Adjuvant Haploidentical Virus-specific T Lymphocytes (VSTs) for Treatment of Disseminated Adenovirus Infection in a Premature Infant (JACI-D-19-00043R1)

Capsule Summary

We report the first therapeutic infusion of haploidentical virus-specific T lymphocytes (VSTs) to treat maternally-transmitted, disseminated adenovirus infection in a premature infant. Infusion of maternal VSTs, isolated using interferon-gamma capture, associated with clinical improvement and viral clearance.

To the Editor:

Human adenovirus has over 50 serotypes that cause infection in immunocompetent and immunocompromised patients. In patients with aberrant or absent adaptive immunity, adenovirus can cause life-threatening, disseminated infection¹. For example, neonates with disseminated adenovirus infection have mortality rates exceeding 85%². Virus-specific T lymphocytes (VSTs) have been used to treat infections in allogeneic hematopoietic cell transplant recipients³ and, most recently, in infants with primary immunodeficiencies⁴. We report the successful isolation and adoptive transfer of maternal adenovirus-specific T lymphocytes enriched by cytokine capture to treat disseminated adenovirus infection in a premature infant.

Details of the methods and analyses are located in this article’s Online Repository at www.jacionline.org. Briefly, maternal haploidentical VSTs were isolated via cytokine capture CliniMACS® Prodigy Cytokine Capture System (IFN-gamma) (Miltenyi Biotec, Auburn, CA) via Emergency Investigational New Drug (eIND) per manufacturer’s instructions. After VST infusion, the infant had at least weekly chemistries and peripheral blood counts as well as graft-versus-host disease (GvHD) assessments to monitor for toxicity. Adenovirus response was monitored by qualitative and quantitative real-time PCR from plasma, urine, stool and endotracheal aspirate/nasopharyngeal swab. Differentiation of maternal and infant’s monocytes and lymphocytes in the infant’s blood was determined by HLA immunophenotype.

A 29 6/7 weeks’ gestation female infant was born by emergent caesarian delivery for breech presentation to a 28-year-old Gravida 4, Para 1 mother with negative screening for human immunodeficiency virus I/II, hepatitis B and C viruses, syphilis, gonorrhea, chlamydia and Group B Streptococcus. Pregnancy was complicated by preterm premature rupture of membranes for six days, preterm labor, cigarette smoking and recreational drug use. Apgar scores at birth were 8 and 9 at 1 and 5 minutes, respectively; and the infant was admitted to the NICU receiving nasal continuous positive airway pressure (CPAP) therapy. Birth weight (1,480 grams, 75%), length (39 cm, 68%), and head circumference (27 cm, 55%) were appropriate for gestational age; and physical examination was normal. Ampicillin and gentamicin were administered for 48 hours until blood cultures were sterile. Two days post-partum (day of life, DOL 2), the mother developed bilateral eye drainage and crusting that persisted through DOL 5 and did not respond to topical antimicrobial therapy. She visited the infant and did skin-to-skin care while providing some maternal milk. The infant also developed bilateral yellow eye discharge with mild injected conjunctivae and periorbital edema on DOL 2. Bacterial and chlamydial cultures were negative, and ocular symptoms resolved without antimicrobial therapy.

The infant did well without respiratory support until DOL 7 when she developed marked lethargy with labored breathing, apnea, bradycardia, and desaturation episodes. Nasal CPAP was restarted, a sepsis work-up was performed, and ampicillin and gentamicin were started. CSF indices were normal; and blood, CSF, and urine bacterial cultures were sterile (Table E2). Head ultrasound was normal. Due to persistent lethargy, hypotonia, desaturation episodes, and hypothermia, another sepsis evaluation was performed on DOL 11 to evaluate for viral etiologies. Antimicrobial therapy was changed to nafcillin, gentamicin, and acyclovir. Blood, CSF, and urine bacterial cultures remained sterile, and PCR testing for herpes simplex virus was negative (Table E2). However, nasopharyngeal respiratory viral PCR multiplex panel was positive for adenovirus species C.

On DOL 13, the infant was transferred to Nationwide Children’s Hospital. The infant remained lethargic on CPAP 7 (30% oxygen) therapy with chest radiograph (CXR) revealing hyperinflated lungs, diffuse bronchial wall thickening, and patchy perihilar and bibasilar opacities. On DOL 14, quantitative plasma adenovirus PCR was >2 million DNA copies/mL, and adenovirus DNA was detected by qualitative adenovirus PCR in nasal secretions and stool (Table 1). The infant also had elevated hepatic enzymes and thrombocytopenia. She received one dose of IVIG (1 g/kg) and started thrice-weekly intravenous cidofovir (1 mg/kg) with probenecid. High-level adenovirus DNAemia continued, and she developed progressive respiratory failure first requiring mechanical ventilation (DOL 17), then high-frequency oscillatory ventilation (HFOV) and inhaled nitric oxide (DOL 19). CXR showed worsening bilateral perihilar and lower lung opacities and small bilateral pleural effusions.

Table 1:

Clinical course, laboratory evaluations, and administered supportive care and antiviral therapies

	Interventions		Adenovirus PCR*				Laboratory Studies
DOL	Antiviral therapy	Supportive care	Plasma copies/mL (log10/mL)	NP/ETT	Stool	Urine	Cr (mg/dL)	ALT (U/L)	AST (U/L)	WBC (x103/μL)	ALC	HGB (g/dL)	PLT (x103/μL)	LDH (U/L)
14	IVIG, Cidofovir#	Nasal CPAP	>2M (>6.3)	Positive	Positive	--	0.54	92	326	10.4	3,120	11	202	--
16	Cidofovir
17		Intubated, MV started	>2M (>6.3)	Positive	--	--	0.42	77	424	10.9	3,488	12	103	--
18	Cidofovir
19		MV→HFOV, iNO started
21	Cidofovir		>2M (>6.3)	Positive	--	Positive	0.50	41	141	16.1	4,699	8.9+	109	3,435
22	VST infusion
23	Cidofovir	iNO stopped		--	Positive	--
24			545,050 (5.7)	Positive	--	Positive	0.41	25	45	23.1	8,547	9.6+	174	1,589
25	Cidofovir	HFOV→MV
26		Extubated→BiPAP
28	Cidofovir
29			16,155 (4.2)	Positive	Negative	--	0.49	16	19	36	10,440	9.6	496	968
30	Cidofovir		--	--	--	Negative	--	--	--	23.7	9,717	9.0+	571	--
31		BiPAP→CPAP	1,951 (3.3)	--	--	--
32	Cidofovir	0.2L O2 NC
34			1,214 (3.1)	--	--	--	0.40	21	23	17.9	9,487	11.3	570	806
35	Cidofovir			Negative	--	--
36			424 (2.6)	--	--	--
37	Cidofovir
43			Negative	--	--	--	0.34	24	26	12.8	7,680	9.1	420	--
57				--	--	--	0.32	29	23	8.3	5,063	7.5	399	623
64			Negative	--	--	--	0.27	36	31	8.8	6,952	7.2	472	806
69			--	--	--	--	0.28	36	22	8.2	6,314	6.9+	483	612
76			--	--	--	--	0.30	35	36	10.8	7,949	11.9	353	697

^*Quantitative (plasma) and qualitative (NP, ETT, stool, urine) PCR results are shown. Dashes represent no sample acquisition at the indicated time.

^#Intravenous cidofovir (1 mg/kg) was infused over one hour thrice weekly starting on DOL 14. Normal saline bolus (10 mL/kg) was administered intravenously over 30 minutes prior to cidofovir infusion. Starting on DOL 18, enteral probenecid (37.5 mg/kg) was administered per oral gastric tube three hours prior to cidofovir and at three and nine hours (each 20 mg/kg) after cidofovir.

⁺Received packed red blood cell transfusion.

ALC indicates absolute lymphocyte count, ALT = alanine aminotransferase, AST = aspartate aminotransferase, BiPAP = bi-level positive airway pressure, CPAP = continuous positive airway pressure, Cr = creatinine, Ct = cycle time, dL = deciliter, DOL = day of life, ETT = endotracheal tube aspirate, g = gram, HFOV = high-frequency oscillatory ventilation, HGB = hemoglobin, IVIG = intravenous immunoglobulin, iNO = inhaled nitric oxide, L = liter, LDH = lactate dehydrogenase, M = million, mg = milligram, mL= milliliter, MV = mechanical ventilation, NC = nasal cannula, NP = nasopharyngeal swab, O₂ = Oxygen, PCR = polymerase chain reaction, PLT = platelets, U = unit, UA = uric acid; mL = microliter, VST = viral-specific T lymphocyte, WBC = white blood cells.

Given the high mortality associated with neonatal disseminated adenovirus infection and the mother’s resolved conjunctivitis presumed due to adenovirus, an eIND application was filed on DOL 17, seeking to infuse maternal haploidentical VSTs enriched via the CliniMACS® Cytokine Capture System (IFN-gamma). After eIND approval, the infant’s mother underwent HLA-typing and donor infectious diseases and medical screenings. On DOL 21, the mother underwent two blood-volume leukapheresis; and her PBMNCs were processed for enrichment of haploidentical VSTs via cytokine capture (Table E3). To this end, the cytokine capture technique yielded 310- and 131-fold enrichment of maternal CD3⁺CD4⁺IFNγ⁺ and CD3⁺CD8⁺IFNγ⁺ T cells, respectively. On DOL 22, the infant (1.9 kg) received 2 mL of maternal VSTs containing 1.29×10⁵ total CD3⁺ and 1.00×10⁵ CD3⁺IFNγ⁺ cells via slow-push intravenous infusion. Thrice-weekly cidofovir and probenecid were continued.

Within 24h after VST infusion (DOL 23), inhaled nitric oxide was discontinued. On DOL 24, plasma viral load decreased to 545,050 copies adenovirus DNA/mL, and hepatic transaminitis and thrombocytopenia resolved (Table 1). The infant’s absolute lymphocyte counts (ALC) doubled from a maximum pre-VST infusion ALC of 4,699 (DOL 21) to a maximum post-VST infusion ALC of 10,440 (DOL 29), coinciding with decreases in viral load and detection of maternal HLA-A9⁺ T-cells in the infant’s blood (Figure 1A). T cells of maternal origin peaked one week after VST infusion, representing 0.6% and 1.9% of total CD4⁺ and CD8⁺ T cells, respectively (Figure 1, panels B and C). Throughout the follow-up period, the CD4-to-CD8 ratios for infant and maternal T cells in the infant’s peripheral blood were age appropriate and differed significantly (Figure 1D). On DOL 25, the infant was transitioned from HFOV to conventional mechanical ventilation and ultimately extubated to bi-level positive airway pressure (BiPAP) on DOL 26.

Figure 1. Biologic and clinical observations associated with infusion of maternal haploidentical virus-specific T cells (VSTs) into a premature infant with disseminated adenovirus infection.

(A) Infusion of haploidentical VSTs correlates with decreased plasma viral load (DNA copies/mL) and increased white blood cell (WBC) and absolute lymphocyte (ALC) counts (cells x10³/mL) in infant blood. (B) Use of flow cytometry to detect maternal human leukocyte antigen (HLA)-A9⁺ monocytes and T-cells in infant blood. A representative dot plot demonstrating maternal cell populations measured in the infant’s blood at DOL 28, six days after haploidentical maternal VST infusion. (K = x10³). (C) Increased numbers of maternal CD3⁺HLA-9A⁺ T cells detected in infant blood associates with increased levels of WBC and ALC and decreased levels of plasma adenovirus measured in infant blood at DOL 28. (D) Infant and maternal CD3⁺CD4⁺-to-CD3⁺CD8⁺ ratios remain distinct at each indicated time of sample acquisition. Indicated p-value calculated using two-tailed paired t test.

On DOL 29, plasma adenoviral load decreased to 16,155 DNA copies/mL, and adenovirus was no longer detected in stool and urine. On DOL 31, the infant was transitioned to nasal CPAP at which time plasma viral load was 1,951 adenovirus DNA copies/mL. On DOL 35, adenovirus PCR of nasopharyngeal secretions was negative. On DOL 36, 14 days after maternal haploidentical VSTs and 22 days after initiating cidofovir, plasma adenoviral load was <500 DNA copies/mL (Table 1). Cidofovir was discontinued on DOL 37. The infant did not experience any recurrence in adenovirus detection or infection nor did she manifest any clinical manifestations of GvHD following adoptive VST therapy. On DOL 78, the infant was discharged home on nasal cannula oxygen for bronchopulmonary dysplasia.

The child is now 10 months of age and is healthy and developing normally. Her newborn screen, which included SCID screening via TREC assessment, revealed presence of TRECs and absence of any metabolic disorders. She has not been hospitalized since having recovered from disseminated adenoviral infection; and she has not had any additional immune work-up performed.

Viruses can cause disseminated disease in neonates, associating with high infant mortality². Although largely undefined, neonatal susceptibility to viruses likely reflects naïve skewing in cellular immune responses to infectious challenge. Specifically, not only do infants lack immune memory, but their lymphocytes also exhibit recent thymic emigrant immunophenotype⁵. Their naïve CD4⁺ T cells also polarize towards Th2 cytokine responses with decreased IFNγ and TNFα production⁶, signature cytokines mediating antiviral immunity. Finally, neonatal tissues possess high proportions of regulatory T cells that can suppress endogenous T cell activation⁷, potentially increasing susceptibility to disseminated viral infection.

Immune response to infectious challenge in the premature infant is less well-characterized, but generally accepted to be less effective than that of full-term infants. Notwithstanding, adjuvant adoptive transfer of maternal VSTs along with concomitant cidofovir resulted in viral clearance and clinical improvement in our premature infant. To our knowledge, this case illustrates the first successful use of haploidentical VSTs to treat maternally-transmitted, disseminated adenoviral infection in a premature infant and supports investigation into the broader applicability of such rapidly generated, antiviral cell therapies beyond the hematopoietic cell transplant setting.

Abbreviations:

CSF

cerebral spinal fluid

HLA

human leukocyte antigen

IFNγ

interferon gamma

IVIG

intravenous immunoglobulin

NICU

neonatal intensive care unit

PCR

polymerase chain reaction

PBMNCs

peripheral blood mononuclear cells

SCID

severe combined immunodeficiency

TNCs

total nucleated cells

TNFα

tumor necrosis factor alpha

TRECs

T-cell receptor excision circles