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 Table of Contents    
RESEARCH ARTICLE
Year : 2022  |  Volume : 54  |  Issue : 4  |  Page : 278-281
 

Immunological factors associated with discordant virological response postcombination antiretroviral therapy in pediatric human immunodeficiency virus infection


1 Department of Pediatrics; Department of Microbiology, AIIMS, New Delhi, India
2 Department of Microbiology, AIIMS, New Delhi, India
3 Department of Medicine, Maharishi Markandeshwar Medical College and Hospital, Solan, Himachal Pradesh, India
4 Department of Pediatrics, AIIMS, New Delhi, India

Date of Submission02-Aug-2021
Date of Decision29-Aug-2022
Date of Acceptance01-Sep-2022
Date of Web Publication04-Oct-2022

Correspondence Address:
Prof. Bimal Kumar Das
Department of Microbiology, AIIMS, New Delhi
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ijp.ijp_616_21

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 » Abstract 


OBJECTIVES: Evaluation of immunological factors responsible for discordant virological responses postcombination antiretroviral therapy (cART) in human immunodeficiency virus (HIV)-positive children aged <5 years.
MATERIALS AND METHODS: Immunological profiling of enrolled 30 HIV-positive children was done at enrollment, 6 and 12 months. Flow cytometric analysis was performed for enumeration of counts and percentage of CD4+, CD8+, and CD19+ cells; expression of CD19, CD86, PD-1, CD3, CD8 and CD28 on lymphocytes was evaluated using whole blood staining technique with monoclonal antibodies. HIV-1 viral load was quantified using a real-time polymerase chain reaction. Serum levels of immunoglobulin G (IgG), immunoglobulin A (IgA), immunoglobulin (IgM), and interleukin (IL)-7 were quantitated using quantitative enzyme-linked immunosorbent assay kits. The HIV-infected children were categorized into virological responders (VRs; HIV-1 plasma viral load <47 copies/mL) and virological nonresponders (VNRs; HIV-1 plasma viral load >1000 copies/mL) following 1-year cART.
RESULTS: The frequency of CD28+ CTLs cells was higher (P < 0.0001), and the frequency of CD28-CTLs cells was lower (P < 0.0001) in VRs than VNRs. CD28+ and CD28-CTLs cells correlated with HIV-1 plasma viremia (r = −0.4695, P = 0.01; r = 0.40, P = 0.03, respectively). VRs had higher CD19 percentage (P = 0.04) and count (P = 0.01) than VNRs. CD19+ B cells in the VRs had lower expression of CD86 (P = 0.03) and PD-1 (P = 0.002) than VNRs. VR had lower levels of serum IgG (P = 0.03), IgM (P = 0.04), and IL-7 (P = 0.01) than VNRs.
CONCLUSIONS: High baseline B-cell counts, lower serum IgG, IgM, IL-7 levels, lower activation and exhaustion of B cells, and higher frequency of CD28+ CTLs are associated with positive virological response, whereas elevated CD28-CTLs are associated with the poor virological outcomes in HIV-infected children.


Keywords: Combination antiretroviral therapy, CD28+ CTLs, human immunodeficiency virus, immunoglobulins, virological responders


How to cite this article:
Singh R, Maurya SP, Das N, Kabra SK, Lodha R, Das BK. Immunological factors associated with discordant virological response postcombination antiretroviral therapy in pediatric human immunodeficiency virus infection. Indian J Pharmacol 2022;54:278-81

How to cite this URL:
Singh R, Maurya SP, Das N, Kabra SK, Lodha R, Das BK. Immunological factors associated with discordant virological response postcombination antiretroviral therapy in pediatric human immunodeficiency virus infection. Indian J Pharmacol [serial online] 2022 [cited 2022 Dec 3];54:278-81. Available from: https://www.ijp-online.com/text.asp?2022/54/4/278/357831





 » Introduction Top


The Joint United Nations Program on human immunodeficiency virus (HIV)/AIDS set a global aim of “90-90-90” for eliminating HIV in 2014. The goal is to achieve viral suppression in 90% of HIV-infected patients using antiretroviral therapy (ART) by 2020.[1] The consolidated antiretroviral guidelines released by the World Health Organization in 2016 define virological suppression as HIV RNA <1000 copies/mL.[2]

The combination of antiretroviral therapy (cART) has successfully led to a reduction in HIV-related morbidity and mortality. The majority of HIV-infected patients shows positive outcome with ART, i.e., a concomitant increase in CD4 count with suppression of viremia to undetectable levels. However, discordant responses do occur in HIV patients despite ART.[3],[4]

Despite achieving viral suppression, some HIV patients do not show a substantial increase in CD4 count. This deviation from the general response is known as discordant immunological response and patients exhibiting such response are known as immunological nonresponders.[5] While other patients failed to achieve viral suppression despite an increase in CD4 count known as discordant virological response and fall into the category of virological nonresponders (VNRs). The later discordant response is indicative of treatment failure as cART is aimed to achieve viral suppression.[3]

Various factors have been known to be associated with discordant immunological and virological failures in HIV-infected children including baseline CD4 count, poor adherence to treatment, and age at ART initiation.[6] However, immunological responses in patients exhibiting discordant responses remain elusive, especially in children <5 years of age. Little or no information is available regarding immunological responses in VNRs in HIV-infected children. The current study aimed at the evaluation of immunological differences in HIV-infected children <5 years of age having discordant virological responses.


