Study of Pathogenesis and Immune Response in Human Puumala Virus Infection

Study of pathogenesis and immune response in human Puumala virus infection

Therese Thunberg

Department of Clinical Microbiology, Infectious Diseases Umeå 2013 Umeå University Medical Dissertations, New series No 1577

Study of pathogenesis and immune response in human Puumala virus infection

Therese Thunberg

Deparment of Clinical Microbiology, Infectious Diseases Umeå 2013

Responsible publisher under swedish law: the Dean of the Medical Faculty This work is protected by the Swedish Copyright Legislation (Act 1960:729) ISBN: 978-91-7459-681-6 ISSN: 0346-6612 Layout: Print & Media, Therese Thunberg Published articles were reproduced with permission from the publishers E-version available at http://umu.diva-portal.org/ Printed by: Print & Media Umeå, Sweden, 2013

To Johan, Klara och & Sofia

ABSTRACT

Hantaviruses can cause two severe human diseases: hemorrhagic fever with renal syndrome (HFRS) and hantavirus cardiopulmonary syndrome (HCPS). Hantaviruses are spread to humans mainly through inhalation of infectious virions, secreted from infected rodents. The human diseases are characterized by an increased capillary leakage syndrome. Hantaviruses are known to infect endothelial cells, but they are non-cytopathogenic. The mechanism behind human disease is not well understood, but an overactive immune response is implicated in the pathogenesis. The aim of my thesis has been to investigate parts of innate and adaptive immune responses in Puumala virus-infected patients.

In paper I we found a sex difference in the cytokine profile during acute infection. Females had significantly higher plasma levels of IL-9, FGF-2, GM-CSF and lower levels of IL-8 and IP-10 compared to males. These differences may affect the activation and function of the immune response.

In paper II we studied the phenotype and kinetics of NK cells. We observed that CD56dim NK cells were elevated during acute infection and that these, predominantly NKG2C+ NK cells, remained elevated for at least two months after symptom debut. Our novel finding of a prolonged NK cell response, implicates that NK cells may possess adaptive immunity features.

In paper III we observed a vigorous cytotoxic T cell (CTL) response during acute infection, which contracted in parallel with decrease in viral load. The CTL response was not balanced by an increase in regulatory T cells. The T cells expressed inhibitory immunoregulatory receptors, known to dampen intrinsic T cell activity.

In paper IV, we found that a low IgG response in patients was significantly associated with more severe disease, while the viral load did not affect the outcome. Our findings support the use of passive immunization as a treatment alternative for hantavirus-infected patients.

In conclusion, my thesis contributes to an increased knowledge about the immune response in hantavirus-infected patients. The findings, combined with future studies, will hopefully lead to a better understanding of the pathogenesis and possible treatment alternatives.

i ii LIST OF PUBLICATIONS

This thesis is based on the following papers referred to in the text by their roman numerals:

Paper I Klingström J, Lindgren T, Ahlm A (2008). Sex-dependent differences in plasma cytokine responses to hantavirus infection. Clinical and Vaccine Immunology, Volume 15: 885-887.

Paper II

Björkström N, Lindgren T, Stoltz M, Fauriat C, Braun M, Evander M, Michaelsson J, Malmberg K. J, Klingström J, Ahlm C, Ljunggren H. G (2011). Rapid expansion and long-term persistence of elevated NK cell numbers in humans infected with hantavirus. Journal of Experimental Medicine, Volume 208: 13-21.

Paper III Lindgren T, Ahlm C, Mohamed N, Evander M, Ljunggren H.G, Björkström, N (2011). Longitudinal analysis of the human T cell response during acute hantavirus infection. Journal of Virology, Volume 5: 10252-10260.

Paper IV

Thunberg T*, Pettersson L*, Rocklöv J, Klingström J, Evander M, Ahlm C (2013). Viral load and humoral immune response in association with disease severity in Puumala hantavirus infected patients-implications for treatment. Clinical Microbiology and Infection. Apr 29. 1111/1469-0691.12259 (E-pub ahead of print)

* equally contributed

iii ABBREVIATIONS

ADCC Antibody dependent cellular cytotoxicity ANDV Andes virus ARDS Adult respiratory distress syndrome APCs Antigen presenting cells CTL Cytotoxic T cell CTLA-4 Cytotoxic T lymphocyte antigen 4 DCs Dendritic cells DIC Disseminated intravascular coagulation DOBV Dobrava virus EIA Enzyme immunoassay FGF-2 Fibroblast growth factor-2 GM-CSF Granulocyte macrophage colony stimulating- factor HCPS Hantavirus cardiopulmonary syndrome HCV Hepatitis C virus HIV Human immunodeficiency virus HTNV Hantaan virus HFRS Hemorrhagic fever with renal syndrome EBV Epstein Barr virus ELISA Enzyme-linked immunosorbet assay ELISPOT Enzyme-linked immunosorbent spot assay ICTV International Committee on taxonomy of Viruses IF Immunoflourescence IFN Interferon IL Interleukin IP-10 Interferon-γ induced protein 10 MDA-5 Myeloid differentiation-associated factor-5 MHC Major histocompatibility complex MΦ Macrophage NE Nephropathia epidemica NK cells Natural killer cells NO Nitrogen oxide NY-1 New-York-1 virus PAMP Pathogen-associated molecular patterns PBMC Peripheral blood mononuclear cells PD-1 Programmed cell death 1 PUUV Puumala virus RANTES Regulated on Activation, Normal T cell Expressed and Secreted RdRp RNA dependent RNA-polymerase RT-PCR Reverse transcriptase polymerase chain reaction SANGV Sangassou virus SARS Severe acute respiratory distress syndrome SEOV Seoul virus

iv SNV Sin Nombre virus TCR T cell receptor TEM Effector memory cells TCM Central memory cells TGF Transforming growth factor TLR Toll-like receptor TNF Tumor necrosis factor VEGF Vascular endothelial growth factor VHF Viral hemorrhagic fever WBC White blood cell count

v Table of Contents

1. INTRODUCTION ...... 1

1.1 BUNYAVIRIDAE ...... 1 1.2 HISTORY OF HANTAVIRUSES ...... 1 1.3 THE VIRION AND VIRUS REPLICATION ROUTE...... 2 1.4 HANTAVIRUS EPIDEMIOLOGY...... 3 1.5 TRANSMISSION TO HUMANS ...... 5 1.6 RISK FACTORS FOR HANTAVIRUS INFECTION...... 5 1.7 PUUV OUTBREAK IN SWEDEN ...... 6 1.8 CLINICAL SYNDROMES CAUSED BY HANTAVIRUSES ...... 6 1.8.1 HCPS ...... 7 1.8.2 HFRS ...... 7 1.9 CLINICAL DIAGNOSTICS OF HANTAVIRUS INFECTION ...... 9 1.9.1 Immunoassays ...... 10 1.9.2 Reverse transcriptase (RT)-PCR ...... 10 1.10 TREATMENT ALTERNATIVES AND PREVENTION ...... 10 1.10.1 Antiviral drugs ...... 10 1.10.2 Other therapeutic approaches ...... 11 1.10.3 Passive immunization ...... 12 1.10.4 Vaccines ...... 12 1.11 INNATE IMMUNE RESPONSES TO VIRAL INFECTIONS ...... 13 1.11.1 Innate recognition of viral pathogens ...... 14 1.11.2 Natural killer cells ...... 14 1.11.3 NK-cell receptors ...... 16 1.11.4 Phagocytic and antigen presenting cells ...... 16 1.11.5 Cytokines ...... 17 1.12 ADAPTIVE IMMUNE RESPONSES TO VIRAL INFECTIONS ...... 17 1.12.1 General features of T cells ...... 18 1.12.2 CD4+ T cells ...... 18 1.12.3 Regulatory T cells ...... 18 1.12.4 CD8+ T cells ...... 19 1.12.5 Memory CD8+ T cells ...... 20 1.12.6 Immunoregulatory receptors ...... 20 1.12.7 Humoral response ...... 21 1.12.8 The balance of an effective immune response ...... 21 1.13 HANTAVIRUS PATHOGENESIS ...... 22 1.13.1 Hantavirus immunopathogenesis ...... 23 1.13.2 The viruses: viral load and virus effects ...... 27 1.13.3 Coagulopathy ...... 27 1.13.4 Genetic host factors ...... 27

vi 1.13.5 What decides the outcome? ...... 28 2. AIMS ...... 29 3. METHODS ...... 30

3.1 MULTIPLEX FLOURESCENT BEAD-BASED IMMUNOASSAY ...... 30 3.2 IMMUNOFLOURESCENCE ANALYSIS ...... 30 3.3 FLOW CYTOMETRY ...... 31 4. RESULTS AND DISCUSSION ...... 32

4.1 DIFFERENCES IN CYTOKINE PROFILE (PAPER I) ...... 32 4.2 NK CELL RESPONSE (PAPER II) ...... 33 4.3 T CELL RESPONSE (PAPER III) ...... 35 4.4 PREDICTORS OF DISEASE SEVERITY (PAPER IV) ...... 37 5. GENERAL CONCLUSIONS ...... 39 6. SAMMANFATTNING PÅ SVENSKA ...... 40 7. ACKNOWLEDGEMENTS ...... 43 8. REFERENCES ...... 45

vii

viii

1. INTRODUCTION

Hantaviruses cause two severe diseases in humans: hemorrhagic fever with renal syndrome (HFRS) and hantavirus cardiopulmonary syndrome (HCPS). Humans are primarily infected by inhalation of rodent excreta and the severity of disease varies between different hantaviruses. Hantaviruses cause a characteristic capillary leakage syndrome, but the pathogenesis of human disease is not well understood. An overactive immune response is believed to be one important factor in disease development. In 2007, Sweden had more than 2000 recognized clinical cases of Puumala virus (PUUV) infection. This was the country´s largest recorded outbreak ever, and the starting point of my thesis.

1.1 BUNYAVIRIDAE

The Bunyaviridae family consists of more than 300 negative-stranded RNA viruses, which are divided into five different genera: the Orthobunyavirus, Hantavirus, Nairovirus, Phlebovirus and Tospovirus. These viruses are mainly arthropod-borne, except for the hantaviruses. Hantaviruses are maintained in nature by small mammals, such as rodents and insectivores [1]. Several members of the Bunyaviridae family can cause hemorrhagic fever in humans; the Rift Valley fever virus (Phlebovirus); the Crimean-Congo hemorrhagic fever virus (Nairovirus); and members of the Hantavirus genus [2]. Recently, a new Phlebovirus was identified in China, causing severe fever with thrombocytopenia syndrome (SFTS) [3].

Causative agents of viral hemorrhagic fevers (VHF) are found worldwide. Clinically important hemorrhagic fevers, outside the Bunyaviridae family, are for example: yellow fever and dengue fever (genus Flavivirus), Ebola and Marburg (genus Filovirus) and Lassa fever (genus Arena virus) [4].

1.2 HISTORY OF HANTAVIRUSES

Two outbreaks led to the discovery of hantaviruses as important human pathogens. The first outbreak was during the Korean War, where thousands of UN soldiers fell ill with hemorrhagic fever (Korean hemorrhagic fever); later denoted hemorrhagic fever with renal syndrome (HFRS). It was not until 1978 when the causative agent was isolated from a striped field mouse; the Hantaan virus (HTNV) [5]. The second outbreak was in the United States in 1993, when a mysterious respiratory disease, resembling adult respiratory distress syndrome (ARDS) was reported. The causative agent, the Sin Nombre virus (SNV) was soon isolated from the deer mouse and the disease is now named hantavirus cardiopulmonary syndrome (HCPS) or hantavirus pulmonary syndrome (HPS) [6, 7]. A few years later the Andes virus (ANDV) was found to cause a large number of HCPS cases in South America [8].

In the 1930s in Sweden, two physicians clinically described a disease later denoted nephropathia epidemica (NE) [9, 10]. The causative agent, Puumala virus (PUUV) was detected 1980 in bank voles. The virus was named after the village, Puumala in Finland, where the animals were captured [11].

Although hantaviruses are not new to mankind, they are described as emerging viruses, because of their increasing significance as human pathogens. Hantaviruses are the most widely distributed zoonotic viruses on the planet. New outbreaks of human infections are still being recorded and there is an ongoing process of discovering novel hantaviruses [1]. The International Committee on Taxonomy of Viruses (ICTV) have to date officially recognised 23 hantavirus species, using a classification considering differences in genetics, serological testing, and the principal reservoir host [12, 13]. There are multiple other hantavirus strains described in literature outside this classification (e.g., more than 40 genotypes in the Americas alone, around half being pathogenic) [14]. The ongoing discovery of new hantaviruses may lead to a revised hantavirus classification in the future [15].

1.3 THE VIRION AND VIRUS REPLICATION ROUTE

Hantaviruses are tri-segmented negative stranded RNA viruses. The virions are spherical in shape, with a diameter of around 100nm (Figure 1). The virus particles consist of a lipid bilayer interspersed with spikes composed of the glycoproteins, denoted Gn and Gc [16, 17]. Inside the virions, there are three gene segments denoted by their size: the large (L) segment encodes an RNA-dependent RNA-polymerase (RdRp), the medium (M) segment encodes the precursor of the two glycoproteins Gn and Gc, and the small (S) segment encodes the nucleocapsid protein [18]. The RNA gene segments are complexed to the nucleocapsid proteins within the virus particle [19, 20]. PUUV, Tuula virus (TULV) and ANDV S-segments also encode for a possibly functional non-structural protein [21, 22].

