Immunomodulatory effect of Artemisia annua and Moringa oleifera on viral load among PLWH on antiretroviral therapy in Uganda

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INTRODUCTION
Acquired Immuno-Deficiency Syndrome (AIDS), caused by the Human Immunodeficiency Virus (HIV), is the most common immunosuppressive disease and one of the major global health problems (Anywar et al., 2020).Reports released by the Joint United Nations Programme on HIV and AIDS (UNAIDS) revealed that 39.0 million [33.1 to 45.7 million] people globally were living with HIV in 2022, two-thirds of whom (25.6 million) are in the WHO African Region (Gouda, 2023), and about 36.3 million people have died from the endemic by the end of 2020 (Kankara et al., 2022).In HIV infection, CD4 cells are targeted, leading to a progressive reduction in their numbers and dysregulation of their function.Over time, sustained and chronic HIV replication can culminate in AIDS, characterized by significantly low CD4 counts, heightened susceptibility to opportunistic infections, associated morbidity, and eventual mortality (Amlogu et al., 2016;Gunda et al., 2017;Aprioku et al., 2022).In the era of highly active antiretroviral therapy (HAART), CD4 T cell depletion is halted, and their numbers may even increase, provided patients are receiving proper HAART.In a few circumstances, for reasons not fully understood, patients on HAART may fail to recover optimal CD4 T cell numbers and or function, leading to a situation known as immunological failure.Patients with HIV infection also exhibit a generalized, non-HIV-specific polyclonal B-cell activation, resulting in hypergammaglobulinemia of all immunoglobulin (Ig) isotypes in addition to HIV-specific IgM and IgG.Serum immunoglobulin concentrations increase as one progresses from asymptomatic to symptomatic HIV infection (Lugada et al., 2004;Nair et al., 2009;Onifade et al., 2017).
As is observed with CD4 T cells, B-cell activation is significantly reversed by HAART, leading to associated reductions in total IgG levels, including HIV-1-specific antibodies (Voltersvik et al., 2003).The effectiveness of humoral responses primarily depends on the affinity and avidity of pathogen-specific antibodies.Antibodies with higher affinity or avidity are capable of binding pathogens at lower concentrations, thereby mediating protective functions more effectively.Conversely, antibody avidity is known to be associated with immune complex-mediated disorders in patients (Dimitrov et al., 2011), complicating the landscape of HIV/AIDS disease and its outcomes.Nevertheless, HIV is recognized for its impact on altering follicular T helper cell function, potentially negatively influencing the affinity maturation process in germinal centers, leading to the production of antibodies with low avidity (Kimuda et al., 2018).While contemporary HAART enhances HIV-1-specific and all immune responses in general (French et al., 2017), the synergistic role of a well-balanced nutritional program cannot be underestimated and is encouraged.In resource-limited countries such as Uganda, maintenance of patients on a balanced diet drains family resources and is often not sustainable for long periods by most families.In Uganda, many PLWH, in addition to HAART, widely use medicinal plants for boosting immunity as well as managing infections (Lubinga et al., 2012;Anywar et al., 2020;Kankara et al., 2022).These medicinal plants represent a source of potential immunomodulating adjuvants that may boost protective HIV-1-specific immune responses (French et al., 2017;Anywar et al., 2020).
Artemisia annua L. (Asteraceae), an annual herb native to Asia and other continents, with deeply grooved branches and leaf color that varies from light green to dark green, has been used for the treatment and prevention of fever, chills, some cancers, and malaria, in traditional and modern medicine (Liu et al., 2013;Mirbehbahani et al., 2020;Ekiert et al., 2021).A. annua leaf powder which increases monocyte and lymphocyte levels (Ogwang et al., 2011;Lubbe et al., 2012), has been used widely by PLWH in Uganda to boost the immune system.Macrophages, representing a key component of the innate immune system, can produce both pro-inflammatory cytokines, such as IL-12/23 P40 and TNFα, and anti-inflammatory cytokines, including IL-10 (Hou and Huang, 2016).
Equally, Moringa oleifera Lam (Moringacaea), a fastgrowing drought-resistant tree, distributed in many countries of the tropics and subtropics, is commonly consumed by PLWH on HAART (Monera-Penduka et al., 2017;Aprioku et al., 2022) as a supplement to enhance immunity and manage opportunistic infections.Besides having anti-inflammatory, antioxidant, anti-hyperglycemic, and hepatoprotective activity, M. oleifera has been reported to exhibit a positive influence on leucocytes, lymphocytes, neutrophils, erythrocytes, hemoglobin, and packed cell volume (Aprioku et al., 2022).However, the synergistic immunological and/or virological effect of the combined use of A. annua and M. oleifera by PLWH in Uganda has not been demonstrated.This study investigated the effect of combined usage of A. annua and M. oleifera powders on CD4 counts, viral load suppression, and on HIV-specific antibodies among PLWH on HAART.

