The second prospective study was a 2-year (1980–1981) schoolbased study involving 1,757 children, ages 4–16 years, in Bangkok, Thailand (3). This was the first study to use active surveillance using school absence as an indicator of potential illness. Antibody titer revealed that 50% of the enrolled students had evidence of dengue antibody, likely indicative of a DENV infection experienced prior to the age of 7 years, and the first to demonstrate that even at this young age the population is already highly flavivirus antibody experienced. Most (87%) of the students who became infected during the study period were asymptomatic as determined by lack of clinical illness. Important study findings were an incidence in dengue-naïve participants of 6.3%, in dengue-experienced participants an incidence of 5.5% and among symptomatic infected children a hospitalization rate of 53%. The symptomatic-to-asymptomatic ratio of DENV infection was 1:8. This study confirmed the importance of subsequent DENV infection as a risk factor for severe infection with an odds ratio for developing DHF in participants with pre-existing dengue immunity ≥6.5. The importance of this prospective study was as the first study to determine the full burden of DENV infection within a cohort, the incidence of infection and the relationship of pre-existing DENV immunity to dengue disease severity.

In the last decade a number of prospective cohort studies on DENV transmission have been performed adding to our knowledge on DENV incidence and risk factors for severe infection. These include an ongoing study in Iquitos, Peru (the first in the Americas), one in Indonesia (the first in adults), an ongoing study in Nicaragua, studies inVenezuela,Vietnam,Malaysia, Philippines, Vietnam, and ongoing studies in Kamphaeng Phet, Thailand.

## **INCIDENCE RATES WILL SUPPORT PHASE III EFFICACY STUDIES THOUGH A HIGH DEGREE OF TEMPORAL AND SPATIAL DIVERSITY IN INCIDENCE OCCURS REQUIRING CONSIDERATION IN CHOOSING POPULATIONS**

The prospective studies from both the America and Asia have demonstrated a high burden of DENV infection and incidence rates that clearly support economically feasible DENV vaccine efficacy studies. With an average incidence of symptomatic DENV infection of 2%, a sample size calculation using an 80% efficacy of the vaccine, an alpha value of 0.05, power of 0.8, will require a total sample size of vaccinated and controls of 1,452 volunteers. Studies in Iquitos, Peru and in Kamphaeng Phet, Thailand (referenced in **Table 1**) demonstrated the heterogeneity of DENV incidence and serotype-specific transmission spatially and temporally and illustrated elegantly in schools in Kamphaeng Phet, Thailand. In Kamphaeng Phet, dengue incidence and serotypespecific transmission was cyclical in each school, with relatively mild years followed by more severe years. A proportion of schools had a severe dengue year while other schools a short distance away had less severe dengue. Similarly, one school would have a DENV-2 outbreak and another a short distance away a DENV-3 outbreak. The diversity of incidence both spatially and temporally is an important observation from these studies in designing dengue vaccine efficacy studies and in estimating and choosing the population and geographic location required to assess statistical efficacy.

## **PRE-EXISTING SEROTYPE-SPECIFIC NEUTRALIZING ANTIBODY DOES NOT PROTECT AGAINST INFECTION SUGGESTS THAT OUR CURRENT ASSAYS TO MEASURE NEUTRALIZING ANTIBODY IS NOT A CORRELATE FOR PROTECTION**

Historically and currently, DENV neutralizing antibody has been a serologic biomarker to measure previous and current DENV serotype-specific infection (1, 17, 18). The role of homotypic and heterotypic antibody as a marker of immunity was first described in studies performed by Sabin in the 1940s in human volunteers (19, 20). The concept as demonstrated was that following a serotype-specific DENV infection, homotypic antibody develops to that specific serotype that last life-long and becomes a biomarker that indicates previous infection to that serotype and thus durable immunity. Heterotypic, cross-reactive antibody to other DENV serotypes following a serotype-specific infection also develops, is transient, and may modify disease severity when infected with another serotype. The development of the plaque reduction neutralization titer (PRNT) assay and its variation for DENV became a relatively high-throughput assay to measure this biomarker for previous and current serotype-specific protection (17). As such it has evolved to be an important biomarker to measure DENV vaccine performance in pre-clinical and clinical DENV vaccine trials. We have demonstrated that this assay has a high degree of variability in its performance depending on the cell lines and prototype viruses used and the detection of heterotypic neutralizing antibody creates confusion in its interpretation of whether an individual is protected or not from a specific serotype (21). In the Kamphaeng Phet cohort studies, the active surveillance for infection and analysis of archived sera from infected individuals allowed the examination of serotype-specific PRNT antibody just prior to severe DENV infection (22). What was demonstrated was the observation that individuals hospitalized for severe serotypespecific DENV had detectable PRNT titers to that serotype using either prototype DENV and to their own isolated virus approximately 6 months prior to infection. The implication of this finding was that the PRNT was not an adequate biomarker for serotypespecific protection. Ten years later this finding was reiterated during the vaccine efficacy studies of the ChimeriVax vaccine (1). Despite high levels of DENV-2 neutralizing antibody as detected by PRNT, only 9.2% were protected. These findings highlight the need for the development of biomarkers that are highly predictive of serotype-specific DENV protection and currently a major obstacle in the development of DENV vaccines.

### **SYMPTOMATIC TO INAPPARENT RATIO IS AN EPIDEMIOLOGIC CORRELATE FOR HETEROLOGOUS PROTECTIVE ANTIBODY AND MAY BE A CORRELATE FOR VACCINE EFFICACY**

The original Bangkok prospective study demonstrated the full burden of DENV infection, documented the occurrence of subclinical infection and introduced the concept of the S:I ratio (3). In the Kamphaeng Pet cohort studies, we have demonstrated that the S:I ratio is not a fixed variable but there is a variance in the ratio that varies spatially and temporally (6). This ratio was fluid with schools experiencing more symptomatic disease, high S:I ratio, than other schools which shifted the following year where schools had more subclinical infections, lower S:I ratios, while other schools experienced high S:I ratios. This variation in the S:I ratio when analyzed closely is not a random event but occurs in a cyclic pattern with one school having a high S:I ratio, more symptomatic infection, followed in the same school by years of lower S:I ratios, less symptomatic infection. This pattern was reflected at the individual school level and also as a population as a whole. An explanation of this variance is that the S:I ratio is an epidemiologic correlate for an undercurrent of heterotypic protective immunity that may not prevent infection but may modify disease severity. At an individual level what this implies is that following a serotype-specific infection there is the generation of heterotypic antibody that modulates severity of infection that fades with time. At the same time there is a heterotypic enhancing antibody that can influence the production of more severe dengue infection. Expanding from an individual level, this effect applies to local populations such as schools, villages and to the population as a whole and will be discussed in more detail in the next section. The data suggest that the S:I ratio is an epidemiologic correlate for this protective or disease enhancing heterotypic antibody and reflects the time-dependant flavivirus experience of a population. For DENV vaccine studies, the S:I ratio may be a very important correlate of the vaccine's ability to confer heterotypic protection or enhancement of disease severity and potentially an important efficacy correlate to follow over time in a population.

## **TIME FROM LAST INFECTION DETERMINES THE S:I RATIO AND PROVIDES EVIDENCE OF A HALF-LIFE OF HETEROLOGOUS PROTECTIVE IMMUNITY WHICH MAY AFFECT THE OBSERVED EFFICACY IN DENV VACCINES DEPENDING ON THE TIME POINT EFFICACY IS MEASURED**

As discussed, the S:I ratio in a population over time is an epidemiologic correlate that reflects an undercurrent of both heterotypic protective and enhancing immunity that is time-dependant. We and others have demonstrated that the time from last infection determines the S:I ratio and that there is a measurable half-life of heterologous protective immunity (23, 24). This was elegantly demonstrated in the Kamphaeng Phet cohort studies and recently published (23). Data from the prospective cohort studies were analyzed for subclinical and symptomatic DENV infections in schoolchildren from 1998 to 2007. Children who experienced one or more DENV infection were selected as the population for analysis (2,169 person-years of follow-up). Demonstrated from the analysis was a shorter time interval between infections was associated with subclinical infection in children who were seronegative for DENV at enrollment; seronegative being an important preexisting condition to document primary to secondary infections as compared to the more complicated second to third infections. This time interval was an average of 1.6 years from first infection that resulted in a subclinical infection, 1.9 years for symptomatic denguefever (DF), and 2.6 yearsfor the onset of severe hospitalized dengue hemorrhagic fever (DHF). These findings indicate that there is a window of cross-protection following DENV infection that last approximately 1.6 years following infection. The implications of these findings for DENV vaccine efficacy studies are subtle but important. The goal for a tetravalent DENV vaccine is to confer durable tetravalent DENV immunity and protection against severe illness. If one serotype in the DENV tetravalent formulation in particular induces a brisk immune response, the results from the prospective cohort studies would suggest that there will be heterotypic protective immunity from that one serotype to other serotypes conferring tetravalent protection that lasts for 1.6 years. If true, then measuring efficacy at 1 year following vaccination as the most recent DENV vaccine trial did, would not be a true measurement of tetravalent protection. A more appropriate efficacy time point would be at least 3 years following vaccination as a true measurement of efficacy. This of course would mean a longer duration of observation of the population following vaccination and cost of the study.

## **THERE IS A HIGH DEGREE OF TEMPORAL AND SPATIAL GENETIC DIVERSITY IN THE DENVs REINFORCING THE NEED FOR A TETRAVALENT DENV VACCINE BUT ALSO CONSIDERATION FOR THE NEED OF GENOTYPIC-SPECIFIC PROTECTION AS WELL**

It is thought that the DENV transmission evolved into four distinct serotypes approximately 1,000 years ago and each of these four serotypes emerged into a cycle of transmission between humans and its mosquito vector approximately 125–320 years ago (25, 26). Phylogenetic analysis suggests that the DENVs are rapidly evolving with major clade replacements and genetic shifts occurring in populations endemic for DENV (25–28). Asia has been pivotal in the evolution of DENV as the location of the first cases of the more severe form of DENV infection, DHF, which made its first appearance in the 1950s first in the Philippines then in Thailand (29). The current Asian genotypes of each serotype are considered more severe, result in more severe dengue illness, than the American genotypes (30). Evidence suggests that DENV circulation in Asia due to its population growth and urbanization, high vector burden, and high level of pre-existing flavivirus seroprevalence, contribute to the increase in genetic diversity which is estimated as increasing at a factor between 14 and 20 in the last 30 years (31). The overall impression of DENV evolution in Asia is the active transmission of viruses in individuals who are highly flavivirus antibody experienced causing evolutionary pressure on the virus to evolve to escape and utilize pre-existing flavivirus immunity. By its nature the current evolving DENVs are adept at escaping heterologous neutralizing antibody and using it as a means to attain

high viral load levels and more severe disease through antibody enhancement.

To understand the evolution and phylogeography of DENV, the prospective cohort studies in Kamphaeng Phet have been isolating and sequencing DENVs from individuals in specific geographic areas and over time. In ongoing studies, viruses are isolated and through the use of global positioning system (GPS) the exact geographic position located allowing specific spatial and temporal analysis. Analysis of isolated viruses demonstrated diverse viral genetic variation in both time and space in the Kamphaeng Phet population with multiple viral lineages circulating within individual schools and villages (32). This implies that there is frequent gene flow of DENV into this microenvironment as individuals move from major urban areas into smaller village. At the microenvironment level, there was clustering of specific viral genotypes within individual schools that are highly conserved from year to year and evidence of frequent viral gene flow among schools closely related in space and time. These results suggest that there is a combination of frequent viral migration into Kamphaeng Phet coupled with population subdivisions of conserved viruses that shape the genetic diversity of DENV at a local and population scale. Taken together is a picture of a virus that is evolving rapidly, spread by population movement, and due to the restricted flight of the vector, genetically conserved at a microenvironment level.