S RNA-dependent RNA polymerase (RdRp)

M Nucleocapsid proteins around negative stranded RNA L Spike complex Gn and Gc

Figure 1. Schematic picture of a hantavirus particle

The glycoproteins mediate virus cell entry into eukaryotic cells, through binding to integrin receptors. In vitro, cell types that can be infected include endothelial cells, epithelial cells, fibroblasts, dendritic cells, mastcells and monocytes/macrophages [23-27]. The β3-integrins mediate cell entry of several human pathogenic hantaviruses, while the β1-integrins of nonpathogenic hantaviruses [28, 29]. The decay-accelerating factor/CD55 found apically on cells might act as an important cofactor for infection at least for HTNV and PUUV [30]. The gC1qR/p32 is found to be another potential virus entry receptor [31]. After binding to a receptor, hantavirus enters the cell by endocytosis and viral genes are uncoated and released into the cytoplasm. The viral RdRp is able to synthesize mRNA from the S, M and L segments. Viral RNA is also used to produce anti-genomic RNA as a template for new viral RNA. New virions are primarily assembled at the Golgi and the infectious particles are thereafter released by exocytosis [1]. In vitro, the whole replication cycle, from infection to the release of new infectious particles, takes more than 24 hours. The amount of hantavirus-virions produced in an infected cell is low, which makes it difficult to obtain high virus titer stocks [18].

1.4 HANTAVIRUS EPIDEMIOLOGY

Hantaviruses are commonly referred to as Old World and New World viruses, due to their geographic distribution, and clinical picture presented in humans [32]. Infection with New World hantaviruses lead to HCPS in the Americas: important examples are SNV in North America and ANDV in South America. SNV, having the deer mouse as reservoir, has a case fatality rate of up to 35% in the United States [33]. Similar case fatality rates (30- 39%) have been reported for ANDV-infection [34, 35]. Notably, there exist several other ANDV-like viruses in South America that can cause HCPS [14, 36].

Old World hantaviruses cause HFRS and are found in Europe and Asia. HTNV and Seoul virus (SEOV) are important human pathogens in Asia. HTNV is found primarily in China, Korea and the eastern part of Russia. SEOV is transmitted by two rat species (rattus rattus and rattus norvegicus) and is distributed worldwide, because of the global dispersal of the reservoir hosts [1]. SEOV has a low case fatality rate (<1%) and for HTNV the case fatality rate was previously recorded to be around 10-15% [1, 37]. In China, at present, the HFRS case fatality rate is much lower (around 1-3%), indicating that HTNV is less fatal than previously believed [38]. In Europe, Dobrava virus (DOBV) is an important causative agent of HFRS mainly in eastern and central parts, with a case fatality rate of 9-12% [1]. Thus, PUUV-infection is the most common cause of HFRS in Europe, spread to humans from infected bank voles. PUUV-infection is diagnosed widely throughout Europe, excluding e.g. the British Isles and southern Mediterranean areas. PUUV- infection is considered a milder form of HFRS, with a case fatality rate less than 1% [39-41].

The distribution of hantaviruses is limited to the geographical range of their respective natural hosts (Table 1). Hantaviruses have a preference to one main host, but spill-over occurs to secondary hosts e.g., ANDV has been identified in at least four rodent species other than the main reservoir (O. longicaudatus) [42]. PUUV spillover is observed from the main reservoir (bank vole) to both the common vole and the wood mouse [43]. In recent years, non-rodent hosts i.e. insectivores (shrews and moles) and bats have been found to be natural hosts for hantaviruses [44-47]. These findings of hantaviruses in bats and insectivores have challenged the view that hantaviruses originated and coevolved only with rodent hosts. There may be additional hantaviruses yet undiscovered, circulating in other small mammal species [48].

Common Rodent Virus Geographic Abbreviation Disease Reservoir name subfamily species distribution (reservoir) Apodemus Korean field Murinae Amur* AMRV HFRS Asia peninsulae mouse Dobrava- Apodemus Yellow-necked DOBV HFRS Europe Belgrade flavicollis field mouse Apodemus Striped field Hantaan HTNV HFRS Asia agrarius mouse Seoul SEOV HFRS Rattus Norvegicus Rat World wide Great Thailand THAIV HFRS Bandicota indica Asia bandicoot rat African wood Sangassou SANGV - Hylomyscus simus Africa mouse Microtus Arvicolinae IslaVista ISLAV - California vole North America californuicus Prospect Microtus PHV - Meadow vole North America Hill pennsylvanicus Puumala PUUV HFRS Myodes glareolus Bank vole Europe

Topografov TOPV - Lemmus sibericus Lemmings Asia European Tula TULV - Microtus arvalis Europe common vole Oligoryzomys Long-tailed Sigmodontinae Andes ANDV HCPS South America longicaudatus pygmy rice rat Oryzomys Bayou BAYV HCPS Marsh rice rat North America palustris Black Creek Sigmondon Hispid cotton BCCV HCPS North America Canal hispidus rat Cano Alston´s CADV - Sigmodon alstoni South America Delgadito cotton rat Oligoryzomys Fulvous Choclo* - HCPS South America fulvescens pygmy rice rat El Moro Reithrodontomys Western ELMCV - North America Canyon megalotis harvest mouse Laguna LANV HCPS Calomys laucha Vesper mouse South America Negra Peromyscus White-footed New York NYV HCPS North America leucopus mouse Sin Peromyscus SNV HCPS Deer mouse North America Nombre maniculatus

Table 1. Selected hantaviruses from the ICTV list. * According to Jonsson et al 2010 [1]. The hantavirus rodent hosts, geographical distribution and human disease. - No known human disease associated with the virus.

Africa was until a few years back considered a missing area on the hantavirus geographical map. This was changed when the Sangassou virus (SANGV), a hantavirus carried by the wood mouse, was found in Guinea [49]. Several studies have followed showing the presence of hantaviruses in both rodent and non-rodent hosts in Africa [47, 50, 51]. To date, there is only a few reports on possible hantavirus human-infections in Africa [52, 53], but it is likely that hantavirus infections are largely under-diagnosed, as infection might resemble other febrile illnesses [47].

1.5 TRANSMISSION TO HUMANS

Humans are infected by hantaviruses mainly through inhalation of aerosolized virus-contaminated rodent feces, urine and saliva [32]. The rodents are seemingly unaffected by the virus, although it is shown that the winter-survival of bank voles is lower for infected versus uninfected rodents [54]. Also, there are subtle histopathological lesions e.g. in the lungs of SNV- infected rodents [55]. The rodents are considered persistently infected, but virus shedding in excreta seems to peak a few weeks after primary infection [56, 57]. Hantaviruses remain infectious outside the rodent host for a long time; PUUV can infect new bank voles up to two weeks after excretion [54] and HTNV can be infectious in cell culture for up to 3 months at 4 ºC under wet conditions [58].

Rodent contact increases the risk of hantavirus-infection and the higher the number of infected rodents, the higher the risk of human transmission [59, 60]. Humans are usually considered to be a dead-end host i.e., do not transmit the virus further. ANDV is so far the only known exception, with documented person-to-person transmission [34, 61-63]. Sex partners to index patients had a higher risk than other household contacts of contracting ANDV-infection, suggesting that close person-to-person contact is needed for transmission [34]. Findings of viral RNA in saliva of PUUV-infected patients support the fact that intimate person-to-person contact, such as deep kissing, might be a potential transmission route for hantaviruses [64].

There are also reports on hantavirus transmission to humans in laboratories, when handling infected animals. Then the likely cause of virus transmission could be inhalation of contaminated air or direct introduction of the virus through damaged skin [65, 66]. Rodent bites can be another possible transmission route of hantaviruses [67, 68], together with transmission through blood products [69].

1.6 RISK FACTORS FOR HANTAVIRUS INFECTION

The obvious source of human infection is virus-contaminated excreta from infected rodents, and there are reports on exposure risk factors. In endemic areas, one important risk factor is household conditions that enable rodent entry, such as holes [70, 71]. Handling firewood and having a house with a fireplace are other risk factors [60, 70]. Persons with a high rodent contact

(i.e. have seen rodents and/or rodent droppings) are of increased risk in several studies [60, 70, 72, 73]. Entering buildings that have been unused for long periods of time, such as summer cottages in endemic areas, are associated with increased risk [70, 74]. Persons living near forests in endemic areas are more exposed [75] and camping is considered a risk activity [76, 77].

Smoking is an important risk factor of PUUV-infection. It is suggested that the condition of the respiratory tract, impaired by smoking, can influence whether the infectious aerosol enters alveoli and remains there long enough to cause infection [70]. Other activities that can stir up hantavirus- contaminated dust, such as cleaning and redecorating houses are also of importance [60]. Occupational risks include farming [78-80] and forestry [70, 80].

1.7 PUUV OUTBREAK IN SWEDEN

In the winter of 2006/2007, Sweden had its largest ever recorded outbreak of PUUV-infection. In total, more than 2000 cases were diagnosed, and more than 90% of the cases were in the four northernmost counties [81]. Usually, outbreaks in Fennoscandia coincide with peaks in the bank vole population, which happens in 3-4 year cycles. During these peak years, most NE cases are seen in autumn and winter [60]. In 2006/2007 there was a peak in the rodent population, although not higher than during previous peaks. The explanation for the large outbreak is believed to be a combination of climate conditions and high number of bank voles. The beginning of the winter was extremely mild, and there was a loss of snow cover. A protective snow cover is believed important for bank vole survival: protecting against predators and for finding food. When missing a snow cover, the bank voles instead sought shelter inside human dwellings, increasing human risk for inhalation of infectious excreta [81, 82].

This outbreak is an example on how climate conditions may affect the rodent reservoir and may lead to higher risk of human exposure to hantaviruses. Climate change may also change the geographical distribution of rodent carriers in the future [83].

1.8 CLINICAL SYNDROMES CAUSED BY HANTAVIRUSES

Hantaviruses cause two known human hemorrhagic diseases, HFRS and HCPS. The hallmark of hantavirus-disease in humans is a capillary leakage syndrome, leading to hypotension, hemoconcentration and vasodilatation. Kidney symptoms are typical of HFRS, while severe cardiac and lung symptoms are typical for HCPS [1]. However, there are reports of pulmonary involvement in HFRS patients [84-86] and renal failure/renal complications in HCPS patients [87, 88], suggesting similarities between the two diseases.

Both diseases have an incubation time that is relatively long, typically 2-3 weeks, but can range from 1 to up to 6 weeks [1]. In humans, hantavirus- infection is transient and believed to give life-long immunity, with high levels of virus-specific antibodies and memory T-cells, decades after primary infection [89-92]. Individuals are believed to be protected against a reinfection, at least against the same serotype [89, 91, 93].

1.8.1 HCPS

HCPS is a severe disease with a rapid onset of respiratory failure and cardiogenic shock. The initial symptoms are flu-like, with a sudden onset of fever, muscle pain, headache, abdominal pains and nausea. The febrile prodrome phase lasts for 2-7 days and is followed by a rapid transition into the cardiopulmonary phase. This phase is characterized initially by cough and shortness of breath that rapidly develops into pulmonary oedema, hypotension and shock. In the lungs, the disease produces a capillary leakage syndrome, which evolves to respiratory failure followed by cardiogenic shock [6, 94]. Patients may need mechanical ventilation or extracorporeal membrane oxygenation (ECMO) during this phase, but despite adequate intensive care, the case fatality rate is high [95]. The cardiopulmonary phase is followed by a diuretic phase with clearance of oedema. Although pulmonary symptoms are the primary cause of HCPS, hemorrhagic and renal manifestations are also reported [87, 88]. The convalescent phase usually leads to full recovery, but many patients report dyspnoea weeks after infection and defects in pulmonary functions can persist for up to two years [96].

1.8.2 HFRS

HFRS is classically divided into five phases: febrile, hypotensive, oliguric, polyuric and convalescent phase (Figure 2) [32, 97, 98]. The disease severity varies between different hantaviruses: In general, HFRS caused by HTNV and DOBV are more severe, while SEOV is more moderate, and PUUV- infection is considered mild. Notably, an individual PUUV-case can be severe and even fatal, while a HTNV-case can be mild or subclinical [1].

The infection starts in the lungs through inhalation of infectious aerosols and symptoms begin with a sudden onset of flu-like symptoms. During the febrile phase (3-5 days) fever, myalgia, headache, backache, abdominal pains and nausea are common features. The hypotensive phase (lasting hours or days) include decreased blood pressure and hemoconcentration due to vascular leakage. In severe cases hypotension and shock may develop. The oliguric phase (few days up to 2 weeks) is characterized by signs of renal impairment (oliguria or anuria) and in severe cases acute renal failure. This renal dysfunction is transient, but may require haemodialysis [97, 99, 100]. Dialysis is reported in around 40% of HTNV-infected patients, in 20% of SEOV-infected patients [1] and in around 0-5% of PUUV-infected patients [100-102]. Around one third of the patients, have bleeding manifestations of

e.g., the conjunctiva, the brain and/or the gastrointestinal tract. Death is usually due to complications from renal insufficiency, shock or bleeding manifestations [1]. During the second week patients enter the polyuric phase, with increased urine production. The convalescent phase can last for several months after acute infection. Most patients recover, but there are reports suggesting an association between previous hantavirus-infection and subsequent hypertension and/or mildly affected renal functions [103-108].

1.8.2.1 PUUV-infection

PUUV-infection is considered a milder form of HFRS (Figure 2), where the different phases are not always clinically evident. One third of PUUV- infected patients develop characteristic blurred vision, due to thickening of the lens [100, 109-111]. Pulmonary involvement and abnormal chest radiography findings are seen in up to 30% of all patients [84, 85, 100, 102]. Renal impairment is evident in the majority of patients and hemorrhagic manifestations occur in approximately one third of PUUV-infected patients: e.g. petechia on the skin, conjunctival bleeding, hematemesis, epistaxis, hematuria, melena, and in severe cases intracranial haemorrhages [41, 99, 100]. Typical laboratory findings include proteinuria, hematuria, increased serum creatinine, thrombocytopenia, leukocytosis and elevated C-reactive protein. Low platelets and proteinuria/hematuria are early signs of infection. When the amount of platelets starts to normalize the creatinine levels usually rises [41].

