as such is renown reference centre for HIV testing Base on this advantage CIRCB organizes free HIVHBVHCV screening tests for several institutions and communities per year therefore a similar free testing campaign will be organized in a nearby secondary school and the present study will be presented to those fulfilling inclusion criteria Potential participants will be invited at CIRCB with their legal guardian for enrolment
Samples will be collected only once at enrolment as well as all relevant socio-demographic and clinical data Samples will be transported to CIRCB the same day for PBMCs isolation DNA plasma and buffy coat isolation CD4CD8 counts and activation inflammatory markers assessment using flow cytometry Plasma will be collected and stored at -80oC for plasmatic viral load PVL genotyping immune activation inflammatory markers assessment using ELISA tests or flow cytometry HIV-DNA will be subsequently extracted from PBMCs and stored at -80oC HIV-DNA will be shipped every three months to the department of experimental medicine University of Rome Tor Vergata Rome Italy for HIV viral reservoir quantification see letter of collaboration in annex section
Laboratory procedures CD4CD8 count CD4CD8 measurements will be determined using flow cytometry machine as previously described 37
Plasma viral load Plasma viral load will be determined using m 2000rt Abbott real time system as previously described 38
HIV-RNA extraction ARN will be extracted from plasma samples using Qiamp viral RNA Mini Kit Qiagen as described by the manufacturer
Pro-viral DNA extraction from buffy coat Pro-viral DNA will be extracted using DNeasy blood and tissue extraction kit Qiagen as described by the manufacturer
PBMCs isolation PBMCs will be isolated by density gradient centrifugation using Ficoll using an in-house protocol
HIV-DNA extraction from PBMCs HIV-DNA will be extracted using QIAmp DNA Mini Kit Qiagen as described by the manufacturer
HIV-1 Genes amplification and sequencing Sanger Sequencing for HIV-1 drug resistance testing will be performed for adolescent experiencing virological failure an in-house protocol as previously described by our research group39 Reverse transcriptase DNA sequences will be analysed for drug resistance mutations using the Stanford University database genotypic resistance interpretation algorithm wwwstanfordedu
Phylogenetic analysis Neighbour Joining phylogenetic trees will be created using MEGA software Kimura 2-parameter model 200 bootstrap replications and FigTree 40 41
Immune profiling Both immune activationinflammatory cytokines IL-1 IL2IL4 IL6 IL10 IL12 and TNF-alpha will be assessed using commercial ELISA kits and flow cytometry
Quantification of HIV viral reservoirs viral reservoirs will be quantified using droplet digital PCR ddPCR as previously described Of note this quantification will be performed in the department of experimental medicine University of Rome Tor Vergata Rome Italy as stated in the letter of collaboration found in annex of this proposal A material transfer and a data sharing agreement will be signed between CIRCB and UTV prior to sample shipment to UTV It is an in-house protocole for HIV-1 B subtype and will be adjusted for HIV-1 non-B subtypes expected from Cameroonian samples
Data analysis Data collected during the study will be analyzed using SPSS software Associated factors will be evaluated using multivariate logistic regression with an estimate approach for the unbiased effect of different parameters The data will be reported as median Non-parametric tests will be used for data not normally distributed comparisons of medians among different groups virological success vs virological failure will be performed by the U-Mann- Whitney test Correlations will be made with Spearman test and p values less than 005 will be considered to be statistically significant
Ethical considerations This study will be conducted according to the declaration of Helsinki on ethical principles for medical research involving human subjects Ethical clearance will be obtained from the Cameroon National Ethics committee for research on human health after an informed notice on the study a written proxy-informed will be obtained from legal guardian and a written assent will also be obtained from the participating HIV adolescent without any coercion Privacy and confidentiality will be ensured through the use of unique identifiers and a password protected database and will be accessed only by authorized staff Participants will be free to deliberately leave the study at any time without any effect on their routine monitoring at the study clinic Phlebotomy will be non-invasive venipuncture and will be performed by a trained nurse
Quality assurance
Our study team will include a quality assurance officer who will be responsible of all SOPs for the study protocol and will manage proficiency testing and data validation during the entire study
Management of potential risks The risks to the patients are minimal since the only procedure the volunteer is subjected to is venipuncture by a phlebotomist or physician The venipuncture may be slightly painful but is practically without risk of complication The potential risks to subjects none of which are likely to occur may include momentary pain bruising at the site or possible but extremely unlikely infection If such complications arise participant will be provided with emergency medical treatment
Study Oversight
Has Oversight DMC:
None
Is a FDA Regulated Drug?:
False
Is a FDA Regulated Device?:
False
Is an Unapproved Device?:
None
Is a PPSD?:
None
Is a US Export?:
None
Is an FDA AA801 Violation?:
None