The Effects of Recombinant Human Lactoferrin on Immune Activation and the Intestinal Microbiome Among Persons Living with Human Immunodeficiency Virus and Receiving Antiretroviral Therapy

Abstract

Lactoferrin modulates mucosal immunity and targets mechanisms contributing to inflammation during human immunodeficiency virus disease. A randomized placebo-controlled crossover clinical trial of recombinant human (rh) lactoferrin was conducted among 54 human immunodeficiency virus–infected participants with viral suppression. Outcomes were tolerability, inflammatory, and immunologic measures, and the intestinal microbiome. The median age was 51 years, and the median CD4⁺ cell count was 651/µL. Adherence and adverse events did not differ between rh-lactoferrin and placebo. There was no significant effect on plasma interleukin-6 or D-dimer levels, nor on monocyte/T-cell activation, mucosal integrity, or intestinal microbiota diversity. Oral administration of rh-lactoferrin was safe but did not reduce inflammation and immune activation.

Clinical Trials Registration: NCT01830595.

Recombinant human lactoferrin treatment in persons living with human immunodeficiency virus and receiving antiretroviral therapy did not significantly change inflammation within blood, nor the intestinal microbiota. Additional strategies are needed to reduce inflammation by targeting intestinal dysbiosis and mucosal immunity.

Effective antiretroviral therapy (ART) has shifted the morbidity spectrum for persons living with human immunodeficiency virus (HIV) (PLWH) from AIDS toward serious non–AIDS-defining conditions, including cardiovascular disease, cancer, and other end-organ diseases [1]. In addition to important behavioral factors, a key contributor to the current disease spectrum includes HIV-associated inflammation [2–5]. Safe and tolerable treatment strategies are needed that can be given in addition to ART and target persistent immune activation. One potential strategy may include targeting irreversible injury to mucosal integrity during HIV infection, with translocation of microbial antigens, such as lipopolysaccharide (LPS) or endotoxin [6].

Lactoferrin is an endogenous iron-binding protein (member of transferrin family) with immunomodulatory properties that reduce inflammation [7]. Lactoferrin binds to LPS with high affinity and interferes with LPS binding of cell surface receptors (eg, CD14), which typically initiates broad proinflammatory effects [7]. Lactoferrin also promotes intestinal cell growth and cell migration, suggesting that it may have potential to restore mucosal integrity and the gut barrier [8]. Lactoferrin has demonstrated the ability to suppress production and release of proinflammatory cytokines [9, 10] from stimulated monocytes, and to enhance secretion of anti-inflammatory cytokines from intestinal myofibroblasts [11]. A recombinant form of lactoferrin has been shown to reduce the mortality rate among intensive care unit patients with sepsis [12].

To our knowledge, there have been no clinical trials of lactoferrin treatment among PLWH. We conducted a pilot study of recombinant human (rh) lactoferrin in 54 HIV-infected participants receiving ART with viral suppression, to evaluate the safety, tolerability, and potential anti-inflammatory effect of such treatment.

METHODS

Research Setting and Target Population

This study was conducted at an HIV clinic within a safety-net hospital in Minneapolis, Minnesota (Hennepin Healthcare). The protocol (NCT01830595) was approved by the institutional review board and received an investigational new drug approval from the US Food and Drug Administration (investigational new drug 118783). All study participants underwent a verbal and written informed consent process before enrollment. Eligibility criteria included PLWH aged ≥40 years receiving continuous treatment with ART and with HIV RNA levels <200 copies/mL for ≥1 year before enrollment. Participants with a gastrointestinal infection within the past month and common diseases known to influence inflammation were excluded.

Study Design and Intervention

We investigated the treatment effects of oral rh-lactoferrin (1500 mg twice daily) versus placebo in a randomized, double-blind, crossover clinical trial design. The crossover design consisted of two 3-month treatment periods separated by a 2–4-month “washout” period. Each period consisted of 3 visits, with period 1 including baseline, month 1, and month 3, and period 2 including months 5, 6, and 8. After informed consent, participants were randomized to a sequence: either active rh-lactoferrin during period 1 and placebo during period 2 or vice versa.