Figure 2. Typical clinical course in PUUV-infected patients (Vapalahti et al. Lancet 2003) [41]. Picture reprinted with permission from the publisher.

1.8.2.2 PUUV-infection: consequences

PUUV-infection is largely underdiagnosed. Based on seroprevalence studies in Finland and Sweden, it has been estimated that only around 20% of PUUV-infected persons get a clinical HFRS-diagnosis [59, 112]. The reported mean age of diagnosed PUUV-infected patients varies between 40-60 years [80, 113]. Children are rarely infected, but they show a similar clinical picture as adults [99, 114, 115]. There are no reports on transmission of virus from mother to child during pregnancy [81, 116].

Acute complications are described during PUUV-infection. Acute tubulointerstitial nephritis and interstitial hemorrhage can be observed in the kidneys during acute infection [117], while mesangiocapillary glomerulonephritis is reported as a complication after infection [118]. CNS involvement, such as seizures, meningitis, encephalitis, cerebral haemorrhage and Guillain Barré has been reported. PUUV-RNA and PUUV- specific IgM have been detected in cerebrospinal fluid [41, 119-121]. Myopericarditis is an acute complication and around half of the patients have abnormal cardiac findings, with signs of impaired contraction of the left ventricle [99, 122]. Severe respiratory symptoms are reported as an acute complication, resembling symptoms of HCPS [86].

Long-term consequences described 5 years after acute infection were hypertension and mild renal dysfunction [105]. However, a follow-up study 10 years later showed that these changes were no longer significant [106]. In addition, no correlation is shown between the severity of renal dysfunction during acute infection and long-term consequences such as hypertension or chronic renal impairment [123]. Cardiovascular disorders are common causes of death during acute PUUV-infection, but also within the first year after infection [124]. Hormonal deficiencies (hypothyroidism and hypopituitarism) can occur during acute phase, but can also be a long-term consequence of PUUV-infection [125].

1.9 CLINICAL DIAGNOSTICS OF HANTAVIRUS INFECTION

Hantavirus-infection can sometimes be misdiagnosed for a bacterial infection such as sepsis, urinary infection or pneumonia. This could lead to e.g. unnecessary antibiotic treatment. It is important to recognize the symptoms and to have reliable and quick diagnostic tools for hantavirus- infections [126]. The routine laboratory diagnostics of hantavirus-infection is based on serology. In Sweden, signs of fever, thrombocytopenia, blurred vision, hematuria and/or proteinuria can indicate a PUUV-infection, but the diagnosis must be confirmed with specific testing. During the first days after symptom debut, both virus specific IgM and IgG antibodies are usually present in patient sera [89, 127].

1.9.1 Immunoassays

Immunoflourescence (IF) and enzyme immunoassays (EIA) are methods based on the binding of patient antibodies to virus-infected cells (IF/EIA) or recombinant proteins (EIA). The methods rely on that the patients have developed antibodies towards the virus, which can sometimes take several days after disease onset [128]. Initially seronegative patients can be verified by an additional serological testing after 2-4 days, or sometimes with quantitative reverse transcriptase (RT)-PCR [129]. Patient sera that are IgM negative but IgG positive can be analysed using IgG avidity testing. This method can be used to discriminate between an acute infection and earlier immunity [130]. In many patients, the sera cross reacts strongly between SEOV, DOBV and HTNV and between PUUV and SNV-like viruses, while cross reactivity can be weak or absent between distantly related viruses e.g. DOBV and PUUV [131]. In certain areas, several hantaviruses are endemic and it is therefore not always possible to determine the causative hantavirus unless specific serology and/or neutralisation tests are performed [132-134].

Serological rapid tests are available for several hantaviruses (e.g., PUUV, DOBV and HTNV). These immuno-chromatographic tests take 5 minutes and are based on IgM recognition of the nucleocapsid-protein antigen of the specific hantavirus [135].

1.9.2 Reverse transcriptase (RT)-PCR

RT-PCR can be used to detect hantavirus-RNA in patient plasma. Real-time RT-PCR quantifies the viral RNA, a technique that can be used to monitor the kinetics of viral load in infected patients. Levels of viral RNA are highest early after disease onset (first days) and gradually disappears associated with an increase in antibody responses [129].

1.10 TREATMENT ALTERNATIVES AND PREVENTION

Hantavirus-infected patients are symptomatically treated, because there is no specific effective treatment available. One of the major problems in the development of vaccines and therapy is the lack of good animal models. The rodent reservoirs are not good disease models, as their infections are persistent and asymptomatic. The first and so far only animal model resembling HCPS in humans is the ANDV-syrian hamster model [136]. The only animal model described that resembles HFRS is PUUV-infected cynomolgus macaques [137, 138].

1.10.1 Antiviral drugs

Ribavirin, a nucleoside analogue that affects the fidelity of the polymerase, resulting in catastrophic errors during viral replication, has antiviral effect against hantaviruses in vitro [139]. In animal models, ribavirin treatment of HTNV-infected suckling mice efficiently lowered the risk of infection

progress and lethal outcome [140]. ANDV-infected Syrian hamsters were successfully protected from lethal outcome using ribavirin administrated the first 3 days post infection. The effect disappeared when ribavirin was administered a week after inoculation of the virus [141].

In humans, ribavirin has been tested as a treatment on HFRS-patients in China. The results of the placebo-controlled study showed significant beneficial effects, by reducing the risk of entering oliguric-phase and experiencing hemorrhage [142]. In Korea, ribavirin given early after disease onset lowered the risk of renal impairment in HFRS patients [143]. Ribavirin has also been tested on American HCPS-patients, but with no significant beneficial effect [144, 145]. The majority of the HCPS-patients were in the cardiopulmonary phase, and it is suggested that ribavirin efficacy is dependent on the phase of disease [146]. Hantavirus RNA can be measured in blood 2 weeks prior to symptom debut [34], therefore ribavirin might have a potential as prophylaxis or as early treatment during the febrile prodromal phase of HCPS [146].

Ribavirin is extensively used by clinicians in combination with recombinant interferon-α (IFN-α) against hepatitis C. One placebo-controlled trial has used IFN-α as treatment of HFRS-patients. The results showed no significant effect on mortality or oliguria/anuria, but reduced the risk of bleeding manifestations [147]. Pre-treatment of cells with IFNs is shown to inhibit subsequent hantavirus-infection in vitro [148-150].

In addition, there are other antiviral treatments tested experimentally against hantaviruses, but not yet on humans [151-154]. An anti-β3-integrin antibody effectively blocks entry of different pathogenic hantaviruses in vitro [153]. The same antibody also decreased viral load in SNV-infected deer mice [155]. Other compounds that target β3-integrin can be potential antivirals: peptides with homology to the β3-integrin receptor have been tested in vitro with promising results; reducing viral infection [29, 153].

1.10.2 Other therapeutic approaches

Hantavirus-infection in humans is characterized by capillary leakage. The endothelium normally forms a fluid barrier through cell adherence junctions, mainly through VE-cadherin. Vascular endothelial growth factor (VEGF) binding to VEGF-receptor 2 (VEGFR2) on endothelial cells can induce tissue oedema by increasing the endothelial permeability [156]. Pathogenic hantaviruses that bind to β3-integrin can sensitize endothelial cells to VEGF, causing enhanced VEGFR2-Src mediated VE-cadherin internalisation, and thereby increased endothelial permeability [157, 158]. Inhibitors/blockers of the VEGF receptor or of VEGF signalling are reported to reduce hantavirus induced endothelial leakage in cell lines and hamsters [29, 154, 158-161]. Several VEGFR2 and Src inhibitors are in clinical trials or are used to treat human cancers. These drugs may have a potential as treatment alternatives for hantavirus-infected patients [162].

A bradykinin-receptor antagonist has been used as therapy in a severe case of PUUV-infection. The patient´s condition improved within 24 hours after subcutaneous administration of the drug. The drug, icatibant, is a bradykinin receptor-2 antagonist, indicated for treatment of hereditary angioedema. The antagonist is believed to reverse bradykinin induced effects, especially vascular leakage [163].

Corticosteroid treatment has been reported for treatment of ANDV- and PUUV-infected patients. Methylprednisolone administered in combination with continuous hemodiafiltration was tested on two PUUV-infected patients with severe pulmonary symptoms. The combination treatment resulted in resolution of shock and respiratory symptoms [164]. For HCPS, the effects of using corticosteroid treatment were inconclusive [165].

1.10.3 Passive immunization

Another approach for treatment and/or for post exposure prophylaxis of hantavirus-infections is passive immunization. In the ANDV-hamster model, passively administered neutralizing sera protected hamsters from disease if used as prophylaxis, but it was also protective against lethal outcome if administered several days after virus challenge [166-168]. In macaques, sera from previously PUUV-infected animals caused sterile protection in animals upon viral challenge [169]. Notably, in SNV-infected patients, a low specific IgG response is associated with a more severe clinical outcome [170, 171]. These results suggest that a strong neutralizing antibody response can be predictive for milder disease and that passive immunization may be useful as therapy [146, 170]. Promising results are reported from South America, using plasma from immune survivors as treatment of HCPS-patients. The results indicate significantly reduced mortality in patients, as referred in Dolgin et al, 2012 [76]. Passive immunization, using neutralizing antibodies, may be limited to patients from the same geographical area, as demonstrated in vitro, when convalescent sera from ANDV-patients showed low or even absence of cross-neutralizing antibodies to SNV [93].

1.10.4 Vaccines

No safe and effective vaccine is available for HFRS in Europe or for HCPS in the Americas. Target groups for hantavirus vaccination represent rural residents in hantavirus-endemic areas and those with risk factors for rodent exposure e.g. forest workers, military personnel and farmers [37]. Medical personnel treating ANDV-infected patients could be another potential group for vaccination [146].

Korea and China are the only countries with hantavirus vaccines in practical use. The vaccines are inactivated HTNV and/or SEOV viral suspensions propagated in rodent brain or cell-culture. In China, about 2 million vaccine doses are given annually and the vaccinations are considered both effective and safe [38]. The usage of HantavaxTM in Korea since the early 1990s has

corresponded with a decrease in the number of HFRS cases. However, the efficacy of HantavaxTM and other inactivated vaccines are questioned due to relatively low neutralizing antibody titers and the need of frequent boosting [172, 173]. The brain-derived vaccine is not approved in most countries, due to safety concerns, regarding autoimmune encephalitis [37, 173].

Promising molecular vaccine approaches are based on recombinant proteins [174, 175], vaccinia-vectored vaccines [176] and DNA-vaccines. DNA- vaccines have been tested using linear DNA [177] or plasmids expressing nucleocapsid and/or glycoproteins [167, 178-180].

Three vaccines have been evaluated in clinical trials in the US: two DNA vaccines based on the M-segment of HTNV and PUUV, and one recombinant vaccinia-virus vaccine against HTNV. The recombinant vaccinia-virus- vectored vaccine expressing genes of HTNV was stopped after clinical evaluation, as less than 30% of previously smallpox-vaccinated individuals produced neutralizing antibodies upon vaccination [176]. The DNA-vaccines generated neutralizing antibodies, at best in 56% of the volunteers, when using a combination of both the HTNV- and PUUV M-segments [181]. A comprehensive DNA vaccine against HFRS is suggested to require two components [173]. Hence, cross-reactive immunity is shown among HTNV, SEOV, and DOBV, but not PUUV vaccinated animals [179]. To date, there is no vaccine tested against HCPS in humans [182].

1.11 INNATE IMMUNE RESPONSES TO VIRAL INFECTIONS

The immune system is a defense system that provides host protection against invading microorganisms such as viruses. It comprises of two branches, innate and adaptive immunity. The innate response is our first line of defense against infection, ready for immediate action when pathogens attack. It is characterized by being unspecific, recognizing a broad range of pathogen molecules and lacking immunological memory properties. The later adaptive immune response is mounted after several days. This response is antigen-specific, and has an immunological memory upon a second encounter with the same pathogen. The innate and adaptive immune systems are highly interactive, as the innate immune response helps shape an effective adaptive immune response.

The first hurdle for a virus or other pathogen is different barriers that protect the host. Such barriers are the skin, and the mucus membranes of e.g. the lungs or gastrointestinal tract. Beyond the primary anatomical barriers innate immunity includes: phagocytic cells such as macrophages (MΦ) and granulocytes, antigen presenting cells (APCs)/dendritic cells (DCs), natural killer cells (NK cells), natural killer T cells, γδ T cells and soluble molecules such as complement, chemokines, cytokines and acute phase proteins [183].

1.11.1 Innate recognition of viral pathogens

When viruses enter the body by passing the protective epithelial barriers and surviving antimicrobial peptides; pattern-recognition receptors (PRRs) of the innate immunity play a central role. These receptors are important in detecting the infection and triggering antiviral responses. PRRs recognize molecules known as pathogen-associated molecular patterns (PAMPs), broadly shared by pathogens. The invaded tissue´s immature DCs and MΦs have a variety of PRRs, including the important Toll-like receptors (TLRs) [184].