Study design and population
This cohort study was nested within a Randomized Controlled Clinical Trial conducted at Mbarara Regional Referral Hospital (MRRH) in Uganda, registered with ClinicalTrials.govunder NCT03366922.The investigation aimed to assess the impact of A. annua and M. oleifera on CD4 count and viral load among people living with HIV (PLWH) HAART.The parent study enrolled 267 participants aged 18 years or older, who were HIV positive and had *Corresponding author.E-mail: itwinomujuni@must.ac.ug.Tel: +256 (0) 776528077.
Author(s) agree that this article remain permanently open access under the terms of the Creative Commons Attribution License 4.0 International License been on HAART for at least one year, with a CD4 count of fewer than 350 cells/µL, and normal haematological and biochemical indices.Exclusion criteria comprised patients with opportunistic infections, those using other herbal medicines, or pregnant individuals.In our nested study, we sampled 37 participants from the parent study.The participants were divided into two groups: a control group consisting of 17 individuals on HAART only and an intervention group comprising 20 participants on A. annua and M. oleifera in addition to HAART.This pilot study had its sample size determined by the availability of sufficient sample volumes for laboratory analyses.

Preparation of plant materials
A. annua and M. oleifera young leaves were obtained from Kabale district, Western Uganda and were authenticated by a botanist from the Faculty of Science at MUST.The leaves were air-dried under the shade, pulverized mechanically, and packaged in 4 and 10 g of A. annua and M. oleifera, respectively following study-specific standard operating procedures and in a setting of good compounding practices.

Administration of treatments and measurement of study parameters
Participants in the intervention arm were given 10 and 4 g of M. oleifera and A. annua powders, respectively to mix and take in their drink of choice (cassava, millet, or posho porridges) every morning at 8 a.m. for 12 months.Each parcel supplied to the participant contained M. oleifera and A. annua packs labelled with participants' study numbers to last one month.Parcels also contained dosing instructions as well as information on storage and safety.To ensure adherence to the M. oleifera and A. annua and HAART treatments, every participant received a reminder SMS every morning between 7 and 8 a.m.Participants were requested to respond with the message 'Taken'' or a call prompt as a proxy to confirm adherence/compliance to the intervention.The study team worked closely with the HIV clinic to ensure that participants were reviewed and accessed routine HIV care as prescribed, including HAART.During the study information sessions before informed consent and throughout the study period, study staff emphasized to all participants that these M. oleifera and A. annua herbal medicines were not replacements for HAART in the treatment of HIV, and as such the study participants remained in the study only while enrolled on HAART as prescribed.This phrase was also added in the consent form, "Please note that these herbal medicines are not being given to you for treatment of HIV, and as such you MUST keep taking your prescribed ARVs as usual and correctly".
Each study participant was followed up for 12 months and reviewed once a month by the study clinician, with additional unscheduled visits allowed in cases of medical emergencies or as needed.The study clinician conducted physical examinations of the participants, documented the findings, and supplemented them with self-reports from the study participants.The evaluation and documentation of performance status and quality of life were also carried out.Case report forms were utilized to capture vital outcome data from participants, alongside laboratory report forms.A blood sample for the measurement of CD4 count, viral load, p24-specific IgG, and IgM antibodies was collected immediately after enrollment but before the initiation of M. oleifera and A. annua treatments.Subsequent samples were drawn at 6 and 12 months following the initiation of the intervention.

HIV-1 p4 ELISA procedure
Each well of an Iimmunlon 4 HBX microtitre plate (Thermoscientific, USA) was coated at 4°C overnight with 200 ng of HIV-1 p24 antigen in 50 μL/well of commercial coating buffer (immunochemistry technologies, USA).The plate wells were then washed five times with wash buffer and blocked with 50 μL/well of blocking solution (5% w/v skimmed milk in PBS) for 2 h at 37°C.Two-fold serial dilutions of serum samples were prepared starting with a dilution of 1/50 and ending at a dilution of 1/200.Each sample dilution (50 μL) was incubated in duplicate wells for 1 h at 37°C, washed as before and then 50 μL/well of anti-human IgG horseradish peroxidase (HRP) conjugate was incubated in the test-well for 1 h at 37°C.The plates were washed again to remove excess conjugate antibodies.Substrate 3.3', 5.5'-tetramethylbenzidine (ImmunoChemestry Technologies, USA) (100 μL/well), was incubated for 30 min at room temperature in the dark.The reaction was stopped by adding 25 μL of 2M sulphuric acid.Optical density (OD) was read in an ELISA plate reader (BioTek ELx 80) at a test wavelength of 450 nm and a reference wavelength of 630 nm.The difference between the two readings was computed to control for background optical interference and was taken as the final OD result.