The study drug (rh-lactoferrin and matched placebo) was provided in capsule form by Ventria Bioscience. The active drug is a recombinant form of human lactoferrin extracted and purified from milled rice flour as partially iron-saturated lactoferrin, and its amino acid sequence is identical to that of human lactoferrin.

Power estimates were calculated for a composite interleukin 6 (IL-6)/D-dimer score that was developed from epidemiologic data of PLWH receiving continuous ART to assess whether a candidate study intervention influences systemic inflammation and/or coagulation activity, and the degree to which any treatment effect would be posited as clinically meaningful [5]. With 80% power, our goal sample of ≥50 participants could detect an effect size in the composite IL-6/D-dimer score that corresponded to a 13% difference in clinical event risk (ie, composite of cardiovascular disease, cancer, end-stage renal disease, end-stage liver disease, or all-cause mortality) [5].

Laboratory Methods for Blood Measures

Participants were fasting for all study blood sample collection. Plasma, serum and peripheral blood mononuclear cell specimens were processed within 30 minutes of collection. Plasma-soluble (s) biomarker levels were measured with enzyme-linked immunosorbent assay and MesoScale multiplex platforms from batched cryopreserved samples. For details in biomarker measurements and immunophenotyping, please see the Supplementary Material (Supplementary Table 1).

Microbiome Substudy Methods

A subset of participants were coenrolled into an intestinal microbiome substudy. A rectal swab specimen was collected before and after study drug exposure for both periods 1 and 2, corresponding to collection at 4 study visits (baseline and months 3, 5, and 8). Microbiome specimens collection and analysis are described in the Supplementary Material.

Statistical Methods

Characteristics and laboratory measures at randomization were summarized, and proportions with unintended effects (assessed subjectively) and adverse events (categorized using the NIH Division of AIDS Table for Grading Severity of Adverse Events, updated 2008) were tallied for rh-lactoferrin and placebo treatment. Treatment effects (rh-lactoferrin vs placebo) on inflammatory and immunologic outcomes were calculated using longitudinal mixed models, with each participant serving as his or her own control. Models included indicators for drug sequence (active drug followed by placebo or vice versa), study period (1 or 2), visit within period (baseline, 1, or 3) ,and drug (rh-lactoferrin or placebo), and carryover effects were examined. For the microbiome substudy, sequence variants and operational taxonomic unit were analyzed for alpha and beta diversity using the R package phyloseq (version 1.22.3). Adonis software (Vegan R project, version 2.5-3) was used to assess the effect size of rh-lactoferrin treatment in the variance. QIIME software (version 2.0) [13] was used for pairwise group comparison and linear mixed-effect model tests.

RESULTS

Study Participants

Supplementary Figure 1 presents the flow diagram for all consented participants through follow-up. The analysis cohort consisted of 54 participants, with 28 randomized to a rh-lactoferrin then placebo sequence and 26 randomized to the countersequence of placebo then rh-lactoferrin. Fifty participants completed period 1, and of those 46 completed period 2. Participant characteristics are presented in Table 1. The median age was 51 years, with 89% of male sex at birth and 72% white. Randomized sequence groups were similar across comorbid disease and HIV risk factors.

Table 1.