For a DENV vaccine these findings have many implications with the most important is that the current vaccines using historical prototype viruses collected in the 1980s may no longer provide the protection to current circulating DENV genotypes and raises the question if protection needs to be both serotype and genotype specific. This question was raised during the ChimeriVax vaccine trial where the DENV-2 vaccine strain did not match the circulating wild-type DENV-2 and failed to produce protection (1).

### **CHANGING POPULATION DYNAMICS AND VIRAL EVOLUTION WITH OLDER INDIVIDUALS BECOMING INFECTED SUGGEST THAT IMMUNOSENESCENCE AND POTENTIAL VIRAL ESCAPE MUTANTS SHOULD BE CONSIDERED WHEN FORMULATING DENV VACCINES**

Dengue illness in endemic countries is considered to be primarily a pediatric disease due to the high degree of transmission and infection that occurs at a young age. Adults are considered highly flavivirus experienced and thus protected from subsequent dengue illness. For this reason, the prospective cohort studies have largely been performed in pediatric populations and illness criteria of severity of disease developed in pediatric populations. In our own cohort studies, we have noted that the age of symptomatic DENV infection has been increasing slowly over the last decade by a median increase of 2–4 years. This has been reflected in Thailand as a whole and interestingly in the rest of Southeast Asia (33). An analysis and mathematical modeling of this phenomenon was performed on data from each of the 72 provinces of Thailand (33). What was described was that the force of infection has declined by 2% each year with the strongest predictor of this change the median age of the population, which was reflective of the changing demographics of the population. The authors' conclusion was that lower birth and death rates decrease the flow of susceptible individuals into the population and thereby increase the longevity of immune individuals. For a DENV vaccine, the implications are despite the decrease in the force of infection the critical vaccination fraction has not changed declining from 85 to 80% (2). A key point in this argument however is the longevity of immune individuals and if protection is life-long. Recently, in our own studies in Kamphaeng Phet we have been documenting a large number of adults who are older than 50 years of age with one 80-year old hospitalized with severe DENV infection. These are individuals who have never moved from Kamphaeng Phet, a highly endemic region for DENV, and defy statistical probability of never having been infected by one or more DENVs in their previous experience. This is under active investigation but does suggest that the aging immune system,immunosenescence,might be a factor in older individuals becoming ill from DENV infection or, and particularly worrisome, that the current strains/genotypes have evolved as escape mutants and individuals are no longer protected from current wild-type DENV strains/genotypes despite infection from another genotype but same serotype in the past. This as noted in the previous section has implications on the current DENV vaccine candidates and whether their vaccine prototype strains/genotypes are applicable to current circulating genotypes.

#### **SUMMARY**

The prospective studies have been invaluable in increasing our understanding on the epidemiology and pathogenesis of this globally important virus and disease. Discussed are important lessons learned from these studies that have direct application to current DENV vaccine development and testing as they move forward in their regulatory development to a licensed vaccine. Identified are critical issues that are challenges for the development of an effective tetravalent DENV vaccine. Additional research is needed to address these areas that are critical to vaccine testing and evaluation. Expanding cohort studies to include those countries where dengue is inadequately characterized is critical for us to further understand the unique viral and host factors that contribute to differences in dengue risk and vaccine response.

#### **ACKNOWLEDGMENTS**

I would like to acknowledge the contributions of many individuals who have been involved in the Kamphaeng Phet, Thailand studies from the University of Massachusetts School of Medicine, the University of Rhode Island, the Walter Reed Army Institute of Research, and the Armed Forces Research Institute of Medical Science who have contributed significantly to their success. These include in alphabetical order: Kathryn Anderson M.D., Buddhari Darunee M.D., Robert Gibbons M.D., Sharone Green M.D., Richard G. Jarman Ph.D., Daniel Libraty M.D., Ananda Nisalak M.D., Alan Rothman M.D., Anon Srikiatkhachorn M.D., Stephen Thomas M.D., and In-Kyu Yoon M.D. I would also like to thank the staff at the Department of Virology, Armed Forces Research Institute of Medical Science (Bangkok, Thailand), the support of the Office of the Provincial Public Health, Kamphaeng Phet Province, and the clinical research nurses at AFRIMS and the support staff at the Kamphaeng Phet Field Station for all their efforts. Though no funding was received for this manuscript, I would like to acknowledge that these studies in Kamphaeng Phet were made possible by grants from the National Institutes of Health (NIH) grant P01

AI034533, R01 GM083224-01 and the Military Infectious Diseases Research Program (MIDRP) and the United States Army Medical Research and Materiel Command, Ft Detrick, MD, USA.

#### **REFERENCES**

**Conflict of Interest Statement:** The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Received: 10 February 2014; accepted: 07 April 2014; published online: 24 April 2014. Citation: Endy TP (2014) Human immune responses to dengue virus infection: lessons learned from prospective cohort studies. Front. Immunol. 5:183. doi: 10.3389/fimmu.2014.00183*

*This article was submitted to Microbial Immunology, a section of the journal Frontiers in Immunology.*

*Copyright © 2014 Endy. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

REVIEW ARTICLE published: 07 March 2014 doi: 10.3389/fimmu.2014.00093

## T-cell immunity to infection with dengue virus in humans

## **DanielaWeiskopf \* and Alessandro Sette**

Division of Vaccine Discovery, La Jolla Institute for Allergy and Immunology, La Jolla, CA, USA

#### **Edited by:**

Scott B. Halstead, International Vaccine Institute, South Korea

#### **Reviewed by:**

Scott B. Halstead, International Vaccine Institute, South Korea William Messer, Oregon Health and Sciences University, USA

#### **\*Correspondence:**

Daniela Weiskopf , Division of Vaccine Discovery, La Jolla Institute for Allergy and Immunology, 9420 Athena Circle, La Jolla, CA 92037, USA e-mail<EMAIL_ADDRESS>
Dengue virus (DENV) is the etiologic agent of dengue fever, the most significant mosquitoborne viral disease in humans. Up to 400 million DENV infections occur every year, and severity can range from asymptomatic to an acute self-limiting febrile illness. In a small proportion of patients, the disease can exacerbate and progress to dengue hemorrhagic fever and/or dengue shock syndrome, characterized by severe vascular leakage, thrombocytopenia, and hemorrhagic manifestations. A unique challenge in vaccine development against DENV is the high degree of sequence variation, characteristically associated with RNA viruses. This is of particular relevance in the case of DENV since infection with one DENV serotype (primary infection) presumably affords life-long serotype-specific immunity but only partial and temporary immunity to other serotypes in secondary infection settings. The role of T cells in DENV infection and subsequent disease manifestations is not fully understood. According to the original antigenic sin theory, skewing of T-cell responses induced by primary infection with one serotype causes less effective response upon secondary infection with a different serotype, predisposing to severe disease. Our recent study has suggested an HLA-linked protective role for T cells. Herein, we will discuss the role of T cells in protection and pathogenesis from severe disease as well as the implications for vaccine design.

**Keywords: DENV,T cells, protection, pathogenesis, HLA, vaccines**

## **WORLD WIDE MEDICAL AND SOCIETAL SIGNIFICANCE OF DENV AND DENV INFECTION**

Dengue virus (DENV) is the causative agent of dengue fever, the most prevalent mosquito-borne viral illness in humans and is primarily transmitted by the mosquitoes *Aedes aegypti* and *Aedes albopictus* (1). The world wide distribution of these two major vectors puts nearly a third of the global human population at risk of infection (2). It was recently reported that as many as 390 million dengue infections occur worldwide each year, thus making this infection potentially even more prevalent than malaria (3). Recent outbreaks in Europe (4) and the US (5, 6), led to the recognition of DENV as a Category A priority pathogen by NIAID and the classification of DENV as a domestic re-emerging disease threat by the CDC.

Disease can be induced by any of the four serotypes of DENV (DENV1–4). DENV-associated disease in most cases ranges from asymptomatic to an acute self-limiting febrile illness. However, in a small proportion of patients, the disease can exacerbate and progress to severe forms of dengue disease [dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS)], characterized by severe vascular leakage, thrombocytopenia, and hemorrhagic manifestations (7). Infection with one DENV serotype presumably results into life-long immunity to the infecting serotype but does only confer short-term protection against the other serotypes (8). In fact, the severe forms of DENV disease are more often observed in individuals experiencing a secondary infection with a different serotype (9, 10). Besides prior infection with one serotype, other factors influencing the disease outcome are the strain of infecting virus, age and gender, nutritional status, and the genetic background of the patient (11–16). No licensed vaccine or

effective anti-viral therapy is currently available and treatment is largely supportive in nature, thus increasing the economic and disease burden on public health systems in tropical and subtropical countries around the globe (17–19).

#### **DENV-SPECIFIC T-CELL RESPONSES T-CELL EPITOPE IDENTIFICATION**

A previous analysis (20) documented the substantial knowledge gaps existing at the level of defined T- and B-cell immune responses. Over the last years, the situation has improved considerably. As of June 2013, the immune epitope database (IEDB)<sup>1</sup> lists 369 antigenic regions identified in humans and 71 identified in HLA transgenic mice. It has been shown that CD8<sup>+</sup> T cells most vigorously and frequently recognized the NS3, NS4B, and NS5 proteins, whereas the capsid, envelope, and NS3 proteins are the dominant targets for CD4<sup>+</sup> T cells (21–25). In parallel, but beyond the scope of this review, significant strides have been made in the definition of DENV-related B cell epitopes.

Despite these efforts, significant gaps yet remain. First, the vast majority of DENV epitopes described in the literature and reported in the IEDB are restricted by HLA MHC class I alleles, and only 13% of the epitopes are restricted by HLA class II and recognized by CD4<sup>+</sup> T cells. Of those epitopes, only a few have defined allele and loci restrictions and characterized functional and phenotypic features of the associated T cells. Thus, a comprehensive analysis of MHC class II restricted responses across all loci is needed in the general population from endemic areas and

<sup>1</sup>www.iedb.org

in patient populations associated with different severity of disease (acute DF versus DHF/DSS). Furthermore, the epitopes recognized after vaccination with experimental vaccines have not been systematically identified or validated. This hampers progress in the field, as the role of MHC class I and class II restricted responses in disease protection and immunopathology cannot be broadly evaluated, and the performance of different vaccines in terms of induction of immune responses in human vaccines remains undetermined.

#### **IMPLICATIONS FOR HLA POLYMORPHISM**

T cells recognize a complex of a particular pathogen-derived epitope presented by a specific MHC molecule. Thus, a given epitope will elicit a response in individuals that express MHC molecules capable of binding that particular epitope. MHC molecules are extremely polymorphic, with several thousand variants known in humans (26, 27). Each variant is present with variable frequency, depending on ethnic lineage and geographic locality. As a result, for basic investigations, diagnostic or vaccine applications and to ensure high and non-ethnically biased coverage of different patient populations, it is necessary that the alleles investigated are carefully selected. This is accomplished by selecting those alleles that are most frequent in the various population groups worldwide.

To address this challenge in the context of HLA class I, we have applied a selection process focused on the 27 most common HLA A and B alleles in the general population (25). As previously described, these 27 alleles allowed us to cover at least three out of four possible HLA A and B alleles expressed per donor in 90% of a cohort from the general population of Colombo, Sri Lanka. In the case of HLA class II, we have recently reported the selection of a panel of 27 different allelic variants that affords high coverage of all four HLA class II loci (DRB1, DRB3/4/5, DQ, and DP) (27). Based on publically available population frequency data (28), this panel of HLA DR, DQ, and DP specificities should allow to cover over 98% of individuals in the general population. Notably, the actual coverage achieved by this panel was similarly high in cohorts of distinctly different ethnic composition that we have previously utilized for our studies (29, 30). For each of these class II molecules, we have established quantitative binding assays (27) and generated a sufficiently large number of measurements to enable derivation of quantitative algorithms for predicting binding capacity (27, 31). Predictive algorithms for the most common HLA class I and class II alleles are now publicly available at several web sites, including the IEDB<sup>2</sup> . Additionally, for each molecule we have produced cell lines transfected with a single HLA class II allele that will be useful for fine mapping of HLA restrictions (30). These approaches now represent efficient and valuable tools for epitope identification, especially in the context of large and complex targets.