Ammonium thiocyanate ELISA for determination of p24-IgG antibody avidity
Immunlon 4 HBX microtitre plates (Thermoscientific, USA) were coated and blocked as described.Samples were individually diluted to give normalized uniform ODs.Then 50 μL/well of the normalized serum dilutions were incubated in duplicate on the HIV-1 p24 coated plates at 4°C overnight.The next morning the assay plate was washed and 50 μL/well of 0.6 M ammonium thiocyanate (LOBA CHEMIE PVT) was added and incubated for 10 min at room temperature.The plates were washed five times and then 50 μL/well of anti-human IgG-HRP conjugate was added followed by incubation for 1 h at 37°C.The plates were washed, 100 μL of the substrate, 3.3', 5.5'-tetramethylbenzidine (ImmunoChemestry Technologies USA), was incubated in each well for 30 min at room temperature in the dark.The reaction was stopped with 2 M sulfuric acid and ODs were read on a BioTek ELx80 plate reader.

Statistical data analysis
The optical densities (ODs) obtained from the reader for both the standards and assay serum samples were exported to MS Excel and subsequently imported into My Assays, an online ELISA tool.A 4-parameter logistic regression analysis was conducted using the My Assays tool.The output from the tool included the standard graph and slope.The concentrations of HIV-1 p24-specific IgG and IgM in the participants' samples were calculated utilizing the standard graph.Additionally, for the avidity ELISA, the ODs were transformed into an avidity index by dividing the average OD of the wells with chaotrope by the average OD of the wells without chaotrope for the same sample.Data were analyzed using GraphPad Prism software version 8.A paired t-test was used to determine the difference between the intervention and control group means of p24-specific immunoglobulin levels, avidity index, and CD4 count.Viral load data were analyzed using STATAv15.The Mann-Whitney (Ranksum) test was used to check for viral load median differences between the test and control groups, while the Wilcoxon matched-pairs signed-rank test was utilized to detect any median differences between the baseline and later concentrations.A p-value of less than 0.05 was considered statistically significant and hence concluded that the means and medians were different.

Baseline characteristics of study participants
Baseline demographics, CD4, and viral load characteristics of the participants in the control and intervention groups were comparable at enrollment.Table 1 describes the demographic and immunological characteristics of the study participants at enrollment.Equally at baseline, the number of patients on the three categories of HAART regimens: AZT + 3TC + EFV, 3TC + EFV + TDF, and 3TC + EFV + others, was statistically similar for both the intervention and control groups (Table 1).The effect of A. annua and M. oleifera was examined on the median viral loads at the 6 and 12-month time points.A gradual decline in viral load was observed from baseline, through the 6 months and up to the 12 months of participant follow-up.However, the viral load was only significantly different between the intervention and control groups at the 12 months' follow-up time point (Table 2).

Effect of A. annua and M. oleifera supplementation to HAART on CD4 counts
At enrollment, participants were marched for CD4 count in addition to other parameters.Therefore, no significant difference in CD4 count at enrolled was observed (Table 1).However, after 12 months of A. annua and M. oleifera supplementation of HAART, the intervention arm showed a significantly higher mean CD4 count when compared with the control group (Figure 1).Data were expressed as Mean ± SD.The level of statistical significance was calculated by comparing the mean CD4 count of the control (A) and intervention (B) groups at baseline and exit.P values less than 0.05 were taken to be statistically significant.