TLRs are present not only on DCs and MΦs, but also on B cells, specific types of T cells and non-immune cells, such as epithelial cells. There are 11 different TLRs in humans, recognizing essential building blocks of bacteria, viruses and parasites. TLRs recognizing viruses are the TLR 3, 7, 8 and 9, all found predominantly inside cells, bound to endosomal compartments. TLR3 recognizes double stranded RNA that appears in cells after infection with RNA viruses. Viral single-stranded RNA is the ligand for TLR7 and TLR8 [185]. TLR9 recognizes CpG unmethylated DNA and can detect e.g. herpes viruses [184]. There are other intracellular PRRs recognizing viruses; retinoic acid inducible protein (RIG-1), recognizing viral single stranded RNA by a 5´triphosphate group; and myeloid differentiation-associated factor 5 (MDA-5), recognizing double stranded RNA [186, 187].

TLR binding to PAMPs activates downstream signalling cascades leading to activation of innate cells and the production of proinflammatory cytokines and interferons (IFNs) [185]. All nucleated virus-infected cells can produce type-I IFNs (IFN-α and IFN-β) at the site of infection, although the plasmacytoid DCs (pDCs) are the main producers. IFNs bind the IFN- receptor and activate the JAK-STAT signalling pathway, thereby warning the body of the dangerous intruders. This pathway induces transcription of several hundreds of interferon stimulated genes (ISGs), the products of which can inhibit viral replication or degrade viral RNA [188].

1.11.2 Natural killer cells

Natural killer cells (NK cells) are one of the major cellular components of the innate immunity, providing an important first line of defense against many viral infections. NK cells, defined as CD3-negative CD56-positive cells, are lymphocytes that lack rearranged antigen-specific receptors. They comprise 10-15% of peripheral blood lymphocytes, but are selectively enriched in other organs, such as the uterus during pregnancy as well as in the liver [189]. NK cells are activated by IFN-α and IFN-β and elevated levels of these cytokines are followed by a rapid increase in the NK cell population (Figure 3). Other NK-cell activating cytokines are interleukin-2 (IL-2), IL-12, IL-15 and IL-18 [190].

NK cells

IFN-α/β Response

0 1 2 3 4 5 6 7 8 9 10 11 12 Days post infection Figure 3. The NK cell response to viral infection

NK cells have the ability to recognize and kill abnormal cells such as virus- infected cells or tumour cells, in a process referred to as “missing self recognition” [191, 192]. Accordingly, NK cells engage in surveillance of the body, recognizing and killing cells that fail to express major histocompatibility complex (MHC) class I molecules on their surface. Hence, infected cells or tumour cells that downregulate their MHC class I in order to escape an adaptive T cell response, might instead be targeted by NK cells [192]. Different NK cells express different sets of membrane receptors. One abundant receptor is CD16, a receptor for the non-antigen binding part of antibodies (Fc-receptor). NK cells can be subdivided into CD56bright NK cells and CD56dim NK cells, depending on the surface density of this antigen. CD56bright CD16-negative NK cells are predominant in lymphoid tissues, whereas the CD16+CD56dim NK cells are predominant in peripheral blood [190, 193]. CD56bright NK cells have an important immunoregulatory function and produce high levels of soluble cytokines such as IFN-γ and TNF-α. On the other hand, CD56dim NK cells have a more cytotoxic profile, containing high levels of cytotoxic granules [194]. Notably, both types can be cytotoxic and produce cytokines [195-197].

NK cell cytotoxicity can be mediated via several mechanisms: I) Natural cytotoxicity, by releasing granzymes and perforin that induce apoptosis in the target cells [198]. II) Interaction between the apoptosis-induced receptors Fas-ligand and/or TRAIL on NK cells with Fas or TRAIL-receptors on target cells [197, 199]. III) NK cell killing of target cells via CD16 and specific antibodies recognizing the target cells, a process referred to as antibody- dependent cellular cytotoxicity.

In humans, NK cell immunodeficiency resulting in complete absence or impairment of NK cells is associated with increased susceptibility to viral infections such as herpes infections [200-202].

1.11.3 NK-cell receptors

NK cells recognize and react against target cells through their expression of different activating and inhibitory receptors. The balance between signalling through these inhibitory and activating receptors regulates NK cell functions, such as lytic capability and/or capacity to produce cytokines. In resting NK cells, at least two different activating receptors need to be engaged to cause NK cell degranulation, a so called synergistic coactivation [203, 204]. In contrast, cytokine-activated NK cells need only to engage one activating receptor to induce its functions [203, 205].

Two of the major groups of activating/inhibitory receptors are the lectin-like family and the immunoglobulin (Ig) super family of receptors. Inhibitory receptor subfamilies are the killer-cell immunoglobulin-like receptors (KIRs) and the lectin-like receptor CD94/NKG2A. The KIRs recognizes MHC class I molecules (primarily HLA-B and HLA-C) on target cell surfaces. The CD94/NKG2A receptor recognizes the non-classical HLA-molecule HLA-E on target cells. This receptor is not found on cell surfaces unless it has bound peptides derived from other MHC class I molecules (HLA-A, HLA-B or HLA- C). Examples of activating receptors are NKG2D, activating members of the KIR family of receptors and also the CD94/NKG2C receptor [190, 193]. Interestingly, ligands to the activating receptor NKG2D such as MIC-A, MIC- B and ULPBs, are stress-induced. Thus, cells that are stressed due to a viral infection or trauma upregulate NKG2D-ligands, which then are recognized by the activating NKG2D receptor. The NKG2D receptor-ligand interaction is considered one of the major mechanisms for NK cell cytotoxicity of infected or stressed cells [206].

1.11.4 Phagocytic and antigen presenting cells

Neutrophils and MΦ can phagocyte extracellular microorganisms and viruses bound to antibodies or complement factors. The engulfed pathogens are killed within the cells by substances such as reactive oxygen and nitrogen intermediates, and these substances can also leak into the surroundings, contributing to tissue damage. Besides killing and clearing pathogens by phagocytosis, activated MΦ play an important role in coordinating other cells of the immune system. They secrete proinflammatory cytokines such as IL-1, IL-6 and TNF-α. These cytokines stimulate immune cells, promote fever by affecting the thermoregulatory center in the hypothalamus, and induce acute phase proteins from the liver.

DCs are an important bridge between the innate and the adaptive immune response. The antigen presenting DCs can present antigens on MHC class I and II molecules. MHC class I presents intracellular proteins, while MHC class II presents extracellular antigens. Immature dendritic cells have phagocytic activity and also have a variety of PRRs. After pathogen recognition, the DCs mature to effective antigen presenting cells that migrate to nearby lymphoid tissues/nodes where they present the antigen to T helper

(TH) cells and cytotoxic T cells (CTLs), thereby activating the adaptive immune response [184].

1.11.5 Cytokines

Cytokines are soluble molecules that can bind to receptors on various cell types, functioning as signals between cells. Chemokines are cytokines that when secreted can induce chemotaxis of many leukocytes. Cytokines are not only produced by several cell types, but they also activate different target cells. A target cell is exposed to a milieu containing several different cytokines that together form synergistic or antagonistic effects. Examples of a few important cytokines are presented below:

IFNs are secreted cytokines with antiviral effects and there are three classes. Type I IFNs are important players of the innate immune response against viral infection. All nucleated virus-infected cells can produce IFN-α and IFN- β. These cytokines induce ISGs and antiviral states in surrounding cells, limiting the spread of a virus [188]. Type II (IFN-γ) is produced mainly by activated T cells and NK cells. Type III IFNs, secreted from epithelial cells, comprise of IFN-λ1 (IL-29), -λ2 (IL-28A) and -λ3 (IL-28B), which all have antiviral effects [207].

Proinflammatory cytokines such as: -IL-1 is produced by monocytes/MΦ, DCs, adaptive immune cells and NK cells, but also by non-immune cells such as endothelial cells. IL-1 stimulates e.g. T and B cells. -IL-6 is secreted by MΦ, B and T cells and from non-immune cells such as endothelium and regulates adaptive T and B cell functions. -TNF-α is produced by MΦ, NK cells, but also by activated B and T cells and other non-immune cells. TNF-α is involved in e.g. increasing vascular permeability and neutrophilic granulocyte phagocytosis [208].

-IL-8 is a chemokine produced by many cell types such as endothelial cells, granulocytes, monocytes and is a chemo-attractant for neutrophils and lymphocytes.

-IL-12 is produced by MΦ and DCs and e.g. stimulates IFN-γ production by activated T and NK cells.

-IL-15 is produced primarily by DCs and mononcytes/MΦ and is important for NK and T cell proliferation and development [183, 208].

1.12 ADAPTIVE IMMUNE RESPONSES TO VIRAL INFECTIONS

The adaptive immune response can recognize and selectively eliminate pathogens and is highly antigen-specific. The adaptive immune response has immunological memory and upon a second encounter with the same pathogen a rapid and heightened immune response is induced. The response comprises of T and B lymphocytes, plasma cells and antibodies. The major

component of the humoral response is the induction of specific antibodies, secreted from plasma cells, while the cell mediated immune response is a result of activated cytotoxic T cells. The adaptive responses are acquired by the recognition of antigens presented by MHC class I and II molecules.

1.12.1 General features of T cells

T cells express the T cell receptor (TCR) and depending on the type of TCR cells can be subdivided into either TCRαβ or TCRγδ cells. The TCR is associated at the cell surface with CD3, making the cells CD3-positive.

The T cells having TCRαβ are divided into two classes, defined by the expression of either CD4 or CD8 molecules. The CD4+ T cells are known as T helper cells (TH), and function as coordinators, promoting humoral or cellular immune responses. In peripheral blood, 60-70% of T cells are CD4- positive and 30-40% CD8-positive cells. The CD8+ T cells are also called cytotoxic T cells (CTLs), which have the ability when activated to kill infected cells [184].

1.12.2 CD4+ T cells

Naive CD4+ T cells can be activated by recognition of antigens presented by MHC class II molecules on APCs. Upon activation the cells start to produce IL-2 and as they continue to respond to cytokines they can differentiate into different T helper cell subtypes:

TH1 cells can be induced by IL-12, secreted from MΦ, NK cells or DCs. These TH1 cells secrete IL-2, IFN-γ and TNF that can evoke cellular immune responses by activating CTLs.

TH2 cells are induced by e.g. IL-4 stimulation of naive CD4+ T cells. TH2 cells can evoke the humoral response by releasing IL-4, IL-5, IL-10 and IL-13. The differentiation into TH17 cells is induced by IL-6 and transforming growth factor-β (TGF-β). TGF-β also helps promote naive CD4+ T cells into regulatory T cells.

Collectively, the cytokine milieu that is created favours either a TH1 or TH2 response. Cytokines involved in TH1 and cellular immunity include IL-2, IFN-γ, TNF, IL-12, and IL-15. Cytokines that activate TH2 and a humoral response are e.g. IL-4, IL-5, IL-9, IL-10, and IL-13 [183].

1.12.3 Regulatory T cells

Regulatory T cells (Tregs) are a group of CD4+T cells that express high levels of surface antigen CD25, as well as the transcription factor, forkhead box protein 3 (FOX P3). There are several types of Tregs and two main subtypes; natural Tregs developed mainly in the thymus, regulating self-reactive T

cells; and inducible Tregs found in peripheral organs, developed from naive CD4+ cells [209].

Tregs can suppress immune responses either through cell contact or via the production of immunosuppressive cytokines such as transforming growth factor-β (TGF-β) and IL-10 [210]. Tregs can inhibit the proliferation of other T lymphocytes and thereby control an overactive immune response and limit immunopathology. On the other hand, an excessive Treg response can limit an effective immune response contributing to e.g. viral persistence and viral expansion/reactivation [209].

1.12.4 CD8+ T cells

An effective cellular response mediated by TH1 cells and activated CTLs are important for clearance of virus-infected cells. The activated TH1 cells produce IFN-γ that stimulate CTLs, but also induce an antiviral state in cells. In most viral-infections, the CTL response peaks during the second week of infection (Figure 4).

NK cells Virus-specific CTLs

IFN-α/β Response

0 1 2 3 4 5 6 7 8 9 10 11 12 Days post infection

Figure 4. Generation of an adaptive CTL response

CTLs recognize antigens presented by MHC class I molecules on infected cells. Virtually all cells express MHC class I and therefore CTLs can kill and eliminate almost any cell type. To avoid self-reactivity by CTLs, the activation from naive precursor CTLs to effector killer CTLs is tightly regulated in several steps. The activation requires both TH1 and CTL recognition of the antigen, costimulatory-receptor activation, as well as, stimulation by IL-2 for proliferation [183, 184].

The effector CTLs can kill target cells by inducing apoptosis. The killing is mainly through exocytosis of cytolytic granules (containing perforin and granzymes) or by Fas/Fas-ligand binding. Activated CTLs also release cytokines such as IFN-γ and TNF-α that can purge the virus from infected cells, without killing them [211].

1.12.5 Memory CD8+ T cells

Following pathogen clearance, effector CTLs undergo contraction by apoptosis, but small numbers survive and mature into long-lived memory T cells. These memory CD8+ T cells persist in the absence of antigen and can give rise to long-term immunity. When re-stimulation occurs, these memory T cells rapidly proliferate and differentiate to effector CTLs.

The memory T cells share many surface molecules with effector CTLs and there is, to date, no single cell-surface molecules that specifically identifies the memory pool. IL-7 and IL-15 mediate survival and self-renewal of memory T cells. High levels of interleukin-7-receptor-α (IL-7Rα/CD127) are associated with long-term survival of memory cells and other surface molecules that co-aggregate with CD127 are CD27 and CD28 [212, 213]. Memory T cells can be divided into central memory cells (TCM) and effector memory cells (TEM). The TCM are CCR7+ cells that can self-renew and are found mainly in secondary lymphoid organs. TEM are CCR7-, found in tissues, and have effector cytotoxic functions. These cells can also express CD45RA and are then referred to as TEMRA [214]. After most acute viral infections, the memory pool of CD8+ T cells gradually redistributes from being mostly TEM cells towards TCM cells. Differences in proportion of these cell-types can influence the immune response to re-infection. The memory T cell phenotype changes over time after an acute viral infection. The memory T cell differentiation is also different when comparing persistent viral infections, such as EBV and HCV [215].