 » Materials and Methods Top


The research was carried out at the All India Institute of Medical Sciences' Departments of Pediatrics and Microbiology in New Delhi. Ethical clearance before enrollment of the study participants was obtained from the institute ethics committee (Ref. No. IESC/T-411/July 22, 2015, RT-01/August 26, 2015). The parents/guardians of HIV-infected children provided written informed consent. All relevant information and demographic data were collected on structured pro forma. Adherence to therapy was assured by pill count at every visit. Follow-up of the enrolled children was done quarterly at the Pediatric Chest Clinic, Department of Pediatrics, AIIMS, New Delhi.

A total of 30 HIV-positive children were included in the study. Follow-up of the enrolled children was done for 1 year, and immunological profiling was done at enrollment, 6 months, and 12 months. Three children could not be followed up and were excluded from the further analysis.

Percentage and counts of CD4+ T helper cells, CD8+ cytotoxic T cells, and CD19+ B cells were enumerated in whole blood as previously described.[7] Acquisition and analysis of samples were done on the FACSCalibur™ system using Multiset software™ (Becton Dickinson, San Jose, California, USA). COBAS TaqMan HIV-1 Test, version 2.0 (Roche Diagnostics, USA) was used to quantify HIV-1 viral load (lower limit of detection: 47 copies/mL).

The serum levels of immunoglobulin G (IgG), immunoglobulin A (IgA), immunoglobulin M (IgM), and interleukin (IL)-7 were quantitated using quantitative enzyme-linked immunosorbent assay kits (all from eBioscience, San Diego, California, USA).

Whole blood staining was performed using the following surface markers: CD19, CD86, PD-1, CD3, CD8, and CD28 (All from BD Biosciences, San Jose, CA, USA). After lysis with BD FACS lyse buffer, the samples were suspended in staining buffer. The stained samples were then acquired and analyzed on a flow cytometer with 50,000 events recorded for each sample. Analysis of flow cytometric data was done using FlowJo software (Tree Star, California, USA).

The HIV-infected children were divided into two groups based on suppression of HIV-1 plasma viremia following 1-year cART: those exhibiting viral suppression (<47 copies/mL) after 1 year of ART were placed in virological responders (VRs) groups, whereas children having HIV-1 plasma viremia >1000 copies/mL were grouped under VNRs.

Statistical analysis

GraphPad Prism software (GraphPad Prism version 6.00 for Windows, GraphPad Software, La Jolla California, USA) was used for statistical analysis. For each study variable, the median and interquartile ranges were calculated. A comparison of different variables between VRs and nonresponders was done using an unpaired t-test or Mann–Whitney test. Differences achieving a P < 0.05 were considered significant. The relationship between the variables was assessed using Spearman or Pearson correlation.


 » Results Top


The baseline characteristics of VRs and VNRs are shown in [Table 1]. At enrollment, there was no difference in the median age of the VR group (32.5 [13–39] months) and the VNR group (34 [21–40] months; P = 0.52). No significant difference was observed in the CD4 percentage (P = 0.96), CD4 count (P = 0.34), CD8 percentage (P = 0.35), CD8 count (P = 0.93), and median HIV-1 viral load (P = 0.20) between the VR group and VNR group. Although VR and VNR had similar percentages and counts of CD8+ T cells, they differed in the frequency of CD8+ CD28+/-T cells in this study. The frequency of CD28+ CTLs cells was higher (P < 0.0001), and the frequency of CD28-CTLs cells was lower (P < 0.0001) in the VR group than in the VNR group [Table 1]. These CD28+ and CD28-CTLs cells were observed to be significantly correlated with HIV-1 plasma viremia (r = −0.4695, P = 0.01; r = 0.40, P = 0.03, respectively) [Figure 1]c and [Figure 1]d.
Figure 1: Immunological profile of viral responders and nonresponder HIV-1-infected children. Immunological markers in HIV-1 infected viral responders (a) and nonresponder children (b) at baseline and follow-up visit. Correlation of CD8+ CD28+ (c) and CD8+ CD28 (d) T-cells with viral load in HIV-1-infected children. HIV = Human immunodeficiency virus

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Table 1: Immunological parameters of viral responders and nonresponder HIV-1-infected children at baseline and follow-up visits

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The children in the VR group had significantly higher CD19 percentages (P = 0.04) and CD19 counts (P = 0.01) than in the VNR group. However, the VR group had significantly lower expression of CD86 (p=0.03) and PD-1 (p=0.002) on the CD19+B cells than VNRs [Table 1].