#### **THE HYPOTHESIS OF ORIGINAL ANTIGENIC SIN AS IT IS RELATED TO A POTENTIAL ROLE OF T CELLS IN DENV PATHOGENESIS**

It has been proposed that cross-reactive T cells raised against the original infecting serotype dominate during a secondary heterologous infection, a phenomenon that has been termed "original antigenic sin" (32, 33). This term was first applied to the humoral response to influenza epidemics (34), but has also been observed in CD8<sup>+</sup> T-cell responses against lymphocytic choriomeningitis virus (LCMV) (35). This hypothesis postulates that during secondary infection, expansion of pre-existing lower avidity crossreactive memory T cells dominate the responses over that of naïve T cells that are of higher avidity for the new DENV serotype. It is further hypothesized that peptide variants derived from the secondary infection serotype can induce a response that is qualitatively different from the response induced by the original antigen, such as inducing a different pattern of cytokine production. Variants associated with this phenotype are often collectively referred to as altered peptide ligands (APLs) (36). It is hypothesized that these altered T-cell responses serotype may contribute to a "cytokine storm" during heterologous secondary infection and thus contribute to immunopathogenesis of severe dengue disease (33). However, this hypothesis is in conflict with the observation that heterologous T-cell responses are not always needed to produce severe disease in infants. DHF or DSS in infants generally occurs between the ages of 6 and 12 months in endemic areas (37). When the maternal antibody titer to DENV decreases below a protective level, infants are actually at an increased risk for the development of severe disease despite the fact that they have never been infected with DENV and lack DENV-specific T-cell memory (38). Furthermore, a recent study has shown a temporal mismatch between the CD8<sup>+</sup> T-cell response and commencement of capillary leakage, suggesting that CD8<sup>+</sup> T cells are not responsible for early triggering of capillary leakage in children with DHF (39).

We have previously reported that "original antigenic sin" is indeed detectable at the level of CD8<sup>+</sup> T-cell responses in the general population (25). However, a potential limitation of those studies was that they were conducted at the level of the general population from an endemic area (i.e., Sri Lanka), and did not measure HLA class II restricted epitopes. Furthermore, it is not known whether the studies could capture *in vivo* impaired or altered T-cell responses during acute infection.

#### **LOW MAGNITUDE T-CELL RESPONSES ARE HLA-LINKED AND ASSOCIATED WITH DISEASE SUSCEPTIBILITY**

The results presented above suggest that antigenic sin does not significantly impair the quality of T-cell responses in the general population. However, lower quality responses might be associated with the relatively few individuals experiencing more severe clinical outcomes. Previous studies highlight that certain HLA alleles are associated with either increased or decreased risk of clinical manifestations (14, 40–45). However, these studies did not determine whether increased risk might be associated with a hyperactive T-cell response, or conversely whether a higher T-cell response might be associated with a decreased risk. Correlations of HLAassociated disease susceptibility with T-cell responses found that weak T-cell responses correlated with disease susceptibility (25). A possible explanation for these observations would be that certain alleles and epitopes are associated with higher magnitude responses, which are in turn associated with higher degrees of multi-functionality, and thus might be most beneficial in protecting from disease. A detailed analysis of cytokines produced by DENV-specific T cells revealed that stronger responses are indeed

<sup>2</sup>www.immuneepitope.org

associated with multifunctional T-cell responses. Thus, it might be possible that while T cells have a protective role in general in the HLA-linked, lack of a multifunctional T-cell response might contribute to pathogenesis in certain individuals.

#### **ROLE OF T CELLS IN PROTECTION AGAINST DENV INFECTION**

The protective role of T cells during viral infection is well established (46). Generally, CD8<sup>+</sup> T cells can control viral infection through several mechanisms, including direct cytotoxicity and production of pro-inflammatory cytokines such as IFN-γ and TNFα. Similarly, CD4<sup>+</sup> T cells are thought to control viral infection through multiple mechanisms, including enhancement of B and CD8<sup>+</sup> T-cell responses, production of inflammatory and antiviral cytokines, cytotoxicity of viral infected cells, and promotion of memory responses (47).

Several lines of evidence suggest that both CD4<sup>+</sup> and CD8<sup>+</sup> T cells may contribute to protection against homologous reinfection or heterologous dengue infection. It has been shown that DENV-specific human CD4<sup>+</sup> T and CD8<sup>+</sup> T cells proliferate, produce IFN-γ, and lyse infected target cells (48–50), suggesting that serotype-specific T cells are activated and functional in humans with primary DENV infection (48, 51). Furthermore, higher frequencies of DENV-specific IFNγ-producing T cells are present in children who subsequently develop subclinical infection, compared with those who develop symptomatic secondary DENV infection (52).

Finally, studies in a murine model of DENV infection demonstrated that both CD4<sup>+</sup> and CD8<sup>+</sup> T cells contribute to protection against DENV challenge (53–56). In parallel to the evidence in the murine model, studies performed previously demonstrated that HLA alleles associated with increased risk of severe disease are also associated with weak CD8<sup>+</sup> T-cell responses, and conversely that strong, multifunctional, T-cell responses correlate with alleles associated with protection from severe disease. These data strongly imply a protective role for CD8<sup>+</sup> T cells against severe DENV disease in humans (25).

## **DENV SEROTYPES AND VACCINE DEVELOPMENT**

The dengue serocomplex consists of four serotypes, each of which is made up of several genotypes (57). The four serotypes share 65–75% genetic homology with each other but are antigenically distinct (58). This high degree of sequence variation in a pathogen, characteristically associated with RNA viruses, poses unique challenges to vaccine development. This is of particular relevance in the case of DENV infections because of the more severe disease and immunopathology associated with prior exposure to a different serotype (9). Consequently, the development of DENV vaccines has been hampered by the potential risk of vaccine-related adverse events and the requirement to induce long-lasting protective immune responses against all four DENV serotypes simultaneously. A recent phase 2b proof-of-concept efficacy vaccine trial (59) of a live-attenuated tetravalent chimeric yellow fever-dengue vaccine (CYD23) showed only 30% overall efficacy, demonstrating partial (60–80%) protection toward three of four DENV serotypes. No protection against DENV2 infection was observed, despite three subsequent immunizations and high neutralization titers against all four serotypes.

As reviewed above, T-cell responses have been implicated to have a protective role in DENV infection. Previous data from our lab and others clearly demonstrate that CD8<sup>+</sup> T-cell responses dominantly target the non-structural proteins NS3, NS4B, and NS5 (21–25). Since these DENV proteins are absent in the recombinant live-attenuated tetravalent dengue-yellow fever chimeric virus vaccine (60), our results perhaps provide an explanation for the low level of vaccine efficacy observed. Further,our data demonstrate the need to accurately assess T-cell responses (and not only antibody responses) in the context of DENV vaccine development.

Five additional and promising vaccine candidates are being tested in human clinical trials. These vaccines rely on technologies spanning from live-attenuated viruses, vectored vaccines expressing certain dengue proteins, replication-defective vaccines to nucleic acid-based vaccines [reviewed in Ref. (61)]. Since our data raise the possibility that T-cell responses against all DENV proteins might be beneficial or even required for vaccine efficacy, it will be of particular interest to study T-cell epitopes induced by multivalent live-attenuated vaccines and compare them to T-cell responses observed in natural infection.

#### **METHODS TO CHARACTERIZE T-CELL RESPONSES AFTER VACCINATION**

Characterization of T-cell epitopes can be performed by a variety of techniques, each associated with distinct advantages and disadvantages. These techniques include: ELISPOT, FACS and ICS assays, cell sorting, and tetramer staining. Though ELISPOT is the most sensitive at detecting low-levels of specific cytokine production, ICS assays are better suited to characterize phenotypes and T cells that are simultaneously producing multiple cytokines. Secretion of particular cytokines such as IFN-γ and TNF-α has been implicated in the induction of DENV-associated immunopathology. IFN-γ has been implicated as having a protective role during DENV infection whereas TNF-α has been implicated as a key mediator of immunopathology (62, 63). Characterization of a broad array of cytokines affords determination of the degree to which the cells responding to a given epitope are polyfunctional effectors. As illustrated in several different systems, T cells with a polyfunctional phenotype capable of secreting multiple cytokines provide the most effective control of viral infection (64–66). Importantly, both ELISPOT and ICS assays can be used to characterize pools of epitopes in conditions where only small amounts of PBMC are available.

An alternative and complementary approach to ELISPOT and ICS involves the use of tetramer staining reagents (67, 68). This approach requires not only the production of specific reagents for each HLA:epitope combination, but also that T cells specific for each combination are present in sufficient frequency in peripheral blood to allow their detection and characterization. In cases where T-cell frequency is low, this limitation can be overcome by the tetramer enrichment technique (69). Because tetramer characterization is in general more technically demanding, tetramer assays are ideally suited for in-depth characterization of a small but representative set of epitope specificities.

Markers associated with memory or activation/exhaustion states are also of interest. For vaccines to be effective, they must promote development of an effective T-cell memory response, in terms of recall of both effector T-cell responses and anamnestic antibody responses (70). T cell can be classified into T<sup>N</sup> (naïve T cells), TCM (central memory T cells), TEM (effector memory T cells), and TEMRA (effector memory T cells re-expressing CD45RA) subsets (71). Activation and conversely exhaustion of T cells are implicated in the regulation of protective immunity and immunopathology (72). Markers such as CD57 (73, 74), CD40L (75), and PD-1 (programed death-1) (76) allow determination of activation/exhaustion states of memory T-cell subsets. All of these techniques are currently available and a detailed analysis of the responses induced by natural or experimental infection with DENV will greatly contribute to the understanding of T-cell immunity in humans and may further contribute to identify robust correlates of protection in natural immunity and vaccination against DENV.

#### **ACKNOWLEDGMENTS**

This work was supported by National Institutes of Health contract Nr. HHSN272200900042C.

#### **REFERENCES**

76. Sharpe AH, Wherry EJ, Ahmed R, Freeman GJ. The function of programmed cell death 1 and its ligands in regulating autoimmunity and infection. *Nat Immunol* (2007) **8**:239–45. doi:10.1038/ni1443

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Received: 27 January 2014; accepted: 21 February 2014; published online: 07 March 2014.*

*Citation: Weiskopf D and Sette A (2014) T-cell immunity to infection with dengue virus in humans. Front. Immunol. 5:93. doi: 10.3389/fimmu.2014.00093*

*This article was submitted to Microbial Immunology, a section of the journal Frontiers in Immunology.*

*Copyright © 2014 Weiskopf and Sette. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

## Control of acute dengue virus infection by natural killer cells

#### **Caroline Petitdemange1,2,3, NadiaWauquier 1,4, Juliana Rey <sup>1</sup> , Baptiste Hervier 1,2, Eric Leroy <sup>3</sup> and Vincent Vieillard1,2,5\***

<sup>1</sup> Centre d'Immunologie et des Maladies Infectieuses (CIMI-Paris), Université Pierre et Marie Curie, Sorbonne Universités, Paris, France

2 INSERM, U1135, Centre d'Immunologie et des Maladies Infectieuses (CIMI-Paris), Paris, France

<sup>3</sup> Centre International de Recherches Médicales de Franceville, Franceville, Gabon

<sup>4</sup> Metabiota Inc., San Francisco, CA, USA

<sup>5</sup> CNRS ERL8255, Centre d'Immunologie et des Maladies Infectieuses (CIMI-Paris), Paris, France

#### **Edited by:**

Simona Zompi, University of California Berkeley, USA

#### **Reviewed by:**

Heinrich Korner, Menzies Research Institute Tasmania, Australia Claire F. Kubelka, Fundação Oswaldo Cruz, Brazil

#### **\*Correspondence:**

Vincent Vieillard, Centre d'Immunologie et des Maladies Infectieuses (CIMI-Paris), Equipe 14 – Cellules NK et Pathologies, Hôpital La Pitié-Salpêtrière, 83 Boulevard de l'Hôpital, Paris F-75013, France e-mail<EMAIL_ADDRESS>
Dengue fever is the most important arthropod-borne viral disease worldwide, affecting 50–100 million individuals annually. The clinical picture associated with acute dengue virus (DENV) infections ranges from classical febrile illness to life-threatening disease.The innate immunity is the first line of defense in the control of viral replication.This review will examine the particular role of natural killer (NK) cells in DENV infection. Over recent years, our understanding of the interplay between NK cells and viral pathogenesis has improved significantly. NK cells express an array of inhibitory and activating receptors that enable them to detect infected targets while sparing normal cells, and to recruit adaptive immune cells. To date, the exact mechanism by which NK cells may contribute to the control of DENV infection remains elusive. Importantly, DENV has acquired mechanisms to evade NK cell responses, further underlining the relevance of these cells in pathophysiology. Hence, understanding how NK cells affect the outcome of DENV infection could benefit the management of this acute disease.