Effect of A. annua and M. oleifera treatment on IgM and IgG antibody responses
The effect of A. annua and M. oleifera treatment was investigated on the antibody responses using IgM and IgG levels, and IgG avidity index as surrogates.Baseline mean IgM and IgG levels were 1,819 and 16,714 ng/mL, respectively.After 12 months of follow-up, when compared with the baseline time point, we found no significant change or difference in mean levels of IgM in both the A. annua and M. oleifera and control groups (Figure 2A and B).The control group equally did not show significant changes in IgG levels at the 12 months of follow-up when compared with baseline (Figure 2C).
However, there was a significant reduction in mean IgG levels in the A. annua and M. oleifera, treatment group when the 12-month time point was compared to the baseline visit (Figure 2D).
Data were expressed as Mean ± SD.The level of statistical significance was calculated by comparing the mean p24-IgM levels of the control (A) and intervention (B) and mean p24-IgG levels of the control (C) and intervention (D) groups at baseline and exit.P values less than 0.05 were taken to be statistically significant.

Effect of A. annua and M. oleifera treatment on avidity of anti-HIV p24-IgG antibodies
Subsequently, we investigated whether supplementation   with A. annua and M. oleifera alongside HAART had any impact on the binding affinity of HIV-specific antibodies, utilizing the avidity index of anti-HIV-1 p24 IgG as a surrogate measure.The selection of p24 was primarily influenced by its status as an abundant HIV-1 antigen in vivo, eliciting high level of IgG targeting the p24 antigen.An increase was observed in the mean anti-p24 IgG avidity indices between baseline and 12 months.This increase was not statistically significant in the control group (Figure 3A) but demonstrated statistical significance in the A. annua and M. oleifera treatment group (Figure 3B).
Data were expressed as Mean ± SD.The level of statistical significance was calculated by comparing the percentage p24-IgG avidity index of the control (A) and intervention (B) groups at baseline and exit.P values less than 0.05 were taken to be statistically significant.