Participant Characteristics at Entry Overall and by Randomized Group

	Participants, No. (%)a
Characteristic	Total Sample (n = 54)	rh-Lactoferrin Followed by Placebo (n = 28)	Placebo Followed by rh-Lactoferrin (n = 26)
Demographics
Age, y, median (IQR)	51 (46–56)	54 (46–60)	50 (46–53)
Male sex	48 (88.9)	26 (92.9)	22 (84.6)
Race/ethnicity
White	39 (72.2)	21 (75.0)	18 (69.2)
African American	11 (20.4)	5 (17.9)	6 (23.1)
Hispanic	2 (3.7)	2 (7.1)	0 (0.0)
Other	2 (3.7)	0 (0.0)	2 (7.7)
Clinical characteristics
BMI, median (IQR), kg/m2	26.8 (24.5–32.4)	27.7 (25.7–35.2)	25.6 (23.6–28.6)
Smoker, current	20 (37.0)	7 (35.0)	13 (50.0)
Hepatitis B or C	8 (14.8)	5 (17.9)	3 (11.5)
Prior CVD	6 (11.3)	4 (14.3)	2 (8.0)
Hypertension diagnosis	18 (33.3)	10 (35.7)	8 (30.8)
Lipid-lowering therapy	17 (31.5)	11 (39.3)	6 (23.1)
Prescribed aspirin	13 (24.1)	9 (32.1)	4 (15.4)
Diabetes	5 (9.4)	4 (14.3)	1 (4.0)
HIV characteristics
Prior AIDS	21 (38.9)	14 (50.0)	7 (26.9)
CD4+ cell count, median (IQR), cells/µL	651 (501–811)	637 (450–771)	687 (557–906)
CD8+ cell count, median (IQR), cells/µL	587 (434–903)	619 (474–928)	570 (430–799)
CD4-CD8 ratio, median (IQR)	1.0 (0.7–1.4)	1.0 (0.7–1.3)	1.0 (0.7–1.6)
HIV RNA undetectable	53 (98.1)	28 (100.0)	25 (96.2)
Tenofovir use	41 (75.9)	19 (67.9)	22 (84.6)
Abacavir use	12 (22.2)	8 (28.6)	4 (15.4)
NNRTI use	15 (27.8)	5 (17.9)	10 (38.5)
PI use	17 (31.5)	9 (32.1)	8 (30.8)
INSTI use	30 (55.6)	18 (64.3)	12 (46.2)
Clinical laboratory values, median (IQR)
Total-C, mg/dL	179 (153–206)	177 (152–206)	180 (161–203)
LDL-C, mg/dL	106 (83–123)	105 (84–126)	106 (82–120)
HDL-C, mg/dL	46 (36–59)	47 (35–61)	42 (36–56)
Ferritin, ng/mL	104.5 (50.0–163.0)	107.5 (59.5–164.5)	93.5 (48.0–153.0)

Abbreviations: BMI, body mass index; CVD, cardiovascular disease; HDL-C, high-density lipoprotein cholesterol; HIV, human immunodeficiency virus; INSTI, integrase strand transfer inhibitor; IQR, interquartile range; LDL-C, low-density lipoprotein cholesterol; NNRTI, nonnucleoside reverse-transcriptase inhibitor; PI, protease inhibitor.

^aData represent No. (%) of participants, unless otherwise identified as median (IQR).

Adherence and Tolerability

The percentage of participants reporting that they took the study drug twice every day, exactly as prescribed, ranged from 72% to 92% during follow-up visits and did not differ between groups (subjective adherence reported in Supplementary Table 2). In addition, the percentage of participants who noted missing ≥3 doses (ie, >20% of potential doses) within the week preceding a visit ranged from 8% to 26%.

Supplementary Table 3 presents a summary of unintended effects and adverse events. Of 50 participants with data available, 31 (62%) reported an adverse effect: 17 while prescribed rh-lactoferrin and 20 while prescribed placebo. The most common adverse effect was gastrointestinal, but the frequencies were again similar in those receiving rh-lactoferrin (n = 15) and those receiving placebo (n = 19). There were no deaths, and none of the adverse events were deemed related to study medication.

Inflammation and Immune Activation Measures

Supplementary Table 4 presents median inflammatory and immunologic blood measurements (with interquartile range) at baseline and month 5 (before starting the study drug at the beginning of periods 1 and 2, respectively). Table 2 presents the primary treatment comparisons, showing that none of the inflammatory or immunologic measures assessed significantly declined with rh-lactoferrin versus placebo treatment (ie, between-treatment comparisons of changes in levels from before to after treatment). There was no evidence of a significant carryover effect from study drug exposure during period 1, through the washout phase and into period 2. We also assessed the treatment effect of rh-lactoferrin on clinical serum iron values. The percentage of transferrin saturation of iron significantly increased with rh-lactoferrin versus placebo, by 2.6% (95% confidence interval, .2–5.0; P = .04) (Table 2). This effect did not reach statistical significance for serum ferritin, serum iron, total iron-binding capacity, or hemoglobin (Table 2).

Table 2.