1.12.6 Immunoregulatory receptors

Activation of naive CTLs to effector CTLs is a tightly regulated process. The activation requires antigen recognition, but also co-stimulation through the interaction between CD28 on CTLs and members of the B7 family on APCs [184]. An antagonist to the stimulatory CD28 is the inhibitory receptor, cytotoxic T lymphocyte antigen-4 (CTLA-4). CTLA-4 competes with CD28 for the binding to B7. The CTLA-4/B7 interaction is inhibitory, providing regulatory braking of T cell activation and proliferation. Another important inhibitory immunoregulatory receptor is programmed cell death 1 (PD-1). These inhibitory receptors on T cells help dampen massive lymphocyte proliferation, needed for maintaining tolerance and for avoiding autoimmunity [216].

Although inhibitory immunoregulatory receptors are important for regulation of T cell activation, they also can cause dysfunctions in T cell immunity. Up-regulation of PD-1 and CTLA-4 on T cells, can cause a so- called “exhausted” phenotype. This exhausted phenotype is associated with viral persistence and disease progression in chronic human infections, such as HIV and HCV [217-219]. Blocking of inhibitory receptor signals can result in partial recovery of T cell function, improving the immune response to infection [220]. It is suggested that blocking of CTLA-4 or PD-1 signals have

a potential as complement treatment of HIV-infected patients [218]. Blocking of CTLA-4 has been tested in patients with hepatocellular cancer and chronic HCV, with promising results [221].

1.12.7 Humoral response

Antibodies of the humoral response can bind viruses, thereby preventing virus entry into cells. Antibody binding of viruses leads to blocking of the physical interaction between viruses and cell receptors, but also to activation of the complement system and of phagocytic cells.

Antibodies are secreted from activated B cells (plasma cells) after recognition of antigens by the B cell receptor (a membrane-bound antibody molecule). Following a primary infection IgM antibodies are secreted first, usually within a few days. Activated B cells mature and isotype switching occurs after stimulation by TH cells and their cytokines. In isotype-switching, the constant part of the immunoglobulin molecule is changed, producing IgA, IgG or IgE antibodies. IgG antibodies are the most abundant class of immunoglobulins in serum, representing about 80%, while IgA are predominant in external secretions such as saliva, tears, and in mucus of the bronchial and gastrointestinal tract. Antigen recognition of B cells forms antibody secreting plasma cells, but also long-lived memory B cells [183].

1.12.8 The balance of an effective immune response

Humans are infected by various viruses during a life-time. Some viral infections are acute and transient, while others develop into persistent infections. Many virus-infections are cleared with no or limited tissue damage, while others induce extensive damage to the host. A virus-infection that for some patients are asymptomatic, can for others be severe or even lethal. Disease outcome in virus-infected patients is influenced by many factors, including properties of the infecting virus, the dose, and the route of infection. In addition, host factors are important such as; genetic susceptibility, the age of the individual, and differences in the immune response. An appropriate immune response is a delicate balance between clearance of the infectious agent and minimizing immune-mediated pathology. An overactive immune response can cause extensive tissue damage through excessive production of pro-inflammatory cytokines and the activation of CTLs. On the other hand, an overweighted activation of Tregs, regulatory cytokines (IL-10/TGF-β) and/or inhibitory immunoregulatory receptors (CTLA-4/PD-1) may lead to ineffective clearance and viral persistence (Figure 5) [222].

Figure 5. A viral infection leads to recognition by PRRs and activation of innate immune responses, such as e.g. IFN secretion. NK cells are important players of the innate immune response, killing virus-infected cells and producing cytokines. Phagocytic cells such as MΦ can be activated, increasing their phagocytic capacity and releasing proinflammatory cytokines. DCs carry viral antigens to local draining lymph nodes. Depending on the cytokine mileu in the lymphnodes, different types of T-helper cells are induced. TH1 cells secrete IFN-γ/TNF and help activate CTLs. These activated CTLs are important for viral clearance, killing virus-infected cells. Tregs secrete IL-10 and TGF-β, which can dampen excessive T cell activity. TH2 cells help activate B cells, which then secrete antibodies that can neutralize viruses. An overactive immune response will clear the virus, but will also gives rise to extensive tissue damage/immunopathology. In contrast, an excess of anti-inflammatory mechanisms (e.g. Tregs, inhibitory receptors) will lead to viral persistence. An effective immune response will not only clear the virus and cause immunity to reinfection, but will also minimize damage to host tissues. The figure is modified from Figure 1 in Rouse et al., 2010 [222], with permission from the Publisher.

1.13 HANTAVIRUS PATHOGENESIS

HCPS and HFRS are considered different clinical manifestations, with a similar underlying pathogenesis. The two human diseases are characterized by vascular dysfunction, thrombocytopenia and increased vascular permeability [1, 223]. Hantaviruses target endothelial cells, but are non- cytopathogenic [224, 225]. So, what then causes this vascular leakage syndrome? The pathogenesis of hantavirus-mediated disease is most likely a complex multifactorial process. It is unclear whether the vascular leakage is

caused by hantavirus dysregulation of endothelial cell functions and barriers and/or by an overactive immune response [1, 226].

1.13.1 Hantavirus immunopathogenesis

Non-cytopathogenic hantaviruses can replicate for at least 2 weeks (sometimes up to 6 weeks or longer) in the human body without causing disease. It is still not known what triggers disease onset and progression, but there is a temporal association between disease onset and an extensive immune response in patients [14, 18]. Although, the immune response might protect against hantavirus-infection, the same immune response, when dysregulated, might contribute to hantavirus-induced immunopathology [223, 227]. A dysregulated, overactive immune response is implicated in disease pathogenesis, especially cell-mediated responses and the release of proinflammatory cytokines [1, 37, 227].

1.13.1.1 Innate immune responses

Hantaviruses are spread to humans through inhalation of infectious virions, hence the first site of infection is the lungs. The virus is known to infect predominantly endothelial cells, but also epithelial cells, monocytes/MΦ and DCs [23, 25-27].

The first line of defense, after anatomical barriers, is the innate immune response. Hantaviruses have strategies to escape recognition of PRRs, inhibit induction of IFNs, and inhibit the induction of ISGs. HTNV can be recognized by TLR-3 [228], but the RNA genome is not recognized by RIG-1 [229]. In vitro, pathogenic hantaviruses (HTNV, ANDV, New-York-1 virus) can suppress the induction of early cellular IFN responses [23, 230]. Hantavirus proteins are known to inhibit or delay IFN-production by different ways (e.g., escaping recognition or inhibiting RIG-1 signalling, disrupting downstream TBK-1-TRAF-3 complex formation or NFκB signalling) [148, 229, 231, 232]. Also, SNV-Gn is involved in inhibiting STAT phosphorylation, thereby potentially interfering with ISG induction [230]. IFN-λ is secreted from hantavirus-infected epithelial cells [233]. The secretion is more rapid in non-human pathogenic Prospect Hill virus (PHV)- infected cells compared to ANDV- and SNV- infected cells [234]. Levels of IFN-α, IFN-β and IFN-λ are not elevated in PUUV-infected patient sera, supporting reports that pathogenic hantaviruses are weak inducers of type I and type III IFN responses [149]. In summary, pathogenic hantaviruses can deregulate the IFN-mediated antiviral state, which is important for viral replication and for viral spread within the endothelium [18].

NK cells are important players of the innate immune response against viral infections, through their cytotoxic activity and cytokine production (IFN-γ and TNF-α). The role of NK cells in hantavirus-infection is largely unknown, but is investigated in Paper II. NK cells are elevated in the lungs and peripheral blood of PUUV-infected patients during acute phase of disease

[235, 236]. In CCHF-infected patients (another member of the Bunyaviridae family), higher NK cell numbers in peripheral blood are associated with severe disease [237], in contrast to in dengue patients, where high NK cell numbers seem protective against a more severe clinical outcome [238].

Dendritic cells have a pivotal role as antigen-presenting cells. Unlike many other viruses that impair the function of DCs, hantaviruses infect and stimulate immature DCs. This stimulation of DCs will increase their expression of MHC molecules and also their cytokine secretion [26]. Through secretion of different cytokines, DCs can help drive the development of different types of TH cells, hence modulating the outcome of infection [223]. Maturing DCs migrate to regional lymph nodes, where they can infect other immune cells. This leads to further spread of the virus, and in turn, the induction of a powerful adaptive immune response. Hence, infection of DCs cells might serve as a vehicle for hantavirus-dissemination [37].

Cytokines are important in shaping the immune response to virus infection. Endothelial cells, MΦ and DCs are important sources of cytokines during hantavirus-infection. An imbalance or overproduction of certain cytokines might induce immunopathological processes. High levels of proinflammatory cytokine (IL-1, IL-6 and TNF-α) producing cells are found in immunohistochemical-stainings of lung tissue from fatal HCPS patients [239]. Increased levels of TNF-α are found in kidney-biopsies of PUUV- infected patients [240]. In peripheral blood, proinflammatory cytokines (IL- 1, IL-6, TNF-α) and nitric oxide (NO) are elevated during the acute phase of the disease [241, 242]. TNF-α and NO are known inducers of vascular permeability and are therefore suggested to be important in hantavirus pathogenesis [242, 243]. IL-6 is also suggested as a potential marker of disease severity in PUUV-infected patients [244], as well as being associated with fatal outcome in HCPS patients [245]. IL-6 and TNF-α can both induce NO, implicated to have a negative inotropic effect on the heart [245, 246].

IL-2, IL-6, IL-8 and TNF-α were reported to be elevated during acute PUUV- infection, while regulatory TGF-β was elevated later (week two) and during convalescent phase of disease [247]. The authors suggest a protective immunoregulatory role for TGF-β, and that an imbalance between proinflammatory and inhibitory cytokines could potentially contribute to disease pathogenesis. In HCPS-patients, high levels of TH1 cytokines were shown to be associated with more severe clinical outcome. The patients also showed low levels of regulatory TGF-β [245]. In DOBV-infected patients, increased levels of both pro-inflammatory TNF-α and regulatory IL-10 were significantly associated with severe disease, suggesting that the balance of produced cytokines may influence disease progression [248].

Hantavirus-infection (HTNV and SNV) of human lung microvascular endothelial cells in vitro induces the expression of the chemokines: IFN-γ- induced protein-10 (IP-10) and RANTES. IP-10 and RANTES are

predominantly chemotactic for mononuclear leukocytes. The authors suggest that T cells, recruited to the site of infection by these chemokines, produce IFN-γ. In turn, IFN-γ induces more chemokine production, contributing to an extensive recruitment of the cell-mediated immune response [249].

1.13.1.2 Cell-mediated responses

CTLs might play an important role in the protective immune response against hantavirus-infection, but are also implicated in pathogenesis [223, 250].

During acute hantavirus-infection, vigorous T cell responses are observed in both HCPS and HFRS patients [251-253] (Paper III). When pulmonary oedema develops in HCPS patients, large immunoblasts are found in the circulation [254]. In addition, high levels of mononuclear cells are found in histopathological analysis on lungs from HCPS-patients and kidney biopsies from HFRS-patients [117, 240, 255]. In vitro, CTLs are shown to increase hantavirus-infected endothelial cell layer permeability [256] and TH1 cytokines are elevated in infected patients [245, 247, 248].

In a study using tetramer staining, high levels of SNV-specific CTLs were significantly associated with severe disease, supporting the notion that CTLs are important mediators of immunopathogenesis [251]. In contrast, higher frequencies of IFN-γ producing HTNV-specific T cells were shown to be protective against severe HFRS [253]. Notably, these seemingly contradictory studies may be explained by the difference in used methods [227, 253]. In a study on PUUV-infected patients, a large proportion of T cells detected during the acute phase using tetramer staining were not readily detected using IFN-γ analysis [252]. This suggests that the two methods show different T cell subsets. Collectively, CTLs with different functions (cytotoxic or non-cytotoxic) increase in hantavirus-infected patients and the quality rather than the quantity of the antiviral T cell response may predict the outcome [227].

An effective CTL response can help clear the infection, but an overactive response may contribute to capillary leakage [37, 227]. It is suggested that CTLs increase vascular permeability by killing hantavirus-infected endothelial cells [223, 250]. This is supported by increased levels of perforin, granzyme B, and of the epithelial cell apoptosis marker caspase cleaved cytokeratin-18 (CK-18), present in patient serum during acute PUUV- infection [257]. However, in biopsies from patients, the infected endothelial cells are not seemingly destroyed [255, 258] and recently it was shown in vitro that hantavirus-infected endothelial cells are protected from cytotoxic lymphocyte mediated killing. The nucleocapsid protein of ANDV/HTNV can inhibit the enzymatic activity of granzyme B and caspase 3, hereby vastly protecting infected cells from apoptosis [259]. It is suggested that the inability of CTLs to kill virus infected cells may explain the overactive CTL response observed in patients. Possibly, these cells contribute to vascular

leakage through extensive release of inflammatory cytokines [227, 259]. Interestingly, in SNV-infected syrian hamsters, an almost total depletion of T cells does not change the course of infection. This study implies that the T cell accumulation is merely correlating and not causing disease in the hamster model; findings that questions the role of CTLs in hantavirus- pathogenesis [260].