DISCUSSION
In this study, the impact of supplementing HAART with a combination of A. annua and M. oleifera leaf powder among PLWH, focusing on p24-specific IgG and IgM antibodies, HIV viral load, and CD4 count was investigated.The average age of the study participants was 39 years (Table 1).This aligns with findings from the Uganda Population-based HIV Impact Assessment (UPHIA 2020-2021), where the age groups with the highest HIV prevalence were 35 to 39 (10.7%), 45 to 49 (12.1%), and 50 to 54 (11.8%), as compared to the national average of 5.8%.This emphasizes that HIV prevalence in Uganda is notably higher among individuals aged 35 and older compared to younger age groups.Our findings align with those of Negin et al. (2016) in Eastern Zimbabwe, where they observed an increasing HIV prevalence among people aged 45 and older, while the prevalence among younger age groups was decreasing.They attributed their findings to the saturation of infection in high-risk groups, behavior change, and a potential impact of widespread HAART on transmission, as well as increased survival of PLWH due to HAART (Negin et al., 2016).
Therefore, the average age observed in this study is supported by various factors, including the context of Mbarara Regional Referral Hospital, the study site, which is surrounded by HIV high-risk groups, including but not limited to commercial sex workers, refugees, and prisoners.
Despite a significant reduction in viral load observed in the intervention group at 12 months compared to the control, this reduction was gradual and not evident at month 6 following the initiation of the intervention (Table 2).This gradual reduction may be attributed to one of the inclusion criteria for this study, which required patients to have been on HAART for at least one year with a CD4 count of 350 cells/µL, potentially causing a delayed rebound of viral load to normal levels.This observation is supported by a study conducted by Liu et al. (2020), who suggested that using the normalization of CD4 counts as the primary evaluation parameter for complete immune restoration in HIV-1 patients under HAART might be insufficient.They noted that the median time for viral load suppression ranges from 3.4 to 4.5 months, irrespective of the HAART regimen, indicating that immune system recovery is a gradual process occurring over an extended period (Liu et al., 2020).
The findings of this study contrast with those of Gambo et al. (2021), who reported that the M. oleifera leaf intervention was not effective in decreasing the viral load of HIV-infected individuals accessing HAART at the S.S Wali Virology Center (Gambo et al., 2021).This disparity can be attributed to the fact that, in this study, M. oleifera was combined with A. annua, which was not the case in their study.Findings of the present study indicate that after 12 months of patient follow-up, there was a statistically significant reduction in viral load combined with an increase in CD4 count in the intervention group compared to the control.This observation aligns with many studies, including the one by Mugo et al. (2022), where most patients experience a reduction in HIV viral load coupled with an increase in CD4 cell count after initiating HAART.In chronic diseases such as HIV and AIDS, adequate micro and macronutrients are vital for normal body functioning, maintaining optimal immunological function, and improving the efficacy of HAART in people living with HIV (PLWH) (Gambo and Gqaleni, 2022).M. oleifera leaves, a rich source of both macro and micronutrients, are often taken as a supplement by PLWH, especially in developing countries, to enhance immunity and manage opportunistic infections (Monera-Penduka et al., 2017).The observed viral load suppression combined with an increase in CD4 cell count in this study can be attributed to the synergistic action of the phytochemicals present in M. oleifera and A. annua leaf powder that was used to supplement HAART among PLWH.
This study observed a decrease in p24-specific IgM antibody titers, although it was not statistically significant compared to the control (Figure 2A and B).Several studies indicate that untreated PLWH exhibit generalized non-HIV-specific polyclonal B-cell activation, resulting in hypergammaglobulinemia of all immunoglobulin isotypes, as well as increased production of HIV-specific IgG and IgM.Following the initiation of HAART, viremia decreases, along with a reduction in IgM titers.However, findings from this study show a further decrease in IgM titers following the initiation of the intervention, although it was not statistically significant compared to the control.This suggests that p24-specific IgM antibodies may not play a significant role in controlling HIV infection.The findings of the present study align with Tomaras et al. (2008), who demonstrated that IgM antibodies induced by transmitted HIV-1 are capable of binding to HIV virions but have little impact on viremia (Tomaras et al., 2008).In contrast, there was a significant decrease in IgG among the intervention group compared to the control (Figure 2C and D).HIV-1 infection results in an increased concentration of circulating IgG, a phenomenon known as hypergammaglobulinemia and dysregulation of B-cell populations.The increased production of capsid proteinspecific IgG antibodies plays an important role in the host immune response to HIV infection (Kardava and Moir, 2019).After HAART, most polyclonal B-cell activation and elevated IgG levels can be restored to levels similar to those in healthy controls, at least in some patients (Song et al., 2020).A similar observation was made in this study in which supplementation of HAART with M. Oleifera and A. annua leaf powder reduced the HIV viremia leading to a reduction in p24-specific IgG antibody concentration, leading to immunological recovery, hence improving the quality of life among PLWH.As noted by Dang et al. (2018), an elevated p24specific IgG antibody concentration coupled with elevated viral load in the control group was observed, indicating poor immune response and therefore treatment failure compared to the intervention group (Dang et al., 2018).In the intervention group, the authors did not only observed a reduced concentration of p24-specific IgG antibodies, but these antibodies also exhibited a higher strength of binding to p24, the most abundant HIV protein essential for the assembly of the capsid that encases HIV genetic material (Anywar et al., 2020), compared to those in the control group (Figure 3).Their findings align with those of Bauer (2021), who stated that antibody avidity increases throughout infection and remains elevated.Upon antigen recognition, immunoglobulin heavy and light chains of Bcell receptor (BCR) are further diversified through rounds of somatic hypermutation, leading to affinity maturation.This process selects B cells in the germinal center with improved antigen-binding properties (Ghraichy et al., 2020).Findings of the current study demonstrate the link between B-cell and T-cell responses, as CD4 T cells are necessary for generating high-affinity and long-lived antibody responses.T follicular helper (TfH) CD4 cells drive affinity maturation, a complex dynamical process enabling the immune system to produce antibodies capable of recognizing antigens (Molari et al., 2020;Bauer, 2021) and directing class switch recombination in germinal center B cells (Kamphorst and Ahmed, 2013;French et al., 2017).This process gives rise to IgG antibodies of high avidity (Kimuda et al., 2018), playing a crucial role in antibody-mediated protection against HIV infection among PLWH.The main limitation of this study was compliance, which was monitored through selfreporting by study participants to the investigators.

Conclusion
This study revealed that supplementing HAART with M. oleifera and A. annua leaf powder diminishes virusspecific immune responses, leading to the suppression of viral replication and an increase in CD4 count.This ensures optimal treatment outcomes in PLWH.

RECOMMENDATION
The authors recommend conducting a similar study in an

Figure 1 .
Figure 1.Effect of A. annua and M. oleifera on CD4 count.

Figure 2 .
Figure 2. Effect of A. annua and M. oleifera on HIV anti-p24-IgM and IgG levels.

Figure 3 .
Figure 3.Effect of A. annua and M. oleifera on the strength of binding between p-24 and IgG antibody among PLWH on ART.

Table 1 .
Baseline characteristics of the study participants.

Table 2 .
Effect of A. annua and M. oleifera on viral load among PLWH on HAART.IQR is inter quartile range.The median viral load readings in the treatment group at the baseline, six, and twelve months were compared with median readings at similar time points in the control.