Inflammatory and Immunologic Outcomes With 3 Months of rh-Lactoferrin Versus Placebo Treatment

		Treatment Effect of Lactoferrin Versus Placebo
Laboratory Outcomes	P Value for Carryover Effects Between Periods	Mean (95% CI)	P Value
IL-6 and D-dimer score	.24	0.01 (−.07 to .06)	.82
Inflammation biomarkers
IL-6, log2 pg/mL	.45	−0.02 (−.20 to .17)	.87
IL-6 receptor, ng/mL	.16	0.03 (−.01 to .06)	.15
IL-1b, pg/mL	.74	0.03 (−.02 to .07)	.26
IL-8, log2 pg/mL	.31	−0.02 (−.11 to .07)	.63
TNF-α, pg/mL	.35	0.02 (−.04 to .08)	.46
TNF-R1, ng/mL	.64	−0.00 (−.04 to .03)	.84
hsCRP, log2 mg/mL	.39	0.09 (−.17 to .36)	.49
Coagulation biomarkers
D-dimer, log2 mg/L	.13	−0.03 (−.14 to .07)	.53
TFPI, log2 ng/mL	.44	0.02 (−.03 to .07)	.52
Mucosal integrity biomarkers
Zonulin, log2 ng/mL	.57	0.00 (−.07 to .08)	.99
I-FABP, log2 ng/mL	.19	−0.04 (−.17 to .10)	.56
Monocyte biomarkers and immunophenotypes
Soluble CD14, log2 mg/L	.23	0.01 (−.04 to .06)	.67
Soluble CD163, log2 mg/L	.24	0.09 (.02 to .17)	.02
CD14+CD16−, %	.08	−0.42 (−1.62 to .79)	.49
CD14+CD16+, %	.07	−0.36 (−1.06 to .33)	.30
CD14dimCD16+, %	.29	0.35 (−.35 to 1.05)	.32
T-cell immunophenotypes, %
MAITS	.51	0.01 (−.09 to .11)	.86
CD8+PD1+	.25	−0.36 (−1.04 to .33)	.31
CD8+A4/B7+	.96	0.33 (−.29 to .95)	.29
CD8+KI67+	0.11	−0.48 (−1.21 to .24)	.19
CD8+CD38+HLA-DR+	.77	−0.52 (−1.10 to .06)	.08
CD8+CD57+CD28−	.27	−0.94 (−2.22 to .34)	.15
CD4+PD1+	.64	−0.14 (−.78 to .51)	.67
CD4+B7+	.96	0.30 (−.39 to .98)	.40
CD4+KI67+	.05	−0.35 (−.92 to .21)	.22
CD4+CD38+HLA-DR+	.97	−0.08 (−.26 to .09)	.36
CD4+ regulatory T cell	.09	0.03 (−.28 to .33)	.87
Clinical laboratory values
Ferritin, ng/mL	.93	5.83 (−3.33 to 14.99)	.21
Serum iron, g/dL	.92	4.00 (−2.85 to 10.85)	.25
Total iron-binding capacity, mg/dL	.46	3.94 (−3.00 to 10.87)	.26
Transferrin saturation, %	.53	2.59 (.15 to 5.04)	.04
Hemoglobin, g/dL	.93	0.06 (−.08 to .21)	.41

Abbreviations: CI, confidence interval; hsCRP, high-sensitivity C-reactive protein; I-FABP, intestinal fatty acid–binding protein; IL-1b, IL-6, and IL-8, interleukin 1b, 6, and 8; MAITS, mucosal-associated invariant T cells; TFPI, tissue factor pathway inhibitor; TNF, tumor necrosis factor.

Intestinal Microbiome

Among 20 participants coenrolled into the microbiome substudy, a subset of 12 participants had adequate specimens for microbiome analysis at all 4 sentinel study visits (baseline and months 3, 5 and 8). There were no significant differences in alpha diversity between participants receiving placebo first and those receiving rh-lactoferrin first. Beta diversity analysis, did not show significant clustering of samples based on treatment group or time point. In contrast, samples from the same participant, at different time points, clustered strongly (Adonis test for placebo-first group, R² = 0.16 and P = .001; rh-lactoferrin first, R² = 0.10 and P = .049), showing stability in the diversity of the gut microbiota over time, independent of treatment status or group sequence (Suppplementary Figure 2). At both phylum and family levels, we did not see any significant within-participant changes after 3 months of treatment with placebo or rh-lactoferrin (Supplementary Figure 3A), nor did we find significant changes in taxa frequencies within participants over all follow-up time points. (Supplementary Figure 3B and 3C).