The cell-mediated response is implicated in the pathogenesis of disease, but is also important in clearing the infection and for development of protective immunity. A robust T cell memory response develops after hantavirus- infection [90, 92]. The frequencies of hantavirus-specific CTLs many years after infection are comparable or even exceeding CTL responses after influenza and EBV [92]. Many years post ANDV-infection, virus-specific T cells have an effector memory profile (being CD127 (=IL-7Rα) negative). This memory cell phenotype resembles that of continuously activated memory cells seen in latent infections, suggesting an intrinsic latent antigenic stimulation of these Gn-specific T cells [90]. Hence, these findings raise the possibility that hantavirus virions or antigens may persist in humans for a long time or that boosting occurs by e.g. asymptomatic reinfection [37, 92].

An overactive T cell response might contribute to human hantavirus-disease. Tregs are important regulators of the cell-mediated T cell response, and a delayed or suppressed Treg response may play a part in hantavirus immunopathology [223]. In contrast to humans, the hantavirus reservoir hosts are persistently infected in the absence of pathological disease. Elevated levels of Tregs and TGF-β are found in the lungs of SEOV-infected rats and SEOV-infected lung endothelial cells help polarize the naive CD4+ T cell differentiation towards Tregs [261, 262]. Elevated Treg responses are also found in SNV-infected deer mice, findings collectively suggesting that upregulation of regulatory responses contribute to viral persistence in the rodent host [263]. In humans, an imbalance between an overactive CTL response and Tregs is supported by studies showing delayed induction of TGF-β in HCPS patients and significantly reduced levels of Tregs during acute HFRS [245, 264].

1.13.1.3 Humoral responses

The humoral response is already present when the first symptoms appear in hantavirus-infected patients. Virus-specific IgM as well as IgG antibodies can readily be detected in sera, although the IgG titers usually rise more slowly [89, 265-267]. In SNV-infected patients, low virus-specific IgG titers were significantly associated with more severe disease [170, 171, 268]. These findings indicate that a strong IgG response may be a predictor of favourable outcome in patients, which is supported by data in our study (Paper IV). The humoral response towards hantaviruses is long-lasting, showing high levels of neutralizing antibodies years after infection in HCPS- and HFRS-patients [89, 93, 268, 269]. Repeated symptomatic infection with the same

hantavirus has not been detected and the disease is believed to cause life- long immunity. In a study on ANDV-infected patients, the neutralizing antibody titers increased months to years after primary infection, independently if the patients were residents in endemic regions or not. The reason for this boosting of the antibody response is unknown, but could suggest a latent antigenic stimulation [90].

1.13.2 The viruses: viral load and virus effects

Viral RNA is readily detected in the circulation at disease onset. RNA is detected in a majority of hantavirus-infected patients for up to at least one week after disease onset [129, 270] (paper IV). Recently, RNA levels were found in patients for up to 30 days in DOBV-infected patients and 16 days in PUUV-infected patients, which means presence of viral RNA in patients, clinically in convalescent phase [265]. It is shown that more virulent hantaviruses (like SNV and DOBV) tend to give higher viral loads than PUUV in infected patients [265, 271, 272]. In addition, a higher viral load is associated with more severe disease in SNV-, HTNV- and DOBV-infected patients [270-273].

Hantavirus-infection does not damage endothelial cells in vitro [25, 224], and in patients the endothelial cellular morphology seem largely intact [258, 274]. Pathogenic hantaviruses are known to bind to β3-integrins, present on endothelial cells. This β3-receptor forms a complex with the VEGF2- receptor. Pathogenic hantavirus infection of endothelial cells enhances the endothelial cell permeability in response to VEGF [157, 159]. VEGF is a potent vascular permeability factor and can be released from endothelial cells under hypoxic conditions [226]. Elevated levels of VEGF are found in hantavirus-infected patients, suggesting that hypersensitivity of infected endothelial cells to VEGF is involved in hantavirus pathogenesis [275, 276].

1.13.3 Coagulopathy

In HFRS patients, hemorrhage is common and is believed to be caused by vascular injury, deficit of functional platelets and/or as a consequence of disseminated intravascular coagulation (DIC) [277]. DIC is an imbalance of thrombosis-fibrinolysis, found in about 25% of all PUUV-infected patients [278, 279]. DIC is also associated with a more severe clinical outcome in the patients [279]. Pathogenic hantaviruses recruit platelets to adhere to endothelial cells, by binding to their β3 receptors. This binding of platelets to the endothelium may be a mechanism for hantaviruses to induce platelet dysfunction and thrombocytopenia [226, 280].

1.13.4 Genetic host factors

MHC class I and II molecules present viral peptides to T cells. Different MHC molecules present different sets of viral peptides. MHC molecules are encoded by HLA-A, HLA-B, HLA-C (MHC class I) and HLA-DR, -DP, and

DQ (MHC class II) genes. There are several hundred alleles of these genes, which means that viral peptides are presented differently depending on the specific HLA-alleles expressed in the infected patient [184]. Certain HLA class alleles are associated with disease severity in hantavirus-infected patients, supporting the role of T cells in immunopathogenesis [227]. PUUV- infected patients with the HLA-B8-DR3 haplotype are more prone to have a severe disease, while HLA-B27 is associated with benign disease [281, 282]. In a Chinese study, the HLA-DRB1*09 allele is significantly more common in HFRS patients than in controls [283]. In HCPS patients, there are contradictory reports showing that the HLA-B3501 allele is associated with an increased risk of severe disease in SNV-infected patients, but with a mild course of disease in ANDV-infected patients [90, 251].

1.13.5 What decides the outcome?

The disease severity of human hantavirus-disease varies between different virus strains and the spectra of affected organs depend on the tropism of the virus. HCPS resembles HFRS except that the lungs are the predominant targets instead of the kidneys. HCPS is usually a severe disease with rapid onset of respiratory failure and cardiogenic shock. The disease severity of HFRS varies from severe for HTNV to mild for PUUV-infection. Notably, there are individual differences in disease severity in both HCPS and HFRS and e.g. an individual PUUV-case can be severe and an HTNV-case mild [1]. So, what may affect the outcome of an individual patient? To conclude, the severity of disease does not only reflect different virus strains, but may also be influenced by individual host factors. Such host factors can be genetic predispositions, age, gender and other underlying chronic diseases (e.g. latent virus infections). It is not known whether the dose of infection is of importance for hantavirus-infected patients, but the dose may for example influence the possibility of the virus to gain access to susceptible target cells in the lungs [222]. A higher age has been associated with an increased case fatality rate for HFRS [113, 284] and the same tendency can be noted for HCPS patients [33, 35]. There are gender differences in hantavirus-infected patients, with more males clinically diagnosed [33, 35, 41, 113, 284]. These host factors may influence the outcome of hantavirus-infected patients through influencing the immune response to infection [222].

2. AIMS

The aim of my work has been to increase the knowledge of the immune response and pathogenesis behind human PUUV-infection. To do so, I have investigated different aspects in innate and adaptive immunity in PUUV- infected patients.

The specific aims have been:

1. To investigate cytokine responses induced upon acute PUUV- infection (Paper I).

2. To study the NK cell response, especially the kinetics and phenotype of NK cells in infected patients (Paper II).

3. To longitudinally analyse the T cell response during acute infection, and to study balancing factors of an overactive response, such as Tregs and inhibitory immunoregulatory receptors (Paper III).

4. To study the viral load and humoral response in PUUV-infected patients and to analyze possible predictors of a more severe illness (Paper IV).

3. METHODS

In our studies we have used different immune-assays, based on antibody- antigen interactions, to study the immune response in PUUV-infected patients. The major assays that we have used are described below:

3.1 MULTIPLEX FLOURESCENT BEAD-BASED IMMUNOASSAY

We analysed different cytokines in plasma during acute and convalescent phase of PUUV-infection using a multiplex bead-assay analysis (paper I). Enzyme-linked immuno sorbent assay (ELISA) has for a long time been the golden standard for analysis of cytokines and other biomarkers [184]. Compared to ELISA, the multiplex bead-assay has the advantage of measuring multiple cytokines simultaneously in one single well and that it requires a smaller amount of plasma volume for analysis. The bead-assay method uses fluorescence as a reporter system instead of enzyme activity. A large set of different fluorescent coloured beads are in solution, each coloured bead linked to a certain cytokine-specific antibody. When the plasma is added to the beads, the cytokines specifically bind to cytokine- specific antibody-linked beads. Next, secondary cytokine-specific reporter antibodies, linked to fluorescent dye, are added. The beads then pass through a detection chamber where a red and green laser excites the coloured beads and the fluorescent reporter antibody. Hereby, the levels of different cytokines in the sample are calculated against standard dilutions of each respective cytokine. The correlation of bead assays with ELISAs for different cytokines are generally good, showing similar trends in cytokine levels, but notably, absolute levels can differ between the methods [285, 286]. In paper II, we used a sandwich ELISA to study different cytokines in patient´plasma.

3.2 IMMUNOFLOURESCENCE ANALYSIS

We used an earlier described IF method to determine IgM, IgA and IgG antibody titers in PUUV-infected patients (paper IV) [129, 287]. We added patient sera to spot-slides covered with fixed PUUV-infected Vero E6 cells (the local strain PUUV Umeå/hu) [288]. For IgM analysis, patient serum was pre-treated with Rf-absorbent to eliminate possible interference of rheumatoid factor. Slides were incubated overnight at 37°C. For IgA analysis, serum was incubated 90 min at 37°C and for IgG analysis, serum was incubated 60 min at 20°C. Presence of PUUV-specific antibodies was determined by using fluorescein-conjugated rabbit anti-human IgM, IgA or IgG. End titration was performed on all samples. In paper II, IF was used to determine the proportion of HTNV-infected HUVEC cells. Human convalescent sera bound to infected cells were visualized using a flourescein- conjugated goat anti-human IgG reporter antibody.

3.3 FLOW CYTOMETRY

Flow cytometry is an effective tool for quantitatively measure different cell phenotypes in peripheral blood. The method is based on laser and light detectors to count and characterize single intact cells in suspension. The cells pass through a laser one by one. The interruption of the laser beam is registered, and thereby cells are differed in size and granular content (forward scatter and side scatter respectively). Cell surface proteins are stained with fluorescently labeled antibodies that can be excited by lasers to emit light, which is subsequently registered by detectors. Using fluorescent antibodies, cells can be characterized as e.g. CD3+ T cells or CD3- CD16+CD56+ NK cells. The flow cytometer gives rise to quantitative data, creating scatter plots, where each dot represents a single cell. We have used a multicolor (9-color) flow cytometer with three different lasers. We stained peripheral blood mononuclear cells (PBMCs) using fluorescent antibodies. These fluorescent dyes were excited by the lasers and as much as 9 different colors were detected in one single sample. The set of flourochromes used were optimized to avoid leakage between detectors. We stained for different cell surface markers and also permeabilized the cells for staining of intracellular components. Apoptotic cells were marked and excluded to avoid non-specific fluorescent antibody binding to the cells. Cytokines were analysed in NK cells after the addition of Brefeldin A, a chemical that prevents protein secretion, followed by intracellular cytokine staining (paper II). Cell proliferation was detected by intracellular staining for Ki67+, a molecule expressed by cells during all active cell-cycle phases [289] (paper II and III). In addition, cell-division was analysed using cell trace violet. This dye covalently binds to intracellular amines and the color intensity is diluted during every cell division (paper II). Below is example of the different antibody-combinations used in paper III to study the T cell response.

Inhibitory Effector Effector CTLs Differentiation receptors molecules Tregs Fluorochrome #1 #2 #3 #4 #5 FITC Ki67 CD28 Ki67 Perforin Ki67 PE Bcl-2 Ki67 PD-1 Ki67 CD4 Qdot605 CD4 (b) CD4 (b) CD4 (b) CD4 (b) - PerCP HLA-DR CD8 HLA-DR HLA-DR CD8 PE-Cy7 CD8 CCR7 CD8 CD8 CD25 PacB CD16 CD45RA CD45RA CD45RA CD127 (b) CasY CD3 CD3 CD3 CD3 CD3 APC CD38 CD127 CTLA-4 Granzyme B FoxP3 APC-Cy7 CD14 +DCM CD14 +DCM CD14 +DCM CD14 +DCM CD14 + DCM

Table 2. Panels (# 1-5) were used to analyse the T cell response in PUUV-infected patients (paper III). The 9-different flourochromes are presented at the left. In each panel, extracellular or intracellular molecules were stained using different flourochrome-linked antibodies. DCM= dead cell marker. (b)= biotinelated antibody used, detected by adding streptavidin

4. RESULTS AND DISCUSSION

4.1 DIFFERENCES IN CYTOKINE PROFILE (PAPER I)

Cytokines are important mediators that for instance shape the immune response to an infection. In this first study the aim was to investigate cytokine levels in peripheral blood of PUUV-infected patients, more specifically, to compare the cytokine profile between females and males.

We studied 39 patients (20 females and 19 males) diagnosed with acute PUUV-infection. The concentration of 27-different cytokines was analysed using multiplex bead-assay analysis. Females had significantly higher levels of IL-1, IL-2, IL-15 and eotaxin when comparing acute and convalescent phase samples. Males had higher levels of IL-1, IL-9, IL-10, IP-10, eotaxin, GM-CSF and IFN-γ during acute phase than in their follow-up samples. The elevated levels of IFN-γ and IL-10 in males are supported by other studies [248, 265]. In contrast to other studies, [244, 247, 248] we found no significant increase of IL-6 or TNF-α, when comparing acute and convalescent phase. One drawback in our study was that we did not analyse healthy controls and therefore we could not compare the convalescent phase samples with normal range. This may affect the interpretation of the findings comparing acute and convalescent phase samples.

Instead, we focused the study on comparing the cytokine profile between males and females. The results showed that females had significantly higher plasma levels of IL-9, fibroblast growth factor-2 (FGF-2), and granulocyte macrophage colony stimulating-factor (GM-CSF). Females showed significantly lower levels of IL-8 and IP-10 than males. To investigate if the observed sex differences for FGF-2, GM-CSF, IL-8, IL-9, and IP-10 were explained by general differences between the sexes, the plasma levels of cytokines were compared also at convalescent phase. No sex differences were observed for any of the 27-investigated cytokines during the convalescent phase.

The chemokines IL-8 and IP-10 are implicated in the pathogenesis of severe acute respiratory distress syndrome (SARS) [290]. IP-10 is also induced upon SNV-infection of human lung microvascular endothelial cells in vitro [249]. We speculate that the higher levels of IL-8 and IP-10 detected in males could indicate a stronger inflammatory response in males versus females. We did not find a sex difference in cytokines inducing a TH1/cellular response nor a TH2 /humoral response, indicating that at least the TH responses do not differ between males and females during acute PUUV- infection.

Generally, males are more susceptible to infectious diseases and show a higher mortality when infected than females [291, 292]. Sex-based differences in immune responses can be important when studying disease

pathogenesis, development of treatments and also in general clinical practice [291]. Males are more often diagnosed with hantavirus-infection than females [33, 41, 113, 284, 293]. For PUUV-infection, the male-to-female ratio ranges from as high as 5:1 to 1.52:1 [41, 113], but the seroprevalence is not significantly different between sexes [112]. This implies that males are more likely than females to develop clinical HFRS. Although the incidence of diagnosed hantavirus-infection is higher amongst males, recent findings suggest that the case-fatality rate is higher amongst females, at least in patients over 50 years of age [35, 284]. Notably, differences between sexes in e.g. occupation and behaviour, leading to increased risk of hantavirus exposure, also play an important role in disease development. Our findings of sex differences in the cytokine profile of PUUV-infected patients suggest potential differences in the activation and function of the immune response between males and females. Findings that might explain parts of the sex differences noticed in hantavirus-disease susceptibility and disease progression.

4.2 NK CELL RESPONSE (PAPER II)

NK cells are important players of the innate immunity against viral infections. In this second paper the aim was to study the NK response in 16 patients during acute PUUV-infection. We studied kinetics and phenotype of the NK-cell response using flow cytometry analysis on patient blood samples. Peripheral blood mononuclear cells were analysed during the acute phase of disease, until 2-3 months later and more than one year after disease onset.

NK cells have been studied extensively in mice, and in humans most NK cell studies have focused on chronic infections, such as HCV and HIV [294, 295]. There are few studies on NK cell responses during acute viral infections. In influenza-infected patients, decreased levels of NK cells are found in peripheral blood, especially in patients with severe symptoms [296-298]. Reduced levels in peripheral blood may reflect homing to sites of infection, such as the respiratory tract [190]. In HFRS patients, it is shown that the proportion of NK cells is elevated in BAL-fluids during acute infection [235, 236]. Upon dengue virus-infection, increased levels of CD56+ NK cells are present in peripheral blood during the first week after disease onset. The levels are significantly higher in patients with mild versus severe illness, suggesting that NK cells are important for the viral clearance [238].

Our results showed that NK cells rapidly expand during the first week after disease onset. This increase in NK cell numbers persisted for a prolonged time, for more than 2 months post symptom debut (Figure 6). Analysis of specific NK cell subsets showed that the expansion consisted of CD56dim NK cells, which peaked after 10 days and remained elevated for at least 2 months. Half of the CD56dim NK cells expressed the proliferation marker Ki67 during the first week, which may contribute to the rapid cell expansion. It was also shown that Bcl-2 was up-regulated, indicating that early

apoptosis is inhibited in the proliferating CD56dim NK cells. The patients had elevated levels of IL-15 in plasma for up to 2 months after disease onset, which might, at least partly, explain the observed NK cell stimulation and proliferation. We also found that the CD56dimNK cells expressed CD94/NKG2C, an activating receptor that recognizes HLA-E. HLA-E was also shown to be expressed on hantavirus-infected endothelial cells. The NKG2C+ cells expanded and persisted for a prolonged time and did account for the significant part of the overall expansion of CD56dim NK cells. The recognition of HLA-E on target cells together with the presence of IL-15 was shown to stimulate NKG2C+ NK cell proliferation. Our findings show that there is a rapid expansion of NKG2C+ NK cells during PUUV-infection. These NKG2C+ NK cells could recognize HLA-E on target cells. This recognition together with IL-15 may contribute to the NK cell response seen in the patients.

More detailed analysis of the NKG2C+ cells in 5 patients, showed that these cells expressed predominantly one inhibitory KIR receptor, in contrast to NKG2C- NK cells, expressing several KIRs. The findings suggested some degree of clonal expansion of the NKG2C+ NK cells. The KIRs expressed on the NKG2C+ NK cells rendered them licensed to be responsive to stimulation, verified using KIR and HLA genotyping. In addition, the NK cells were shown to be responsive, as they rapidly secreted cytokines (IFN-γ, TNF) after stimulation in vitro.

NKG2C+ NK cells are known to expand in response to human CMV- infection: in CMV-seropositive patients, levels of NKG2C+ NK cells are higher than in CMV-seronegative individuals [299, 300]. In our study, we found that 3 out of 16 patients were CMV IgG negative. All CMV seropositive patients were shown to be CMV-DNA negative in plasma during acute phase of disease, hereby not showing signs of CMV-reactivation. In addition, there was no expansion of CMV-specific T cells during the course of infection. The CMV-negative patients had lower levels of NKG2C+ NK cells and no subsequent expansion occurred in these three patients. Our results imply that it is possible that previous CMV-infection can prime NK cells for expansion upon hantavirus-infection. A predominant proportion of the NKG2C+ cells expressed the maturation marker CD57. This CD57 marker has in CMV-infected patients suggested to be a memory NK cell marker [301]. In line with our results, expansion of NKG2C+ NK cells has been reported also in chikungunya virus-infected patients [302]. However, this NK cell expansion was transient and not prolonged as observed for PUUV- infected patients in our study.

Figure 6. The NK cell response in 16 PUUV-infected patients (Figure 2a from paper II)

In mice, long-term persistence of NK cells with memory like features have been described [303-306]. Our novel finding of a long lasting NK cell response in PUUV-infected patients supports that NK cells might possess traits of both the innate and adaptive immunity [307].

4.3 T CELL RESPONSE (PAPER III)

Previously, an overly vigorous CTL response has been implicated in the pathogenesis of human hantavirus-infection [223, 227]. An imbalance between an overactive CTL response and insufficient immunoregulatory mechanisms such as Tregs, has been suggested to contribute to immunopathology in patients [223]. However, there are contradictory reports on the role of T cells in human hantavirus-infection. A positive correlation is shown between high levels of virus specific CTLs and disease severity in HCPS patients [251], while for HTNV-infection a protective effect is reported [253]. The aim of this study was to analyse the primary T cell response, especially the CTL response in PUUV-infected patients. We also wanted to study balancing mechanisms of the immune response such as Tregs and inhibitory receptors on T cells during the course of infection.

The primary T cell response was longitudinally analysed in 15 PUUV-infected patients, from their first presentation at the hospital, during acute infection, until convalescent phase more than 2 months post symptom debut. Flow cytometry analysis was performed on peripheral blood T cells, staining for the expression of different cell surface- and intracellular markers. The virus- specific T cells were identified as being triple positive for Ki67+, HLA-DR+ and CD38+, according to Miller et al. in 2008 [308]. Our results showed that the triple positive CD8 T cells peaked during the first week after disease onset, in average representing 26% of total CD8 T cells. These responding effector CD8 T cells declined during the second week, in parallel with the decrease in viral load. To further ensure that the triple positive CD8 T cells represented effector cells we characterized the proliferating Ki67+

responding cells by different cell surface markers and for the presence of cytotoxic granule contents (granzyme B and perforin). We found that the responding cells were CCR7-CD45RA-CD127-CD28+/-, a phenotype different from Ki67- cells and from healthy controls. We also saw that >90% of responding cells contained perforin and granzyme B, showing that the triple positive cells were effector CTLs specifically responding to the infection.

To evaluate mechanisms that would potentially balance the vigorous CTL response, we studied Tregs and inhibitory receptors on T cells in PUUV- infected patients. Tregs were present in peripheral blood during acute infection, but no significant increase of the Treg population was observed. The inhibitory receptor CTLA-4 was found primarily on proliferating CD8 T cells during the first week after disease onset. More than 50% of proliferating CD4 T cells expressed both CTLA-4 and PD-1 inhibitory receptors during the first week. Both CD8 and CD4 T cells expressed inhibitory immunoregulatory receptors on their surface, which might downregulate their activity during acute infection.

We conclude that there is an extensive activation of CD8 T cells during acute PUUV-infection, representing up to as much as 50% of all CD8 T cells. Our study is limited in assessing the effector cells by activation markers in a non- antigen specific way. Some of the activated CD8 T cells might be unspecific bystanders (i.e non-hantavirus specific T cells), but the earlier study, using the same algorithm of markers, observed minimal bystander activation of T cells [308]. Reactivation of chronic latent infections could contribute to bystander activation, but in paper II, we saw no signs of CMV-reactivated T cells. Reactivation of EBV cannot be excluded, but EBV-specific CD8 T cells have been shown to increase late, 2-3 weeks after PUUV-disease onset [252]. In mice studies, virus-specific CD8 T cells have shown to represent up to 50- 80% of all CD8 T cells upon acute infection [309]. Hantavirus-infections induce a strong and long-lasting CD8 T cell response. High levels of PUUV- specific CD8 T cells is found up to 13 years after disease [92]. In addition, 15 years post SNV-infection, as much as 17% of all CD8 T cells were still specific for a single SNV-epitope [90]. One explanation to the high proportion of activated CD8 T cells that we found in our study could be the use of phenotypic markers. This method can detect a larger proportion of the activated virus-specific T cell response than tetramer staining and detection of IFN-γ cytokine production [310].

When analyzing for possible balancing mechanisms of the CTL response, we found that Tregs remained stable during the course of infection. In another study on HTNV-infected patients, the Tregs were reduced during acute infection [264]. In contrast to what is observed in these studies, Tregs are elevated in SNV- and SEOV-infected rodent reservoirs [261, 263]. In the rodent host, Tregs are believed to be important in controlling the immune response, hindering immunopathology and enabling viral persistence [261- 263].

We conclude that there is an extensive CTL response in PUUV-infected patients. Such an overactive CTL response may contribute to the clinical picture and disease pathogenesis in hantavirus-infected patients.

4.4 PREDICTORS OF DISEASE SEVERITY (PAPER IV)

In paper IV, we analysed viral load, humoral response and different clinical laboratory parameters in 105 patients with PUUV-infection. The aim was to analyse potential predictors for a more severe clinical outcome. We followed the patients under acute phase of disease until convalescent phase, around 1- 3 months after disease onset. All patients had signs of acute HFRS, and 15 of the 105 patients were classified as having moderate/severe illness [279]. The majority of all patients had thrombocytopenia and almost 1/3 bleeding manifestations. Symptoms from the lower respiratory tract were common, found in 26% of all patients. Kidney impairment (with creatinine levels more than four times normal) was found in 33% of the patients, but only one patient needed haemodialysis.

PUUV RNA was detected in the majority of patient plasma samples up to 9 days after disease onset. Previous studies have shown an association between viral load and more severe disease in SNV, HTNV and DOBV-infected patients [270-273]. In our study, the RNA level was not significantly associated with moderate/severe illness. One explanation might be possible differences in virulence between hantavirus species. Hantaviruses causing more severe disease than PUUV also tend to give rise to higher viral loads in patients than PUUV [265, 271, 272], a finding supported by our study. Notably, the first 0-3 days after disease onset, we found that PUUV-RNA levels were higher in patients with moderate/severe illness compared to in patients with mild illness. Also, when stratifying for time intervals in the statistical analysis, no significant association was observed, perhaps due to the small sample size (only 4 samples in the moderate/severe group at day 0- 3). The tendency of a higher viral load in moderate/severe ill patients during the first 3 days after disease onset support earlier studies that antivirals, such as Ribavirin can be a potential treatment alternative, at least early after disease onset in hantavirus-infected patients [142, 145].

When analysing the humoral response we found that 93% of the patients were IgM positive, 80% IgG positive and 86% IgA positive, in their first available serum sample. There was an inverse association between the presence of IgG and IgA titers and viral load. Using logistic regression analysis we observed that the IgG response was significantly associated with moderate/severe illness, while the IgM and IgA response were not. Our findings support other studies, showing that that a low IgG response is associated with a more severe disease outcome in SNV-infected patients [170, 171]. Together, these findings suggest that a strong IgG response might protect against severe disease. Promising clinical studies using passive immunization as a treatment alternative in hantavirus-infected patients, are referred to in Dolgin, 2012 [76].

In clinical settings, a low IgG titer could potentially be used as a predictor for suspected later severe clinical outcome in PUUV-infected patients. We also found that patients with moderate/severe illness had lower platelet counts, higher creatinine levels, higher white blood cell counts (WBC), and especially higher neutrophils than patients with mild illness.

A higher age was significantly associated with moderate/severe illness (p<0.05), supporting studies of increased case-fatality rate with age for hantavirus-infected patients [113, 284]. The gender of the patients was not associated with disease severity or hospitalization frequency. We found that respiratory symptoms were common in the PUUV-infected patients. These symptoms were usually mild, but show similarities to the respiratory symptoms observed in HCPS patients. A low IgG response was significantly associated with respiratory symptoms and need for oxygen treatment in patients.

We found a significant association between a low IgG response and a more severe clinical outcome in PUUV-infected patients. The viral load, per se, was not significantly associated with the outcome. We conclude that passive immunization might be a potential treatment for hantavirus-infected patients.

5. GENERAL CONCLUSIONS

My thesis provides knowledge regarding aspects of the innate and adaptive immune response in PUUV-infected patients.

In my studies I found that:

 The cytokine profile in PUUV-infected patients differed between males and females. These sex differences may affect the activation and function of the immune response to infection (Paper I).

 NKG2C+ CD56dim NK cells rapidly expanded after disease onset and remained elevated for at least 2 months after symptom debut. This novel finding of long-term persistent NK cells resembles adaptive immune response features (Paper II).

 The CTL response in PUUV-infected patients was vigorous and contracted in parallel with decrease in viral load. An overactive cell- mediated response may contribute to hantavirus pathogenesis (Paper III).

 Low IgG titers were associated with a more severe clinical outcome, while the viral load was not significantly associated with disease severity. These findings support the usage of passive immunization as a possible treatment alternative (Paper IV).

Virus Innate NK cells T cells

0 5 10 15 20 50 100 Days post infection

Data in this thesis provides new insights into the immune response of hantavirus-infected patients. Hopefully, these findings, together with future studies, will lead to a better understanding of disease pathogenesis and to possible treatment alternatives.

6. SAMMANFATTNING PÅ SVENSKA

Vad är hantavirus och hur smittar det människan?

Hantavirus är en grupp med virus som finns över stora delar av världen. Dessa virus sprids till människa från kroniskt infekterade gnagare, genom att viruspartiklar utsöndras i deras avföring, urin eller saliv. Den huvudsakliga smittvägen tros vara inandning av viruskontaminerat damm, som bildas från gnagarnas utsöndringar. Människor som bor eller vistas i områden där det finns infekterade gnagare, riskerar att drabbas.

Hantavirus kan orsaka två sjukdomstillstånd hos människa: Hemorrhagisk feber med njurengagemang (HFRS) samt Hantavirus sjukdom med hjärt- och lung-påverkan (HCPS). Hantavirus i Europa och Asien orsakar HFRS, medan hantavirus på den amerikanska kontinenten orsakar HCPS. Sjukdomens svårighetsgrad varierar mellan olika hantavirus, men kan också bero på olika individfaktorer hos de smittade. Dödligheten varierar från <1% till uppemot 15% för HFRS, medan HCPS är en mycket allvarlig sjukdom med en dödlighet på ca 35-40%.

I Sverige finns Puumala-virus (PUUV), ett hantavirus som orsakar en mildare form av HFRS (dödlighet på <1%). Denna blödarfeber med njurengagemang kallas sorkfeber eller nephropathia epidemica. PUUV sprids till människa via skogssork och är endemisk i sorkpopulationen i norra Sverige.

Sorkfeber - en blödarfeber

Sorkfeber, precis som andra former av HFRS kännetecknas av ett plötsligt insjuknade med influensa-liknande symptom. Hög feber, muskelvärk, huvudvärk, allmän sjukdomskänsla, buk- och ryggsmärta är vanligt första dagarna. Dimsyn är ett karakteristiskt tecken på sorkfeber och beror på en förtjockad lins i ögat. Blödningssymptom förekommer hos ca en tredjedel och kan vara blödningar i huden, blod i urinen, blödningar från mag- tarmkanalen och i allvarliga fall även hjärnblödning. Huvuddelen av patienterna får efter några dagar till en vecka påverkan på njurarna. Detta märks genom minskade urinmängder och ökad vätskeansamling i kroppen. I de flesta fall återhämtar sig njurarna efter några dagar, men i undantagsfall kan det krävas dialys under denna period. När njurarna sedan återhämtar sig så har patienterna ofta en övergående fas av kraftigt ökad urinproduktion, där flera liter urin kan bildas på ett dygn. Efter 1-3 veckor är patienten i återhämtningsfas/konvalescensfas, som ofta kännetecknas av en uttalad trötthet i flera veckor upp till månader. De allra flesta återhämtar sig helt, även om det finns studier som talar för en viss risk för senare högt blodtryck och kronisk njurskada.

Sorkfeber anses ge en livslång immunitet, eftersom det inte finns några rapporterade fall av återinsjuknande. Det finns idag ingen specifik behandling eller något vaccin mot sorkfeber.

Varför blir vi sjuka av hantavirus?

Inkubationstiden för hantavirus är lång, ofta 1-3 veckor. I samband med insjuknandet finns ett kraftigt immunsvar hos patienterna. Hantavirus orsakar en blödarfeber, och infekterar kärlväggens endotelceller. Infektionen i sig orsakar ingen uppenbar kärlskada, däremot ses ett ökat kärlläckage. Detta kärlläckage anses vara bidragande orsak till symptombilden hos människa. I dagsläget vet forskare inte riktigt hur detta kärlläckage sker, men det tros bero på flera faktorer. Viktiga sådana faktorer är virusets inverkan på de infekterade endotelcellerna, men även hur kroppens eget immunförsvar svarar på infektionen.

Syftet med min avhandling

I mitt avhandlingsarbete har jag studerat hur olika delar av kroppens immunförsvar reagerar vid sorkfeber. Syftet har varit att förstå mer om sjukdomsmekanismerna dvs. varför människor blir sjuka.

Under vintern 2006/2007 hade Sverige det största någonsin tidigare rapporterade utbrottet av sorkfeber, med över 2000 drabbade patienter. 90% av dessa patienter diagnosticerades i de fyra nordligaste länen och Västerbotten stod för över 800 fall. Vi startade vår studie under denna period och inkluderade patienter som sökte vård vid infektionskliniken på Norrlands Universitetssjukhus. Över 100 patienter blev det patientmaterial som är utgångspunkten för mitt doktorandprojekt. Vi följde patienterna från den dagen de sökte vård, under hela sjukdomsfasen fram till ca 1-3 månader efter sjukdomsdebuten.

Resultat och slutsatser av min forskning

I studie 1 undersökte vi olika cytokiner, små proteiner som används av immunförsvarets celler för att signalera till varandra. Dessa cytokiner formar hur immunförsvaret agerar genom att exempelvis aktivera olika immunceller. Vi undersökte en mängd (27-stycken) olika cytokiner i plasma hos 39 patienter. Syftet var att jämföra om det fanns någon skillnad i cytokinprofil mellan män och kvinnor. Vi fann en könsskillnad i vilka cytokiner som var stegrade i blodet vid sjukdomens akutfas. Däremot fanns inga skillnader mellan någon av de 27 undersökta cytokinerna vid konvalescensfasen. Denna könsskillnad i cytokin-koncentrationer, kan innebära att immunförsvaret formas olika mellan könen. Detta skulle kunna innebära könsskillnader i hur kroppen tar hand om infektionen.

I studie 2 undersökte vi såkallade naturliga mördarceller (NK-celler). Dessa celler är en typ av vita blodkroppar, lymfocyter, som har en viktig funktion i

det tidiga ospecifika immunsvaret mot virusinfektion. Vi fann att NK-celler var förhöjda i blodet hos de 16 undersökta sorkfeberpatienterna. Huvuddelen av dessa förhöjda NK-celler, bar en aktiveringsreceptor NKG2C på sin yta. Dessa NKG2C+ NK-celler ökade i antal under första veckan efter insjuknandet, och var som högst vid dag 10. Överraskande, fortsatte dessa NKG2C+ NK-celler att vara förhöjda i blodet även efter 2 månader, dvs. även vid konvalescensfasen av sjukdom. Normalt sett ska NK-celler, som del i det tidiga immunförsvaret, försvinna efter dagar men i detta fall liknar NK- cellsvaret mer ett specifikt immunförsvar.

Studie 3 innebar att undersöka T-celler, en viktig del av det specifika immunförsvaret vid virusinfektion. Cytotoxiska T-celler (CTLs) kan döda virus-infekterade celler och har ansetts viktiga mediatorer till kärlläckaget vid hantavirusinfektion. I denna studie undersökte vi T-cellssvaret i blodet hos 15 sorkfeberpatienter. Vi såg att CTLs var kraftigt förhöjda under den första veckan efter insjuknandet. Dessa CTLs minskade sedan under den nästkommande veckan, tillsammans med att virusnivåerna i blodet sjönk. Ett sådant kraftigt CTL-svar kan bidra till sjukdomsbilden vid hantavirusinfektion.

I den sista studien, studie 4, undersökte vi om antikroppsvaret och/eller virusmängden i blodet hos 105 patienter kunder prediktera svårighetsgraden av sorkfeber. Femton av våra 105 patienter klassificerades ha en medelsvår/svår sjukdom. Vi fann att låga IgG antikroppsnivåer var signifikant kopplat till en svårare sjukdom hos patienterna. Däremot fann vi att virusmängden inte påverkade utfallet. Slutsatsen var att tillförsel av antikroppar/passiv immunisering skulle kunna vara ett alternativ till behandling av hantaviruspatienter.

Sammanfattningsvis har jag studerat olika delar av immunförsvaret vid sorkfeber. Förhoppningsvis kan mina resultat öka förståelsen av vad som händer i kroppen vid hantavirusinfektion. Ökad förståelse om bakomliggande orsaker ökar chansen att utveckla specifik behandling i framtiden.

7. ACKNOWLEDGEMENTS

Firstly, I would like to thank all of you that have read this far……

There are lots of people that have contributed to this work and I would like to thank ALL of you and especially:

My supervisor Clas Ahlm: Thank you for your unfailing support in good times and in bad. Thank you for all your help during these years, and for a really nice collaboration. It´s great that you always find time for discussion, even if I know you have lots and lots to due. Without you, there wouldn´t be a thesis. Thanks again for being there.

My co-supervisor Jonas Klingström: thanks for being so supportive and full of new ideas. Thanks for always having time for questions, and for all your help during these years. It was great, spending time with you in Glasgow!

Niklas Björkström, thank you so much for all your help, and for taking care of me during my stay at CIM. During this time I really learned a lot from you! You are a really great person and scientist. Thank you for introducing me to the world of NK cells . Professor Hans-Gustaf Ljunggren, thank you for giving me the opportunity to do research at Center for Infectious Medicine, Karolinska Institutet, Huddinge, Stockholm.

I would like to thank members of our Bunyaviridae group:

Lisa- it has been great working with you, in the lab, with data analysis and during writing. Think about all those hours that we have spent on this last project. We finally made it, 4 kids later! Johan- thanks for being such a nice colleague in science and in the clinics. You have taught me a lot in both fields! By the way, your computer skills are invaluable and hopefully you were rewarded in Beijing. Ann-Marie, thanks for always being so helpful, positive and nice. Look forward continuing to work with you, both with science and maybe as clinical colleagues? Nahla, thanks for all your laboratory help and nice discussions. May Bylund, and Irene Eriksson, thanks for all your technical assistance. Magnus Evander, thanks for all the help and supervision during these years. I would also like to thank Karin and Shawon, working at Karolinska institutet. It has been great getting to know you! Greg Rankin, I really appreciate your help in the last minute!

Thanks to all of you, working with me at FOI! Fredrik Elgh, thanks for letting me do my exam work in your lab in 2000/2001. Göran Bucht and Patrik Johansson, thanks for introducing me to hantavirus-research. I am really proud of that study we made on linear-DNA as an alternative strategy for DNA vaccination!

Gunborg! You always have solutions to technical and practical issues. Thank you for always being so helpful and nice!

I would also like to thank the Infectious clinic, at Umeå University hospital, and my boss Carolina Lundberg, for giving me the time and opportunity to do research, even though this prolongs my residency. After the dissertation, I´ll be back! I would also really like to thank all my colleagues at the clinic, for being so supportive. I am so glad that I work in such a great place, with such talented and nice people. Stephan Stenmark, Johan Wiström, Lena Skedebrant and Denise Jarvis: thanks for letting me work with you, it is always a pleasure! Jens Boman, thank you for introducing me to “Smittskyddet”.

I would also like to thank my supporters outside of work....

All the members of the girl mafia: Sofie, Hanna, Ida, Sara, Sara and Åsa. Thanks for being such great friends all these years, and I look forward to our next girl weekend! Sofie, an extra super thanks for your support and help .

Eda och Oskar! Thanks for being such great people and neighbours, and Eda, now when your back home, let´s go to IKSU! All other great neighbours and “förskole” friends at Sofiehem, it is always great seeing you all.

A big hug goes to Johan´s family, now being my family too. Kristina, Åsa and Anna, thanks for a great time in Paris! Hope we can do another trip some day. It is really great that we have such tight family bonds, especially for the kids. A special thanks to Kristina and Arne for all your help in our daily family-life puzzle.

Thanks to mamma and pappa and my little sister Camilla. Mum and dad, you have always supported me and helped me out. I know I can always count on you and I love you! Mum, sorry for being so distracted lately. My wonderful Sis, you are the best! David, our “bus-David”-thanks for all the good times and please marry my sister soon. Thanks for making me an ant and good-mother to Viktor, and I hope for more kids . Let us meet in “Rag” during lasy summer days! Who will get the biggest fish?

Last and mostly, I would like to thank my own family. My wonderful two children, Klara and Sofia! Mummy loves you, and you are the best that has ever happened to me! Johan, my husband: You are the love of my life! Thanks for all your support and for taking care of me and the kids. Yoy make me happy, and I am looking forward spending the rest of my life with you.

Kram Therese

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60 Department of Clinical Microbiology, Infectious Diseases ISBN (printed): 978-91-7459-681-6 Umeå University, SE-901 87 Umeå, Sweden ISBN (digital): 978-91-7459-682-3 www.climi.umu.se ISSN 0346-6612 Serial No: 1577