The serum IgG, IgA, IgM, and IL-7 levels were quantitated in VRs and VNRs. At baseline, the median serum IgG (P = 0.03), IgM (P = 0.04), and IL-7 (P = 0.01) levels were lower in VR than in VNRs. No difference in the serum IgA levels was observed between the two groups [Table 1].

Similar results were obtained at 6 months and 12 months after initiation of cART. The two groups differed in the count of CD19+ B cells, percentages of CD19+CD86+, CD19+ PD-1+ B cells, and CD8+CD28+/- T cells [Figure 1]a and [Figure 1]b. The VR group had higher CD19 B-cell count (P = 0.04), lower expression of CD86 and PD-1 on CD19+ B cells (P = 0.04 and P = 0.01, respectively), lower frequency of 28-CTLs (P = 0.01), and higher frequency of CD28+ CTLs (P = 0.01) than VNR group. At 12 months of cART, the VNR group had higher IgG (P = 0.02), IgM (P = 0.003) serum immunoglobulins, and IL-7 (P = 0.001) compared to the VR group. The frequency of CD86 and PD-1 expressing CD19+ B cells was higher in the VNR group than in the VR group. The CD19 B-cell count and frequency of CD28+ CTLs were lower in the VNR group accompanied by the higher frequency of CD28-CTLs when compared to the VR group [Table 1].


 » Discussion Top


The present study evaluated the immunological response in HIV-infected children < 5 years exhibiting discordant virological response to cART. HIV-1 infected children exhibiting high baseline B-cell counts, lower serum IgG, IgM, IL-7 levels, lower activation and exhaustion of B-cells, and higher frequency of CD28+ CTLs turned out to be VRs at 12 months of cART. Conversely, children exhibiting low B-cell counts, higher IgG, IgM, IL-7 levels, highly activated, functionally exhausted B-cells, and lower frequency of CD28+ CTLs turned out to be VNRs. In the present study, the VRs displayed a regulated immune response for the clearance of HIV viremia. The probable cause of regulated response may be the higher percentage of CD28+ CTLs in VRs which is key players in the HIV-1 viral clearance as reflected by the negative association of CD28+ CTLs with plasma viremia. On the other hand, the CD28-CTLs that are associated with poor virological outcomes were highly elevated in VNRs.[8] This might have led to the persistence of HIV viremia in VNRs.


 » Conclusion Top


Taken together, HIV infection led to several abnormalities in immunological response. The cART is effective in the suppression of HIV viremia and restoration of CD4 counts; however, individual differences among HIV-infected patients may lead to discordant outcomes. Although the study limits its findings in terms of a small sample size, the findings could pave a path to improving the outcomes of cART.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
 » References Top

1.
UNAIDS. 90-90-90 An Ambitious Treatment Target to Help end the AIDS Epidemic; 2014.  Back to cited text no. 1
    
2.
WHO. Consolidated guidelines on the use of antiretroviral drugs for treating and preventing HIV infection: Recommendations for a public health approach, 2nd ed. Geneva: World Health Organization; 2016. url: https://apps.who.int/iris/handle/10665/208825. [Last accessed on 2019 Feb 28].  Back to cited text no. 2
    
3.
Kiweewa F, Esber A, Musingye E, Reed D, Crowell TA, Cham F, et al. HIV virologic failure and its predictors among HIV-infected adults on antiretroviral therapy in the African cohort study. PLoS One 2019;14:e0211344.  Back to cited text no. 3
    
4.
Kelly C, Gaskell KM, Richardson M, Klein N, Garner P, MacPherson P. Discordant immune response with antiretroviral therapy in HIV-1: A systematic review of clinical outcomes. PLoS One 2016;11:e0156099.  Back to cited text no. 4
    
5.
Shete A, Dhayarkar S, Sangale S, Medhe U, Panchal N, Rahane G, et al. Incomplete functional T-cell reconstitution in immunological non-responders at one year after initiation of antiretroviral therapy possibly predisposes them to infectious diseases. Int J Infect Dis 2019;81:114-22.  Back to cited text no. 5
    
6.
European Pregnancy and Paediatric HIV Cohort Collaboration (EPPICC) and Early-treated Perinatally HIV-infected Individuals: Improving Children's Actual Life with Novel Immunotherapeutic Strategies (EPIICAL) Study Groups. Predictors of faster virological suppression in early treated infants with perinatal HIV from Europe and Thailand. AIDS 2019;33:1155-65.  Back to cited text no. 6
    
7.
Singh R, Mukherjee A, Singla M, Vajpayee M, Negi N, Kabra SK, et al. Impact of HIV infection and highly active antiretroviral therapy (HAART) on B cell subpopulations in children. J Med Virol 2018;90:1222-31.  Back to cited text no. 7
    
8.
Vivar N, Ruffin N, Sammicheli S, Hejdeman B, Rethi B, Chiodi F. Survival and proliferation of CD28 – T cells during HIV-1 infection relate to the amplitude of viral replication. J Infect Dis 2011;203:1658-67.  Back to cited text no. 8
    


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