DISCUSSION

We report results from the first study of a rh-lactoferrin treatment among PLWH, given in addition to ART as a strategy to reduce systemic inflammation. In this pilot placebo-controlled, double-blind, crossover clinical trial, we did not detect a significant treatment effect from rh-lactoferrin on blood measures of inflammation and immune activation, nor on changes to the intestinal microbiota or mucosal integrity. In fact, among a subset of 12 participants, we describe remarkable stability in the intestinal microbiota with repeated assessment over 8 months. Consistent with its iron-binding functionality, treatment with rh-lactoferrin did result in a small, significant increase in transferrin saturation of about 3%. Finally, the intervention was well tolerated.

Prior studies of recombinant forms of lactoferrin have demonstrated potential anti-inflammatory and immunologic benefits. In a placebo-controlled phase 2 trial of 194 patients with severe sepsis, enterally administered talactoferrin (rh-lactoferrin produced by Aspergillus niger) reduced the all-cause mortality rate [12]. Posited mechanisms include the ability of lactoferrin to bind and modulate LPS signaling, thereby reducing bacterial-mediated inflammatory response [9]. Among pregnant women with iron-deficiency anemia, treatment with bovine lactoferrin reduced plasma IL-6 levels and increased hemoglobin and ferritin levels more than ferrous sulfate supplementation [14]. In that study, it was hypothesized that the anti-inflammatory effect of bovine lactoferrin improved iron homeostasis and facilitated the release of sequestered iron that occurs in states of chronic inflammation. In addition, lactoferrin may also directly improve iron stores, and we did detect a modest increase in the percent transferrin saturation with 3 months of rh-lactoferrin treatment.

Differences in the potential anti-inflammatory effect of lactoferrin between prior published reports and our findings may be explained, in part, by differences in the target populations studied. Compared with HIV disease, the degree of systemic inflammation attributable to microbial antigen-mediated innate immune activation may be much greater during states of sepsis. Although iron sequestration is a consequence of chronic inflammation, iron deficiency per se is not a central driver of HIV-associated inflammation, and our study population had normal serum ferritin levels at entry and low overall levels of inflammatory markers. Finally, it is also possible that the treatment effects of oral lactoferrin may be most apparent within the mucosal tissue effector site, and therefore an immunologic signal may not be present, or at least may be very modest, within the peripheral circulation. To this point, data from a murine model suggests that rh-lactoferrin may improve regulatory T cells within the intestinal lamina propria, but this effect was not seen within peripheral blood [14].

Our pilot study had several limitations. The small sample size is unable to fully rule out a small but potentially meaningful treatment effect. The choice of rh-lactoferrin preparation could affect the outcome. Our immunologic assessments were limited to blood samples and did not directly assess the mucosal tissue. Despite this limitation, our findings suggest that rh-lactoferrin is unlikely to have a clinically meaningful impact on systemic inflammation and associated end-organ disease risk among the target population of individuals with ART-treated HIV infection. We only characterized the intestinal microbiota among a limited subset of participants, although there was remarkable stability in the microbial community over time. Finally, approximately one-quarter of participants acknowledged suboptimal adherence, but sensitivity analyses restricted to optimal adherence did not change results.

In summary, rh-lactoferrin treatment among ART-treated PLWH did not result in significant changes to inflammation or immune activation within blood, or to the intestinal microbiota. Additional strategies to reduce inflammation by targeting intestinal dysbiosis and improving mucosal immunity are needed to improve the health of PLWH.

Supplementary Data

Supplementary materials are available at The Journal of Infectious Diseases online. Consisting of data provided by the authors to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the authors, so questions or comments should be addressed to the corresponding author.

Notes

Acknowledgments. We thank all the study participants and staff at the Hennepin Healthcare Positive Care Center.

Financial support. This work was supported by Hennepin Health Services (career development award), the intramural research program of the National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH), the National Cancer Institute, NIH (contract HHSN261200800001E), the intramural research program of NIAID/NIH and the NIAID microbiome core facility, and Ventria Bioscience (provision of study drug, including both active rh-lactoferrin tablets and matching placebo).

Potential conflicts of interest. After study conduct and manuscript development, R. P. was employed by Gilead Sciences, which had no involvement in the development or conduct of this study. All other authors report no potential conflicts. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed.