Delayed viral clearance despite high number of activated T cells during the acute phase in Argentinean patients with hantavirus pulmonary syndrome

Abstract

Background: The hallmarks of HPS are increase of vascular permeability and endothelial dysfunction. Although an exacerbated immune response is thought to be implicated in pathogenesis, clear evidence is still elusive. As orthohantaviruses are not cytopathic CD8⁺ T cells are believed to be the central players involved in pathogenesis.

Methods: Serum and blood samples from Argentinean HPS patients were collected from 2014 to 2019. Routine white blood cell analyses, quantification and characterization of T-cell phenotypic profile, viral load, neutralizing antibody response and quantification of inflammatory mediators were performed.

Findings: High numbers of activated CD4⁺ and CD8⁺ T cells were found in all HPS cases independently of disease severity. We found increased levels of some proinflammatory mediators during the acute phase of illness. Nonetheless, viral RNA remained high, showing a delay in clearance from blood up to late convalescence, when titers of neutralizing antibodies reached a high level.

Interpretation: The high activated phenotypic profile of T cells seems to be unable to resolve infection during the acute and early convalescent phases, and it was not associated with the severity of the disease. Thus, at least part of the activated T cells could be induced by the dysregulated inflammatory response in an unspecific manner. Viral clearance seems to have been more related to high titers of neutralizing antibodies than to the T-cell response.

Funding: This work was supported mainly by the Administración Nacional de Laboratorios e Institutos de Salud (ANLIS) "Dr. Carlos Malbrán". Further details of fundings sources is included in the appendix.

Keywords: Andes virus; Hantaviridae; T cells; hantavirus pulmonary syndrome; orthohantavirus.

Figure 1

Incubation period and the clinical course of hantavirus pulmonary syndrome patients. (a) Patients reported during the Andes virus-caused person-to-person transmission outbreak in southwestern Argentina. Each patient is represented as a horizontal bar, stacked in order according to the date of fever onset. The first case was reported in November 2018 and the last in February 2019 (n = 34). The most probable incubation periods are represented in dark grey; the symptomatic period between fever onset (Day 0) and hospital admission is shown in light grey, the stay at low-complexity rooms in pink, and the period required in intensive care unit in red. Severity grade is indicated after each bar (Roman numerals). (b) Distribution analysis of the incubation period of person-to-person infected patients. Southwestern Argentina person-to-person events (salmon dots, n = 33) were plotted with other person-to-person events during the period and historical person-to-person data (turquoise symbols, n = 33) in a quantile–quantile plot, indicating that together they fit to a normal distribution model (p < 0·001) with a mean of 22 d. Comparing both groups, the 2018–2019 outbreak data showed greater deviations from normality than clusters previously reported. Grey lines show 100 simulated normal data sets with equal sample size, mean, and standard deviation (using the Visual Studio bootstrap method) and compares the theoretical curve of perfectly normal data with a slope equal to standard deviation and an intercept equal to mean.

Figure 2

Hematological findings during the person-to-person transmission outbreak in southwestern Argentina. (a) Representative smear from a hantavirus pulmonary syndrome patient (cells were subjected to May Grünwald-Giemsa staining and analysed by microscopy, Axio Scope A1); the black arrow indicates an immunoblast. (b) Frequency (%) of immunoblasts along the acute phase (n = 7, 55 samples). (c) Platelet count during the acute phase (n = 30; 180 samples). (d–e) Lymphocyte and neutrophil absolute numbers during the acute phase (n = 30; 180 samples). Pink areas in each graph show the reference range of normality.

Figure 3

Quantification of leukocyte subpopulations in peripheral blood in hantavirus pulmonary syndrome (HPS) patients during the Epuyén person-to-person transmission outbreak in southwestern Argentina. (a) The percentage of different cell types are represented in each ring graph in different colors in the cardiopulmonary phase. From left to right, healthy volunteers (HV, n = 12) and HPS cases grouped as mild or severe according to disease severity grade: mild (II, n = 5), severe that survived (III, n = 7), and severe that were fatal (IV, n = 10). Cell quantification was performed by an automatic hematological counter. (b) Maximum neutrophil-to-lymphocyte (NLR) ratios in mild (I and II, n = 12) and severe patients (III and IV, n = 18), Mann–Whitney– Wilcoxon test (p = 0.0006). (c) Maximum lactate dehydrogenase (LDH) activities (unit per Litre [U/L]) recorded in survivors with mild (n = 12) and severe (n = 18) disease. (d) Correlation of LDH activity (U/L) in serum with neutrophil absolute numbers (cells per µL) in acute patients; Spearman's ranked correlation (ratio: 0·69, p < 0·0001). (e) Correlation of LDH activity (U/L) in acute serum samples with lymphocyte absolute numbers (cells per µL); Spearman's ranked correlation (ratio: 0·44, p < 0·0001).

Figure 4

T lymphocyte subpopulations and viral load in acute hantavirus pulmonary syndrome (HPS) patients. (a) CD4⁺ to CD8⁺ ratio in acute and convalescent HPS patients (red dots) and healthy volunteers (green dots). (b–c) Comparison of the frequency (%) of activated CD8⁺ T cells (week 1, n = 14; week 2, n = 13; week 3, n = 8; > 4 weeks, n = 8) or CD4⁺ T cells (week 1, n = 16; week 2, n = 11; week 3, n = 8; > 4 weeks, n = 8) between HPS patients (red dots); and healthy volunteers (green dots, n = 8) according to time after symptoms debut. (d) Comparison of the frequency of active cytotoxic CD8⁺ T cells in mild and severe patients during the prodromal and cardiopulmonary phases, Mann–Whitney–Wilcoxon test (ns: not significant). Viral RNA in blood was quantified in patients (e) in different weeks after fever onset: week 1, n = 117; week 2, n = 46, week 3, n = 6, week 4 n = 5, and weeks > 4 (up to 233 days). n = 44 and (f) during different phases of disease: acute, in patients with IgM>IgG (n = 169); early convalescence, IgM<IgG (n = 28); and late convalescence, no IgM and high IgG (n = 21). Kruskal–Wallis H test followed by Dunn's Multiple Comparison test (a, b, c, and f, *p < 0·05, **p < 0·01, ***p < 0·001, ****p < 0·0001).

Figure 5

Neutralizing antibody (nAb) responses in hantavirus pulmonary syndrome (HPS) patients infected by different Andes-virus -like viruses. Neutralizing antibody titers against Andes virus (ANDV) in cases grouped by viral variant: Buenos Aires virus (BASV; light blue), Lechiguanas virus (LECV; purple), Orán virus (ORNV; orange) and ANDV (green). Samples from survivors were divided in three different group of periods, from left to right: (a) 10–60 d (BASV: n = 6, mean: 29 d; LECV: n = 4, mean: 30·8 d; ORNV: n = 5, mean: 35·5 d; ANDV: n = 10 mean: 33·7 d), (b) 61–365 d (BASV: n = 3, mean: 108·3; LECV: n = 6, mean: 121 d; ORNV: n = 4, mean: 176 d; ANDV: n = 17, mean: 170 d) and (c) more than 365 d after onset of disease (BASV: n = 2, mean: 1,904 d; ANDV: n = 2, mean: 1,495 d). Kruskal–Wallis H test (p = 0·0018); Dunn's multiple comparison test (**p = 0·0023).

Figure 6

Concentrations (pg/mL) of over-expressed and under-expressed cytokines, chemokines, and growth factors in hantavirus pulmonary syndrome (HPS) patients (red boxes), compared to healthy volunteers (green boxes, n = 6). HPS patients were divided into three groups according to the days after symptoms debut. HPS 1–4 d n = 23; 5–10 d n = 28; >10 d n = 6 (range 16–28 d). Mann–Whitney–Wilcoxon test; (*p < 0·05; **p < 0·01 ***p < 0·001; ****p < 0·0001). Abbreviations: IL, interleukin; IL1B, interleukin 1 subunit beta; IL2RA, interleukin 2 receptor subunit alpha; TNFA, tumour necrosis factor alfa; TNFB, tumour necrosis factor beta; INFG, interferon gamma; IFNA, interferon alpha; CXCL, Chemokine C-X-C motif ligand; CCL, Chemokine C-C motif ligand; CSF, colony-stimulating factor; LIF, leukaemia inhibitory factor; HPS: hantavirus pulmonary syndrome; HS: healthy volunteers. * indicates cytokine concentrations calculated from only one read (see Methods).