**Keywords: NK cells, dengue viral infection, cytokines, cytotoxicity, viral escape mechanisms**

#### **INTRODUCTION**

Dengue virus (DENV) is the most widespread arbovirus worldwide transmitted by mosquitoes of the Aedes genus and is responsible for major outbreaks leading to serious health and economical problems (1). Approximately 500,000 DENV cases progress to be a life-threatening disease each year causing up to 20,000 deaths (2). DENV is a member of the genus Flavivirus and is divided into four different serotypes (DENV1–DENV4). Early 1970, Halstead and Simasthien (3) suggested that primary infection with one serotype of DENV only confers short-term partial cross-protection against other serotypes. Furthermore, secondary heterologous infections contribute to the development of severe forms of dengue fever (DF) [dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS)] (4).

The clinical picture of primary DENV infection includes fever, headache, myalgia, arthralgia, and petechial rash. Patients rapidly develop high viremia for up to 6 days after the onset of fever. The rapid initiation of the hosts' innate defense mechanisms might be a limiting factor in the development of DENV infection. In rare cases, patients may develop complications including plasma leakage and coagulation disorders, which may lead to a fatal outcome (5, 6). One of the most important questions in regards to dengue pathogenesis is the identity of the cells that play a crucial antiviral role during the innate immune response to DENV at the earliest stages of infection (7). Natural killer (NK) cells are a major component of the innate arm of the immune system. Although NK cells hold an early and central role early after

number of viral infections, not only for viral containment but also for timely and efficient induction of adaptive responses, their role in the control of DENV infection is still poorly documented. The observation in the late 1970s that viral infections induce NK cell-mediated killing stimulated a frenzy of research focused on evaluating the role of these cells in defense against several viral infections and cancer. The clearest demonstration of this role derives from the growing number of cases of NK cell deficiency. Since the initial known case of a girl with multiple severe or disseminated herpesvirus infections, about 40 other unrelated cases have been described and were mainly associated with severe herpesvirus infections, and EBV- or HPV-related cancers (8). Major progress has been achieved since, in several important fields related to NK cells. NK cells represented a wonderful biological paradox in that they appeared fully competent to kill target cells and yet were clearly self-tolerant. As such, NK cells were "armed" but not dangerous. Scientists focused their attention on understanding how these potent killers were contained and controlled. We now appreciate that a precise balance of inhibitory and activating signals mainly regulates the functional outcome of these cells (9–11). A flood of information concerning multiple types of negative receptors on NK cells was gathered. Many of these receptors respond to stimulation by major histocompatibility complex (MHC) class-I molecules expressed on the surface of target cells and this is considered to be the predominant mechanism responsible for NK cell tolerance to self. These inhibitory NK receptors include ILT-2 and the CD94/NKG2A complex, which recognize

HLA-G and HLA-E, respectively, whereas the inhibitory killercell immunoglobulin-like receptors (KIRs) recognize polymorphic MHC class-I molecules; in particular: KIR2DL1/KIR2DL2 and KIR2DL3 bind group 2 (C2) and group 1 (C1) HLA-C alleles, respectively, while KIR3DL1 recognizes HLA-Bw4 epitopes (12). Numerous studies have shown that these factors likely synergize to generate susceptibility or resistance to pathogens and disease (12, 13). In patients from southern Brazil, the susceptibility to DF is positively associated with the presence of KIR3DS1- Bw4, KIR3DL1-Bw4, KIR2DL1-C2, and KIR2DS1-C2 genes, and negatively associated with KIR2DL3-C1/C1 (14).

To destroy a target, NK cells also present several cell-surface activating receptors such as: NKG2D, DNAX accessory molecule-1 (DNAM-1), an adhesion molecule physically and functionally associated to Lymphocyte function-associated antigen 1 (LFA-1), and the natural cytotoxicity receptors (NCRs: NKp30, NKp44, and NKp46) (15, 16). Simultaneous interactions of certain of these activating receptors on NK cells with their specific ligands on the target cells, lead to the integration of different signals including the modulation of the cell-cycle, and together dictate the quality and intensity of the effector NK cell response (17). The relative contribution of each of the activating receptors to NK cytotoxicity against target cells differs, indicating the existence of an array of specific ligands that will be presented and their role in DENV infection further developed by Beltrán and Lopez-Verges, under the research topic "Protective Immune Response to Dengue Virus Infection and Vaccines: perspectives from the field to the bench."

In this mini-review, we focused on what is currently known about the mounting of the NK cell response with its role in cytotoxicity and immunoregulation during DENV infection as well as the mechanisms acquired by the virus to evade NK cell killing.

#### **ACTIVATION OF NK CELLS AFTER DENV INFECTION**

Very early after infection, interferon-alpha (IFN-α) is the mainstay of host defenses. This type I, IFN, is a crucial mediator of the antiviral response directly inhibiting viral replication and modulating downstream immune responses to counteract viral spread (18). Elevated IFN-α plasmatic levels are observed shortly after onset of symptoms in children and adult DENV-infected patients (19, 20). Recently, Gandini et al. (21) have shown that DENV2 efficiently activated IFN-α production by plasmacytoid dendritic cells (pDCs), which produced up to 1000-fold more IFN-α than other cell types in response to virus exposure. The infected pDCs could then decrease DENV infection of monocytes. The importance of the IFN-α response is also illustrated by the increased lethality of IFN-α/β receptor knockout mice when administered DENV2 by intraperitoneal injection (22). Although it was shown that the *in vitro* pretreatment of cultured cells with IFN-α/β dramatically reduces DENV replication, type I IFN has little effect on DENV replication after viral replication has been established (23). Indeed, DENV can reach high titers (<10<sup>9</sup> infectious doses per milliliter) in humans despite the induction of high levels of circulating IFN-α (19, 24). Therefore, it seems likely that DENV has evolved mechanisms to counteract the IFN response, a characteristic that is shared by many pathogenic viruses (25).

It has long been established that one of the main mechanisms accounting for the efficacy of the type I IFNs is their ability to activate NK functions (**Figure 1**); thus, they promote the accumulation and/or survival of proliferating NK cells by the STAT1 dependent induction of IL-15 secretion (26). This early activity of NK cells may be important for clearing primary DENV infection. In a sensitive mouse model, acute infection with DENV showed a rapid increase of NK cell levels (27). A significant increase in the frequency of NK cell circulation was also shown in patients who developed an acute DF (28). In addition, patients with a mild DF have elevated NK cell rates when compared to those with severe DF (29). Interestingly however, levels of circulating MIP-1β are higher in mild acute DF and are associated with higher NK cell frequencies (30). To characterize the primary NK response to DENV infection in mice, the phenotype of NK cells in the spleen was assessed by flow cytometric analysis. Three days after infection, the DENVinfected mice had twice as many NK cells than the mock-infected mice, and more than 50% of these NK cells expressed the early activation marker CD69, although only 5–10% of NK cells in the mock-infected animals expressed CD69 (27). Concomitantly, NK cells from DENV-infected patients display simultaneously high levels of CD69, HLA-DR, and CD38 (28, 29). For example, a significant increase in the percentage of CD69+-expressing NK cells was observed in DENV-infected patients at the early and acute phase of infection (days 1–5 with 29 vs. 13%), maintained at days 6–10 (24 vs. 18%), but decreased after 11 days (13 vs. 5%) (28). Altogether, these observations support the concept that DENV infection induces the selection and proliferation of a subset of activated NK cells further reinforcing their potentially important role during the early stages of the disease.

## **NK CELL FUNCTION IN THE PROTECTION AGAINST DENV CYTOKINES PRODUCED BY ACTIVATED NK CELLS**

Upon activation, NK cells may produce cytokines that favor the complete elimination of the disease and the infectious agent during the adaptive response, such as IFN-γ, TNF-α, G-CSF, and GM-CSF, as well as chemokines, such as MIP-1α, MIP-1β, and Rantes (31) (**Figure 1**). Numerous investigations have shown that DENV-infected patients presented significantly elevated levels of IFN-γ, G-CSF, and GM-CSF (20, 29, 32), whereas other reports suggest that TNF-α elevation could be associated with disease severity (33, 34). However, the results were obtained by measuring cytokine levels in sera collected from DENV-infected patients, and do not reveal which specific cells are activated and involved in their production. To date, the role played by NK cells in the production of these soluble factors is unknown. Deeper investigations will be necessary to precisely determine the implication of the immune-regulation by NK cells during the acute DENV infection.

#### **NK CELL AND THEIR RECEPTORS DURING DENV INFECTION**

One of the most prominent functions of NK cells is the capacity to lyse virus-infected cells. When the balance of inhibitory/activating signals is in favor of activation, the engagement of activating receptors on the surface of NK cells leads to directed exocytosis of granules containing perforin and granzymes, which in turn elicit the disruption of the target cell membrane and/or the activation of apoptosis pathways within the infected cell (11). It has been shown that activated NK cells are cytotoxic against DENV-infected cells (35). Furthermore, a marker of cytotoxicity, the granule cytotoxic

T cell intracellular antigen TIA-1, as well as two adhesion molecules, CD44 and CD11a, both involved in NK cell migration to various tissues and NK cytotoxicity, have been found to be significantly elevated on NK cells collected from acutely infected patients (28).

Hershkovitz et al. (36) have demonstrated the existence of a direct protein–protein interaction between recombinant DENV soluble envelope E protein and NKp44 (but not NKp30 or NKp46) that could be involved in the triggering of cytolysis (**Figure 1**). Using West Nile virus (WNV) like particles (VLPs) and WNVinfected cells, they have shown that E–NKp44 interaction triggers the secretion of cytotoxic granules contained in NK cells, the lysis of target cells, and the increased production of IFN-γ suggesting that flavivirus E proteins activate NK cytotoxic activity through NKp44 engagement.

Overall our understanding of the mechanisms involved in the cytolysis of target DENV-infected cells by NK cells is in its infancy. The receptors and signaling pathways essential to this major function are yet to be clearly identified. Recent investigations focusing on the various target ligands involved in the cytotoxic response by Beltrán and Lopez-Verges, under the Research topic "Protective Immune Response to Dengue Virus Infection and Vaccines: perspectives from the field to the bench" may bring some insight as to these questions.

### **ACTIVATION OF NK CELLS BY ADCC RESPONSES DURING EARLY DENV INFECTION**

Antibody-dependent cell-mediated cytotoxicity (ADCC) is another known mechanism by which NK cells recognize and lyse antibody-coated target cells, through the engagement of antibody binding to the Fc gamma receptor IIIA (CD16) (37). In an *in vitro* model, Kurane et al. (35) reported that human blood NK cells are cytotoxic against DENV-infected cells via direct cytolysis but also via ADCC (**Figure 1**). Indeed, PBMC collected from a DENVinfected patient successfully lysed DENV-infected cells that did not express MHC class-I molecules, and lysis of infected cells was significantly increased upon addition of anti-DENV1 and DENV2 monoclonal antibodies. The addition of sera from an individual without DENV antibodies did not lead to an increase in lysis of infected cells. More recently, García et al. (38) tested acute and convalescent patients' sera for ADCC activity, differentiating mild and severe forms of DF. ADCC activity was observed with acute sera only in cases of DHF/DSS but not DF. However, using convalescent sera collected 1 year later, all samples induced ADCC activity. This suggests that the development of ADCC activity during the acute phase of the infection could be associated to the pathological manifestations of the severe syndrome and that the systematic development of ADCC activity after a primary infection, in convalescent patients could be associated with the development of severe forms during a subsequent heterologous DENV infection. However, other studies suggest a protective role for ADCC against DENV secondary infections. Indeed, higher ADCC activities have been associated with higher plasma neutralizing antibody levels and lower viral loads during secondary infection but solely if the patient was secondarily infected by DENV3 and not DENV2 (39).

Altogether, this data support a strong implication of ADCC after DENV infection, however, careful investigations are needed to determine its exact contribution both in the viral clearance during the initial acute phase of this infection and DENV pathogenesis in secondary infections.

## **DENV EVASION OF NK CELLS**

Co-existence of viruses and their infected hosts imposes an evolutionary pressure on both the virus and the host's immune system. The host has developed an immune system able to attack viruses and virally infected cells, and viruses have developed an array of immune evasion mechanisms to escape being killed by the host's immune system (40, 41). A wide variety of viruses have developed mechanisms to evade the NK cell response, and among these the flaviviruses are known to have developed particularly evolved mechanisms to escape NK cell-mediated lysis. Many viruses evade T-cells recognition by down-regulating MHC class-I restricted antigen presentation, whereas flaviviruses induce their cell-surface expression. This leaves infected cells less susceptible to NK lysing, MHC class-I molecules being able to effectively engage NK cell inhibitory receptors (11, 42, 43). Regarding DENV infection, a number of studies have demonstrated that DENV replicon expression is sufficient to enhance membrane expression of MHC class-I inducing a reduced susceptibility to NK lysis (42, 44). The way by which DENV induces MHC class-I up-regulation on the surface of infected cells has been associated with an activation of NF-κB, independently of IFN and without an increased synthesis of MHC class-I molecules (45). The exact mechanism(s) and especially the viral components involved are still obscure yet several hypotheses were proposed. The up-regulation of MHC class-I expression could be the result of: (i) an increase in the supply of peptides to the endoplasmic reticulum mediated by the transporter associated with antigen processing (TAP) (46) (ii) the expression of DENV non-structural protein(s) and/or viral RNA replication (44), and (iii) the accumulation of uncleaved C-prM protein during viral assembly (47).

The major biological consequence of DENV-induced MHC class-I increase is a profound reduction of NK cell lysis as shown in *in vitro* cytotoxicity assays (35, 44). Moreover, an aggregation of MHC class-I molecules at the surface of infected cells seems to lead to increased affinity with NK cell inhibitory receptors (44). Given that the kinetics of DENV viremia coincide with the peak of NK cell response, the modulation of MHC class-I expression may have physiological relevance in counteracting host NK cell defense. It is likely that there is a complex balance between DENV-induced activation of NK cells and subversion of NK killing by DENV-induced MHC class-I expression on infected cells. An additional component to take into account is the genetic background of the host since KIRs/HLA molecules combinations play a crucial role in the strength of NK cell inhibitory function (13), and this could clearly play important role on DENV infection susceptibility, protection, and severity.

These viral escape mechanisms certainly reflect evolutionary pressure exerted by the host immune response on the pathogen but also highlight the importance of NK cells in the defense against DENV infection.

## **CONCLUSION**

Overall, several lines of evidence converge to suggest that NK cells are activated early during an acute DENV infection, and produce major cytokines, such as IFN-γ, which participate in the control of the viral replication while promoting the development of an efficient adaptive immune response. In the future, it will be interesting to determine if and how DENV leaves an imprint on the NK receptor repertoire possibly favoring cells with a strong cytolytic potential, as previously shown, by us and other groups, with several other viruses, including chikungunya virus, which is yet an other arbovirus (48). Numerous studies have also shown that DENV increases MHC class-I and adhesion molecule expression, allowing the virus to escape NK cell lysis. Whilst underlining the importance of NK cells in DENV infection, these observations must be interpreted with great caution. Due to the lack of extensive phenotypic and functional studies in DENV-infected patients, we cannot conclude in favor of a beneficial or a deleterious role of the NK cells in the control and/or the evolution of the disease. Future studies based on the depletion of NK cells in a relevant animal model for DENV infection, will be able to unravel this fascinating topic.

## **ACKNOWLEDGMENTS**

This work was supported in part by grants from the Institut National de la Santé et de la Recherche Médicale (INSERM) and the Université Pierre et Marie Curie (Paris-6), France.

### **REFERENCES**

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Received: 12 February 2014; accepted: 27 April 2014; published online: 13 May 2014. Citation: Petitdemange C, Wauquier N, Rey J, Hervier B, Leroy E and Vieillard V (2014) Control of acute dengue virus infection by natural killer cells. Front. Immunol. 5:209. doi: 10.3389/fimmu.2014.00209*

*This article was submitted to Microbial Immunology, a section of the journal Frontiers in Immunology.*

*Copyright © 2014 Petitdemange, Wauquier, Rey, Hervier, Leroy and Vieillard. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

## NK cells during dengue disease and their recognition of dengue virus-infected cells

## **Davis Beltrán1,2,3 and Sandra López-Vergès <sup>1</sup>\***

<sup>1</sup> Department of Research in Virology and Biotechnology, Gorgas Memorial Institute for Health Studies, Panama City, Panama

2 Institute for Scientific Research and Technology Services (INDICASAT-AIP), Panama City, Panama

<sup>3</sup> Department of Biotechnology, Acharya Nagarjuna University, Guntur, India

#### **Edited by:**

Simona Zompi, University of California, Berkeley, USA

#### **Reviewed by:**

Birgit Strobl, University of Veterinary Medicine Vienna, Austria Alexis DeHaven Dunkle, Lawrence Livermore National Laboratory, USA

#### **\*Correspondence:**

Sandra López-Vergès, Department of Research in Virology and Biotechnology, Gorgas Memorial Institute for Health Studies, Ave. Justo Arosemena and Calle No. 35, Panama City 0816-02593, Panama e-mail<EMAIL_ADDRESS><EMAIL_ADDRESS>
The innate immune response, in addition to the B- and T-cell response, plays a role in protection against dengue virus (DENV) infection and the degree of disease severity. Early activation of natural killer (NK) cells and type-I interferon-dependent immunity may be important in limiting viral replication during the early stages of DENV infection and thus reducing subsequent pathogenesis. NK cells may also produce cytokines that reduce inflammation and tissue injury. On the other hand, NK cells are also capable of inducing liver injury at early-time points of DENV infection.  In addition, NK cells may directly recognize DENV-infected cells through their activating receptors, although the increase in HLA class I expression may allow infected cells to escape the NK response. Recently, genome-wide association studies have shown an association between MICB and MICA, which encode ligands of the activating NK receptor NKG2D, and dengue disease outcome.This review focuses on recognition of DENV-infected cells by NK cells and on the regulation of expression of NK cell ligands by DENV.

**Keywords: dengue, NK cell, NK receptor, NK ligand, innate immune response**

## **INTRODUCTION**

Dengue is a major public health problem in tropical and subtropical regions world wide and is caused by four serotypes of dengue virus (DENV-1, -2, -3, -4), a flavivirus transmitted to humans by *Aedes* mosquitoes (1). DENV infection can be asymptomatic or induce mild to severe disease, traditionally referred to as dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS), which can lead to death (2). While there have been important advances in elucidating dengue pathogenesis, it is difficult to foretell if an acutely infected individual will develop the disease and to predict its severity; however, disease outcome appears to depend on the virus as well as on host genetics and prior immunity (3–5). For this reason, it is crucial to understand the immune response during DENV infection. Many studies have focused on the adaptive response, as antibodies and T cells play a crucial role in protection against infection, as well as in the pathogenesis of dengue disease (4–6). The innate immune response plays an intrinsic role at the level of the infected cell (7), but also recruits and activates innate immune cells that can eliminate the virus at early stages and induce the development of the adaptive response (8, 9). Indeed, the extent of DENV replication during the early period of infection correlates with dengue disease severity (10–14). Interstitial dendritic cells (DCs) are believed to constitute the first line of host defense against invading DENV at the anatomical sites where it replicates after the initial bite by infected mosquitoes (8). Type-I interferon-dependent immunity is known to play a critical role, and early activation of natural killer (NK) cells may also be important in limiting viral replication at the early stages of DENV infection (6, 8).

## **NK CELLS IN VIRAL INFECTIONS**

Natural killer cells are innate lymphocytes specialized in defense against viral and intracellular bacterial infections and tumors (15). NK cells share some characteristics with the adaptive immune system and may possess specific memory features against some viruses and antigens (16). They can be rapidly recruited into infected organs and tissues by chemoattractant factors produced by virus-infected cells and activated resident macrophages and DCs, which are a major source of the interferon IFNα/β that induces NK cell proliferation and activation (17, 18). Reciprocally, NK cells can shape DCs activation and subsequently the adaptive response (17). Once activated, NK cells fight infection by producing chemokines and anti-viral cytokines, mainly IFNγ and MIP1-β, and by recognizing and eliminating infected cells by antibody-dependent cell-mediated cytotoxicity (ADCC) or by direct recognition through their activating receptors (15). NK cells have activating and inhibitory receptors that allow them to recognize stressed cells, tumors, and pathogen-infected cells and to differentiate them from healthy cells (15, 19). Most of the inhibitory receptors recognize classic and non-classic major histocompatibility complex class I (MHC I) molecules, and many viruses decrease the expression of MHC I molecules in infected cells to escape the CD8<sup>+</sup> T-cell response, thereby becoming more vulnerable to NK cell recognition (19–21).

Virus-infected cells often induce or increase the expression of ligands at their surface, allowing for recognition by NK cell activating receptors, including NKG2D, DNAM-1, CD94-NKG2C; the NCR receptors NKp46, NKp30, NKp44; and others (19, 20, 22). The ligands include host stress-induced molecules and viral

**Table 1 | Summary of the published findings related to ligands for receptors on NK cells and dengue virus infection**.

proteins (20). To date, the ligands for many of the activating receptors (for example the NCRs) are still unknown, and their expression has been detected indirectly by cell staining with recombinant receptors or by blocking of killing with receptor-specific antibodies. A better characterization of NK ligands is needed. NKG2D is the most well-characterized NK activating receptor, and ligand binding leads to target killing and cytokine production (22). NKG2D ligand expression is increased by "stress" conditions, including viral infections (22, 23). In humans, eight ligands have been described for NKG2D, including MICA, MICB, and ULBP1-6. In addition to expression of NKG2D ligands on the surface of infected cells, soluble isoforms can be released into the serum, although the physiological relevance of the soluble ligands is controversial (22, 24). These soluble forms have been described in cancer patients, as well as in HIV-1-infected patients without therapy (25, 26) suggesting that soluble NKG2D ligands might be released in other viral infections.

#### **ROLE OF NK CELLS IN DENGUE DISEASE**

Many studies suggest that NK cells play a role in the response against DENV infection, principally in the early infection stages by limiting DENV replication. A higher absolute number of NK cells was observed in patients with mild dengue fever (DF) compared with children who developed DHF (27–29). However, the percentage of NK cells and CD8<sup>+</sup> T cells expressing CD69, a marker of activation,was higher early during infection of children who developed DHF (27, 30). Homchampa et al. found evidence of NK cell cytotoxicity against non-infected K562 target cells from children with acute dengue, and the cytolytic activity was increased on a per-cell basis in the early disease stages of dengue compared with healthy controls, and was even higher in the most severe form of the disease. It was suggested that this NK cell activity was associated with higher viremia in the more severe cases. Studies in adults showed that patients with mild disease had higher numbers of NK cells, with the majority of cells having increased expression of activation markers (CD69, CD38, and HLA-DR), adhesion molecules (CD11a and CD44) (**Table 1**), and markers of intracellular cytotoxic granules (TIA-1), in contrast to severe dengue where reduced NK cell numbers were observed (31). The authors suggest that higher NK cell percentages and activity might indicate a good prognosis of disease. In a genome-wide association study (GWAS), it was shown that the transcriptome of blood cells from children with DSS was characterized by decreased abundance of transcripts related to T and NK responses, probably not due to a difference in lymphocyte counts but to an impaired response (32). The differences observed in NK cell numbers and percentages in these studies may be explained by differences in age (children versus adults), ethnicity, time of infection, and the experimental methods to define NK cells and their activation. Nonetheless, all these observations point to the importance of NK cells and their activation during early DENV infection. The activation and phenotype of NK cells during dengue acute infection is further developed by Petitdemange et al., under the research topic "protective immune response to dengue virus infection and vaccines: perspectives from the field to the bench."

The protective role of NK cells in the response against DENV is supported in mice models of the disease. In immunocompetent A/J mice, the early activation of NK and B cells was associated with the control of the viral load and the prevention of disease (33, 34). In C57BL/6 mice, the recruitment of NK and NKT cells by mast cells to the site of infection was also crucial for viral clearance (35), underlining the importance of these cells during the host early response against DENV. The early recruitment of NK cells to the liver was induced in part by CXCL10 (IP-10), and in the liver NK cells produce effector molecules (perforin, granzyme A, and granzyme B) needed for viral clearance (36). On the other hand, NK cells appear to play not only a protective role in dengue disease, as it was shown that after intrahepatic infiltration, NK cells were responsible for cell death in the liver at the early phase of infection, whereas CD8<sup>+</sup> T cells were responsible for damage later (37). The mechanism of this cell death has not been elucidated, but the authors suggested that NK cells were killing DENV-infected cells. It is possible that under some conditions, the elimination of DENV-infected cells by NK cells can be exacerbated in a way that the immune effector cells become responsible for organ injury. Similarly, NKT cells in some conditions can be detrimental during dengue pathogenesis, in part by inducing NK cells and neutrophils activation (38).

Natural killer cells may play an important role in early DENV infection *in vivo* also by producing together with γδ T cells, IL-22 and IL-17A, which may influence dengue disease outcome (39). NK, NKT, and T cells that can respond in a non-TcR-dependent fashion are a major source of IFNγ in immune responses induced by inactivated DENV (40); however, in this context, the frequency of IFNγ-producing cells observed was very low. Using C57BL/6 mice, a study confirmed the role of NK cells in the early IFNγ production induced by IL-12 and IL-18 in response to DENV, key for the control of viral load and DENV-2-associated disease severity and lethality (41).

Natural killer cells can mediate ADCC against DENV-infected cells (42, 43) and this mechanism may be important during secondary infections when antibodies to DENV are present. Indeed, ADCC activity in plasma obtained before secondary DENV-2 or DENV-3 infection correlated with serotype-specific neutralizing antibody titers, anti-DENV IgG1 levels, and a multitypic PRNT<sup>50</sup> pattern (44). Interestingly, a higher level of ADCC activity measured before secondary DENV-3 infection was associated with lower subsequent viremia, which suggests a protective role for antibodies and NK cells; however, this association was not observed for secondary DENV-2 infection. In another study,ADCC was correlated with DENV surface antigen expression, suggesting recognition by anti-DENV antibodies (42), whose Fc region is then recognized by the activating low affinity CD16 (FcγRIII) receptor on NK cells (45).

Human NK cells (CD3−CD16+CD11b<sup>+</sup> cells) can lyse DENVinfected cells to a greater level than uninfected cells even in the absence of antibodies, suggesting a mechanism of direct recognition as well (42, 43). These studies were performed using DENVinfected Raji cells as targets. The NK receptors and their ligands implicated in the direct recognition of DENV-infected cells have not been fully elucidated, indicating the need for future studies.

#### **EXPRESSION OF NK CELL LIGANDS DURING DENGUE DISEASE**

Viruses try to escape the immune response of the host. As NK cells are crucial players in the anti-viral response, many viruses induce the up-regulation of MHC I, which serve as ligands for NK inhibitory receptors, in order to dampen the NK cell response even if enhancing expression of MHC I might increase their recognition by CD8<sup>+</sup> T cells (20). DENV and other flaviviruses induce the up-regulation of MHC I (46–51). Expression of flavivirus pr-M protein in hamster cells induced increased surface expression of MHC I, although the authors suggested this could be an incidental consequence of viral assembly rather than a specific mechanism of immune evasion (52). It has also been shown that human cell lines expressing the non-structural (NS) proteins of DENV up-regulate MHC I (HLA-A, -B, -C) expression at their surface by TAP-dependent and TAP-independent pathways (46). This resulted in a lower sensibility to lysis by NK cells, probably due to recognition by the corresponding inhibitory receptors (e.g., KIR2DL1 for HLA-C) and the inhibitory receptor LIR-1 that recognizes all MHC I proteins (**Table 1**). Further studies are needed to determine if MHC I up-regulation by DENV is the response to a particular viral protein, viral replication itself, or type-I IFNs induced by viral infection, and whether it plays a role in immune evasion of NK cells *in vivo*. Neurotropic flaviviruses, such as West Nile virus (WNV), can transiently activate and then suppress NK cell activity (53). Future studies are needed to determine if this happens during DENV infection by analyzing NK cell activity at different time points post-infection.

Even if DENV NS proteins induce MHC I up-regulation, NK cells can kill DENV-infected cells (28, 42, 43), suggesting that the signals for NK cell activation overcome the inhibitory signals. As described above, NK cells can kill DENV-infected cells by ADCC mediated by CD16 (42, 44) (**Table 1**), which is expressed on resting human NK cells and can induce a strong activating signal leading to cytolysis (20, 54). Interestingly, NK cells can also kill DENVinfected cells in the absence of antibodies (42), implicating a direct recognition by NK cell activating receptors. It has been reported that the activating receptor NKp44 can interact with the envelope protein (E) of DENV (55) (**Table 1**). NKp44 has also been implicated in killing WNV-infected cells after blocking the inhibitory receptor LIR-1 on NK cells, and this also induced IFNγ production. Further studies are necessary to determine if recognition of DENV-E by NKp44 on NK cells can induce a similar response against DENV-infected cells. NKp44 is expressed on activated, but not resting, NK cells (56) and can trigger NK cell killing of both tumor and virus-infected cells (57, 58).

Recently, a sequencing-based typing method and genotyping of asymptomatic DF and DHF patients in Cuba uncovered an association of certain alleles of the *MICA* and *MICB* genes (*MICA\*008* and*MICB\*008*) with symptomatic DENV infection (59) (**Table 1**). The importance of MICB in dengue susceptibility was also indicated by GWAS with a large number of pediatric cases in Vietnam, where certain *MICB* and *PLCE1* alleles showed a significant association with DSS (60). These results were confirmed by a study showing that *MICB* rs3132468 and *PLCE1* risk genotypes were also associated with less severe clinical phenotypes of dengue in adults as well as with DENV infection in infants (61). This strongly suggests a role for this *MICB* variant in susceptibility to overall clinically apparent dengue disease. It still has not been determined whether the association between these NKG2D ligands and dengue clinical responses is directly due to the function of MICA/B molecules in dengue pathogenesis. Nonetheless, the importance of NKG2D ligands in the NK cell response against other viral infections (22) supports this hypothesis. Given the role of MICB in activation of NK, NKT, and CD8<sup>+</sup> T cells through the NKG2D receptor, these findings support a central role for these cell types in shaping the outcome of DENV infection. It

is plausible that the *MICB* risk-associated phenotype is associated with an impaired NK cell response, potentially resulting in a higher *in vivo* virus titer and an increased risk of developing both symptomatic and severe dengue. Furthermore, inefficient induction of cytokines secreted by NK cells might result in dysregulated T-cell responses that may also shape the clinical phenotype (62). NKG2D ligand (MICA/B) expression on DENV-infected cells may allow direct recognition by NK cells, which might be important for the early innate immune response against DENV infection, leading to either more effective control of viral infection or alternatively contributing to the disease pathology. The expression pattern of these NKG2D ligands in DENV-infected cells *in vitro* and *in vivo* needs to be determined. It is also necessary to determine if in acute viral infections such as DENV, the production of soluble NKG2D ligands can also be observed and if this impacts dengue clinical manifestations. Because ligands of other NK activating receptors, such as DNAM-1, can be induced by "stress" (23), further studies are needed to characterize all the NK receptor ligands induced during DENV infection, and the results may depend on the cell-type analyzed, as well as the DENV serotype. A complete characterization of DENV-infected cell recognition by NK cells (**Figure 1**) is crucial to better understanding the role of these cells in dengue disease.

## **CONCLUSION**

The innate immune response, and particularly type-I IFN and NK cells, plays a key role during the early infection events due to its ability to rapidly limit viral dissemination and to affect the antigen-specific, adaptive immune responses to effectively clear pathogens (8, 9). More studies in animal models and human populations will enable deciphering NK cell responses during DENV infection *in vivo*. Furthermore, *in vitro* experiments will also be needed to determine which NK receptors and ligands are implicated in DENV-infected cell recognition and NK cellmediated killing and cytokine production. To date, only the upregulation of MHC I and the induction of a putative NKp44 ligand (DENV-E protein) have been reported in DENV-infected cells. Are there other ligands that play a role in NK cell recognition induced during DENV infection? Genetic studies suggest an important role for the NKG2D ligands MICA and MICB; however, as yet no functional experiments have validated this hypothesis. There is still much to do to determine if ligands of other NK receptors, for example HLA-E for CD94/NKG2A-C and the ligands for the activating receptors 2B4 and DNAM-1, are induced or repressed during DENV infection, and whether this has an implication in the immune response to DENV and its clinical outcome. Finally, the characterization of NK receptor ligands and the NK cell phenotype in patients' blood cells will provide insights into the NK cell subsets activated during dengue disease. It will also establish possible associations between NK cell activity or NK cell ligand expression and protection from disease and/or increased dengue severity. Whether DENV interferes with innate anti-viral immunity mediated by NK cells at early times of infection and whether DENV virulence might be associated with its ability to counter the host cell defenses are critical issues that remain to be elucidated.

## **ACKNOWLEDGMENTS**

The authors thank Eva Harris (University of California Berkeley) and Lewis Lanier (University of California San Francisco) for helpful comments and scientific discussion of the manuscript. The authors are supported by grants from Secretaría Nacional de Ciencia, Tecnología e Innovación, Panama (270-2013-288 to Davis Beltrán and 71-2012-4-CAP11-003 to Sandra López-Vergès).

#### **REFERENCES**

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Received: 21 February 2014; accepted: 18 April 2014; published online: 05 May 2014. Citation: Beltrán D and López-Vergès S (2014) NK cells during dengue disease and their recognition of dengue virus-infected cells. Front. Immunol. 5:192. doi: 10.3389/fimmu.2014.00192*

*This article was submitted to Microbial Immunology, a section of the journal Frontiers in Immunology.*

*Copyright © 2014 Beltrán and López-Vergès. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

## Dendritic cells in dengue virus infection: targets of virus replication and mediators of immunity

## **Michael A. Schmid<sup>1</sup>\*, Michael S. Diamond2,3,4 and Eva Harris <sup>1</sup>\***

<sup>1</sup> Division of Infectious Diseases and Vaccinology, School of Public Health, University of California Berkeley, Berkeley, CA, USA

<sup>2</sup> Department of Medicine, Washington University School of Medicine, St. Louis, MO, USA

<sup>3</sup> Department of Molecular Microbiology, Washington University School of Medicine, St. Louis, MO, USA

<sup>4</sup> Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, MO, USA

#### **Edited by:**

Stipan Jonjic, University of Rijeka, Croatia

#### **Reviewed by:**

Dina Weilhammer, Lawrence Livermore National Laboratory, USA Katja Fink, Singapore Immunology Network (SIgN), Singapore

#### **\*Correspondence:**

Michael A. Schmid, Division of Infectious Diseases and Vaccinology, 510D Li Ka Shing Center, School of Public Health, University of California, 1951 Oxford Street, Berkeley, CA 94720-3370, USA e-mail: michael.a.schmid@ berkeley.edu; Eva Harris, Division of Infectious Diseases and Vaccinology, 185 Li Ka Shing Center, School of Public Health, University of California, 1951 Oxford Street, Berkeley, CA 94720-3370, USA e-mail<EMAIL_ADDRESS>
Dendritic cells (DCs) are sentinels of the immune system and detect pathogens at sites of entry, such as the skin. In addition to the ability of DCs to control infections directly via their innate immune functions, DCs help to prime adaptive B- and T-cell responses by processing and presenting antigen in lymphoid tissues. Infected Aedes aegypti or Aedes albopictus mosquitoes transmit the four dengue virus (DENV) serotypes to humans while probing for small blood vessels in the skin. DENV causes the most prevalent arthropodborne viral disease in humans, yet no vaccine or specific therapeutic is currently licensed. Although primary DENV infection confers life-long protective immunity against re-infection with the same DENV serotype, secondary infection with a different DENV serotype can lead to increased disease severity via cross-reactive T-cells or enhancing antibodies. This review summarizes recent findings in humans and animal models about DENV infection of DCs, monocytes, and macrophages. We discuss the dual role of DCs as both targets of DENV replication and mediators of innate and adaptive immunity, and summarize immune evasion strategies whereby DENV impairs the function of infected DCs. We suggest that DCs play a key role in priming DENV-specific neutralizing or potentially harmful memory B- and T-cell responses, and that future DC-directed therapies may help induce protective memory responses and reduce dengue pathogenesis.

**Keywords: dengue virus, dendritic cells, monocytes, macrophages, innate immunity, antibody-dependent enhancement, immune evasion**

## **INTRODUCTION**

## **DENDRITIC CELLS, MONOCYTES, AND MACROPHAGES: LOCATION AND FUNCTION**

Dendritic cells (DCs) reside and migrate into barrier tissues such as the skin and mucosal epithelium that are the sites of pathogen invasion. In the steady state, DCs display high levels of phagocytic activity, take up antigen, and probe for pathogens via patternrecognition receptors. DCs express Toll-like receptors (TLRs) and C-type lectins as transmembrane proteins as well as intracellular sensors, such as retinoic acid-inducible gene I (RIG-I), melanoma differentiation factor 5 (MDA-5), and cyclic GMP-AMP synthase (cGAS) that recognize conserved microbial patterns (1, 2). Upon pathogen recognition, DCs become activated, produce inflammatory cytokines and chemokines, migrate to lymph nodes (LNs), and present antigen to prime naïve T-cells (3).

Subsets of DCs, monocytes, and macrophages (MΦs) reside in different tissues and fulfill distinct functions. Classical DCs (cDCs) display the characteristic DC dendrites, are present in LNs, spleen, and bone marrow as well as skin, lung, liver, and intestine, and have the greatest ability to stimulate naïve T-cells (4). All cDCs can present endogenous antigen from the cytosol via MHC I to CD8<sup>+</sup> T-cells (5). In addition, most subsets of cDCs present exogenous antigen via MHC II to CD4<sup>+</sup> T-cells, whereas only specialized subsets can cross-present exogenous antigen via

MHC I to CD8<sup>+</sup> T-cells (6, 7). In addition, plasmacytoid DCs (pDCs) are another DC subset that reside in the spleen, bone marrow, and liver and circulate in the blood. During viral infections, pDCs migrate to infected tissues and secrete up to 1,000-fold higher amounts of interferon (IFN)-α/β than other cell types (8), although their capacity for antigen presentation is still debated (9). Nevertheless, the role of DCs in priming protective immune responses against many human pathogens and their potential contribution to pathogenesis and development of disease need further investigation.

Monocytes circulate in steady-state blood, patrol lymphoid, and non-lymphoid organs, and are recruited to inflamed tissues, where they phagocytize pathogens as well as infected or damaged cells (10). During inflammation, monocytes can differentiate to monocyte-derived DCs (moDCs) (11, 12). *In vitro*generated human moDCs are used widely to study DC biology (13). Monocytes are isolated from human peripheral blood, differentiated in the presence of GM-CSF and IL-4 first to immature moDCs and after further stimulation with inflammatory cytokines or pathogen-associated microbial patterns (PAMPs) to mature moDCs (13, 14). The ability of moDCs to prime naïve T-cell responses remains controversial, as this function initially was attributed solely to cDCs (15). Nevertheless, recent studies demonstrated that moDCs can migrate to LNs and prime naïve

T-cells during *Leishmania major*, influenza virus, and bacterial infections (12, 16, 17). In contrast to DCs, MΦs have limited ability to migrate and prime naïve T-cells. MΦs reside within tissues, where they phagocytize, secrete cytokines, present antigen to effector and memory T-cells, and contribute to the healing of injured tissue (18).

#### **DENGUE EPIDEMIOLOGY AND PATHOGENESIS**

Female*Aedes aegypti* and*Aedes albopictus* mosquitoes transmit the four dengue virus serotypes (DENV1–4) while feeding on blood vessels in the skin (19). The positive-sense RNA genome of the flavivirus DENV encodes three structural (C, prM/M, E) and seven non-structural (NS) proteins (20). DENV causes the most prevalent arthropod-borne viral disease of humans, with an estimated 390 million infections and 96 million apparent cases per year (21).

The acute febrile illness dengue fever (DF) can progress to a potentially life-threatening vascular leakage syndrome, dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS), the latter characterized by hypotension and circulatory failure (22). At present, no vaccine or therapeutic against dengue is approved for use in humans. A major challenge in the development of vaccines and therapies is that although infection with one DENV serotype leads to long-lasting immunity against the same serotype, subsequent infection with a different (heterotypic) serotype is the major risk factor for severe disease (19). To date, the mechanisms by which the host immune response to DENV provides either protection or enhancement in secondary infection remain poorly understood. Antibodies can neutralize infection or conversely trigger "antibody-dependent enhancement" (ADE) (23, 24), whereby cross-reactive anti-DENV antibodies facilitate entry of DENV into Fcγ receptor (FcγR)-bearing cells and thus increase viral load and ultimately disease severity. Some DHF/DSS cases occur during primary (1°) infections, especially in infants 6– 9 months of age (25). In this case, it is thought that maternal DENV-specific antibodies transferred via the placenta wane to levels that can enhance a newly acquired DENV infection (26). Thus, the quantity and quality of the antibody response influences the severity of a secondary DENV infection. Similarly, T-cells can provide protection (27–29), but cross-reactive T-cells have been implicated in disease pathogenesis (30–32). Nevertheless, most secondary DENV infections are asymptomatic or mild, suggesting that the immune system can mount protective responses against dengue.

*Aedes* mosquitoes that take a blood meal from a human with acute dengue viremia become infected and, after DENV spreads to the salivary glands, transmit the virus when feeding on a new individual. Mosquito saliva contains components that counteract the host hemostatic response and modulate immunity (33, 34). The addition of saliva from *Ae. aegypti* mosquitoes was found to decrease DENV infection of moDCs*in vitro* (35). In contrast, mosquito saliva or transmission via infected mosquitoes prolonged DENV serum viremia and fever in "humanized" mice as compared to inoculation with DENV alone (36). Furthermore, saliva that was inoculated by non-infected mosquitoes prior to needle inoculation of DENV blocked the upregulation of genes involved in innate pathogen recognition and increased serum viremia in mice deficient in IRF3 and IRF7 (37). Although certain *in vivo* studies suggest that mosquito saliva can facilitate DENV infection by generating an environment that favors early virus replication, the impact of saliva on skin DCs requires further study.

#### **TARGETS OF DENV REPLICATION**

#### **DENV INFECTION IN THE ABSENCE OF ENHANCING ANTIBODIES**

Identifying the targets of DENV infection is crucial for understanding virus spread and disease pathogenesis. Human autopsies revealed staining for DENV structural proteins and negative-sense viral RNA, indicative of virus replication, in MΦs in LNs, spleen, lung, and liver and monocytes in clotted blood from patients with lethal dengue disease (38, 39). Staining of the non-structural protein NS3 confirmed DENV replication in phagocytes (including monocytes, MΦs, and DCs) in LNs and spleen, as well as in MΦs in the lung in other autopsy studies (40, 41). In earlier stages of the disease, most DENV-infected cells in the peripheral blood of acute dengue patients were identified as CD14<sup>+</sup> CD11c<sup>+</sup> activated monocytes, with higher proportions of monocytes and DENV-infected total cells in the blood in DHF compared to DF patients (42).

Although in humans, DENV efficiently suppresses the IFN response, replicates, and causes disease, DENV fails to antagonize mouse IFN responses, and thus wild-type (WT) mice generally do not sustain DENV replication or develop disease (43). In comparison, mice deficient in IFN-α/β receptor (*Ifnar*−/−) and also -γ receptor (AG129) are susceptible to DENV infection and display a tropism similar to humans (40, 44–46). DENV replicated in murine MΦs that were isolated from the peritoneum (47), as well as in MΦs in LNs and spleens of AG129 mice (48, 49). Treatment with clodronate liposomes that deplete monocytes and MΦs decreased viral load in AG129 mice on day 2 but increased viral load on day 4 post-inoculation with DENV2 (48). Monocytes and MΦs thus play an important role as targets for early DENV replication as well as in subsequent control of DENV infection.

Human moDCs generated *in vitro* support DENV infection (50), with immature moDCs being more susceptible to DENV infection than mature moDCs, monocytes (51), or MΦs (52). Analogously, CD11chigh cells in the spleen of AG129 mice (47) that likely comprised both moDCs and cDCs supported DENV replication *in vivo*. Recent studies in *Ifnar*−/<sup>−</sup> mice have shown that early after infection, monocytes are recruited to the dermis and differentiate to moDCs, where they become primary targets for DENV replication (53). Although DENV can infect monocytes and MΦs directly, these studies emphasize the greater permissiveness of DCs to DENV infection in the absence of enhancing antibodies, such as during 1° infection conditions.

Surface expression of viral attachment factors determines the susceptibility to DENV infection. DC-SIGN (dendritic-cellspecific ICAM3-grabbing non-integrin, CD209) is a C-type lectin expressed on the surface of DCs and MΦs that recognizes mannose-type sugars on the surface of bacterial, fungal, and viral pathogens. Signals via DC-SIGN induce the phagocytosis of pathogens and contribute to host defense (54). However, DC-SIGN also interacts with carbohydrates on DENV glycoproteins and mediates the attachment of DENV to moDCs (55–57). Human immature moDCs express high levels of DC-SIGN and are highly susceptible to DENV infection (50, 58, 59). Of note, DC-SIGN mediates virus attachment to the cell surface, but not endocytosis into moDCs (60). Activation of immature moDCs via inflammatory cytokines results in downregulation of DC-SIGN, explaining in part why mature moDCs are less susceptible to DENV infection (51, 58). Further, human cDCs freshly isolated from blood do not express DC-SIGN and, accordingly, become highly susceptible to DENV infection only after culture with GM-CSF and IL-4 that induces DC-SIGN expression (61). Similarly, treatment of monocytes with IL-4 or IL-13 increased DC-SIGN expression and DENV infection (62), but it remains unclear whether these monocytes had differentiated to moDCs or MΦs. Consistent with these findings, higher levels of DC-SIGN expression on cDCs of different blood donors correlated with higher DENV infection (61). Furthermore, a polymorphic variant of the DC-SIGN promoter with a decreased transcriptional activity correlated with protection against DF in humans (63). In summary, changes in DC-SIGN expression on different myeloid cell subsets through differentiation correlate with DENV infection (**Figure 1**).

Although *Aedes* mosquitoes transmit DENV when probing for blood vessels in the skin, most studies have focused on DENV infection in tissues after the virus has spread via the blood. Few studies have examined DENV infection and the immune response in the skin. DENV infects epidermal Langerhans cells (LCs) in healthy human skin explants *in vitro* (58, 64). Infection of LCs was confirmed in AG129 mice after intradermal inoculation of DENV2 (48). However, the dermis of intradermally inoculated *Ifnar*−/<sup>−</sup> mice contains 100-fold more DENV-infected cells than the epidermis (53). Recent studies indicate that dermal cDCs, and to a lesser extent MΦs, are the initial targets of DENV replication after intradermal inoculation of *Ifnar*−/<sup>−</sup> mice (53) or infection of skin explants from healthy human donors (65). Subsequently, *de novo*-recruited monocytes differentiate into moDCs, which become primary targets for DENV replication in the dermis (53).

Regarding the source of infectious virus, DENV produced in mosquito cells interacts with DC-SIGN and infected human immature moDCs *in vitro* (66). In contrast, DENV that was produced in human moDCs did not bind to DC-SIGN or infect moDCs but instead was infectious for cells expressing the homolog L-SIGN, such as monocytes and endothelial cells (66). The difference in binding to DC-SIGN or L-SIGN was likely due to different N-linked glycosylation patterns present on DENV particles produced in mosquito or mammalian cells. This may explain how DCs in the skin are the initial targets for DENV infection immediately after transmission. Characterization of the initial targets and immune response to DENV in the skin may foster new strategies to block DENV replication and abort pathogenesis.

#### **ANTIBODY-ENHANCED DENV INFECTION**

Dengue virus-specific adaptive immune responses, in particular subneutralizing concentrations of antibodies, can enhance DENV

**FIGURE 1 | DENV infection varies among macrophages, monocyte, and dendritic cell (DC) subsets in the presence or absence of enhancing antibodies**. DC-SIGN expression correlates with high infection in the absence of enhancing antibodies (i.e., 1° infection conditions), whereas FcγR expression modulates antibody-enhanced infection during ADE. Macrophages (MΦ) and monocytes express low levels of DC-SIGN, and show little DENV infection in the absence of enhancing antibody, but are highly infected in the presence of enhancing antibody. Under inflammatory conditions, monocytes differentiate to immature monocyte-derived DCs (moDCs) and, further, to mature moDCs after stimulation via PAMPs or inflammatory cytokines. While immature moDCs express high levels of DC-SIGN and can be infected with

DENV in the absence of antibodies, mature moDCs express lower levels of DC-SIGN and show moderate permissiveness under these conditions. Accordingly, mature moDCs show a capacity for enhanced infection in the presence of subneutralizing anti-DENV antibodies. Classical DCs (cDCs) that are freshly isolated from human blood do not express DC-SIGN, but express high levels of DC-SIGN after stimulation with GM-CSF and IL-4 in vitro (\*), which renders them highly susceptible to DENV infection without antibody, similar to immature moDCs. Plasmacytoid DCs (pDCs) do not express DC-SIGN or support DENV replication in the absence of antibody. cDCs and pDCs express FcγRs, but DENV infection of cDCs and pDCs during ADE has not been determined (n.d.).

entry and infection. In cell culture, subneutralizing amounts of DENV-immune human serum or monoclonal antibodies enhance infection of monocytes (67, 68) and mature moDCs, but not immature moDCs (51). Human splenic MΦs showed low levels of DENV infection at baseline *in vitro*, but at least 10-fold greater infection in the presence of enhancing concentrations of diluted DENV-immune human (69). *In vivo*, CD14<sup>+</sup> monocytes in the blood of acute dengue patients contained significantly higher levels of DENV genomic RNA in severe DHF compared to DF cases, and in secondary compared to 1° infections (70). Thus, ADE mediates efficient DENV infection of monocytes, mature moDCs, and MΦs.

FcγR expression determines the susceptibility of cells to ADE during DENV infection via uptake of virus-antibody complexes (**Figure 1**). Most myeloid cells express FcγRs, which bind the Fc region of antibodies and thus are an important link between cellular effector functions and antigen recognition via the antibody. Different types of FcγRs recognize distinct isotypes of IgG with varying affinity and can transmit activating or inhibitory signals to the cells (71). Attachment of DENV-antibody complexes to ectopically expressed FcγRI (CD64) (72) and FcγRIIA (CD32) (73) in fibroblast cell lines mediated ADE, independently of FcγRsignaling. DENV2 infection in primary human monocytes was increased 50-fold in the presence of enhancing DENV-immune human serum and depended on binding of antibody-virus complexes to FcγRI or FcγRIIA (68). Consistent with these data, blocking of FcγRIIA, but not of FcγRIIB, abrogated ADE of mature moDCs (51). Although mature and immature moDCs express similar levels of FcγRIIA, only mature moDCs sustain antibody-enhanced DENV infection because immature moDCs express high levels of DC-SIGN and thus do not require FcγR for DENV attachment or entry (51). Inflammation may lead to activation and differentiation of monocytes and moDCs, as well as altered expression of FcγRs and DC-SIGN, which modulates ADE of DENV infection in a cell-type specific manner.

Different classes of FcγRs transmit activating or inhibitory signals and play different roles during ADE. Although activating FcγRIIA and inhibitory FcγRIIB similarly bind DENV-antibody complexes, only FcγRIIA mediates enhanced DENV infection (74). In contrast to activating FcγRs, the inhibitory FcγRIIB may help prevent ADE. DENV-antibody aggregates cross-linked the inhibitory low-affinity FcγRIIB, which inhibited the phagocytosis and infection that would have occurred through activating FcγRs (75). This study showed evidence that the size of antibody-DENV aggregates may contribute to the neutralizing versus enhancing capacity of DENV-immune sera. More detailed studies on the expression of activating and inhibitory FcγRs are needed to understand the neutralization or enhancement of DENV infection of diverse cell subsets in different tissues. Along with greater infection *in vitro*, ADE may contribute to severe dengue disease in humans. Secondary DENV infection with a heterologous serotype is associated with an increased risk of DHF/DSS (76–79). Consistent with a possible role for ADE *in vivo,* polymorphisms in FcγR genes affect binding affinities for IgG subclasses and may influence the susceptibility to severe disease. Homozygotes for the arginine variant at position 131 (R/R131) of the FcRIIA gene, who have less capacity to opsonize IgG<sup>2</sup> antibodies, showed reduced risk of developing DHF (80,81). In contrast, the histidine variant H/H131 of FcRIIA was associated with an increased risk of developing DHF (81). Binding affinity of FcγRs to DENV-antibody complexes and the ratio of activating and inhibitory receptors likely determine DENV infection and disease outcome in the setting of pre-existing anti-DENV antibody.

Animal models also have been used to study ADE *in vivo*. AG129 mice develop mild disease after intravenous or intradermal inoculation with DENV2 in the absence of pre-existing antibody and lethal disease after passive transfer of subneutralizing levels of DENV-immune mouse or human serum prior to infection with otherwise sublethal doses of DENV (53, 82–84). The vascular leakage syndrome that develops in DENV-infected AG129 mice during ADE is similar to that observed after high-dose lethal DENV infection and recapitulates many features of severe dengue disease in humans (40, 44). Monoclonal antibodies directed against DENV protein E or prM can mediate ADE *in vitro* and *in vivo* (82, 83, 85). Addition of antibodies that block FcγR binding, F(ab)'<sup>2</sup> fragments that lack the Fc domain, or recombinant monoclonal antibodies that lack the ability to bind FcγR all prevented ADE (82, 83). DENV-specific monoclonal antibodies with modified Fc domains that do not mediate ADE show therapeutic potential *in vivo* (85–87).

Mouse models have helped to define possible cellular targets for antibody-enhanced DENV infection. While the same cell types become DENV infected in the presence or absence of enhancing antibodies, DENV infection increases during ADE (82). In addition, MHC II<sup>+</sup> cells in the intestinal lamina propria and sinusoidal endothelial cells in the liver of AG129 mice were infected by DENV mostly in the presence of enhancing concentrations of antibody (83).

## **INNATE FUNCTION OF DENDRITIC CELLS AND EVASION BY DENV**

As first line of defense against virus infection, host cells recognize PAMPs (e.g., viral nucleic acids) and induce cell-intrinsic and cellextrinsic innate immune responses. DENV infection stimulates responses via TLR7, TLR3, MDA5, and RIG-I (88–90) and induces the secretion of IFN-α/β that renders other host cells resistant to subsequent DENV infection (91, 92).

Plasmacytoid DCs recognize DENV via TLR7 in endocytic vesicles (61, 89), become activated, produce high amounts of IFN-α (93, 94), and may thus limit DENV replication. Further, pDCs sense DENV-infected cells by direct cell-to-cell contact, and immature DENV particles containing uncleaved prM were found to trigger higher IFN responses in pDCs compared to mature particles (95). Nevertheless, DENV does not infect human pDCs efficiently *in vitro*, and IFN-α production in pDCs appears independent of active viral replication within pDCs (61). This suggests that pDCs combat DENV without being susceptible to infection or to immune evasion mediated via cytosolic viral proteins. Because DENV cannot infect pDCs productively, its proteins cannot block the production of pDC-derived IFN-α that promotes transcription of IFN-stimulated genes (ISGs), which induce an antiviral state in neighboring cells. Patients with non-severe DF produced high levels of IFN-α in the serum (93, 96) and had an increased frequency of circulating pDCs compared to steady state (94). In contrast, numbers of pDCs declined early (day 3 or 4 of illness) in the blood of children who subsequently developed DHF (94) and,correspondingly,accumulated less in severe compared to nonsevere adult cases (93). Furthermore, serum of patients with severe dengue contained less IFN-α than patients with non-severe manifestations (96). Consistent with this observation, peripheral blood mononuclear cells of patients that subsequently developed DSS expressed fewer transcripts of a set of ISGs than patients with mild disease (97, 98). Overall, pDCs protect against DENV pathogenesis by producing high amounts of IFN-α that prevent infection of additional target cells.

Dengue virus actively blocks the production and action of IFNα/β in cell types that are susceptible to infection. The DENV non-structural protein NS2B/3 cleaves the human protein STING (also known as MITA) (99, 100), which is a key adaptor molecule in the cellular response to virus infection and in establishing the basal set-point of IRF3 signaling and IFN-α/β production (101). DENV NS5 protein induces targeted degradation of STAT2 via the proteasome (102–105) and NS4B blocks STAT1 activation (102, 106) and thus inhibits IFN-α/β and likely IFN-γ and IFN-λ receptor signaling in DENV-infected cells. However, non-infected cells remain capable of IFN production and IFNAR signaling, which may induce resistance to subsequent DENV infection. Indeed, pretreatment of cells with IFN-α/β or IFN-γ prevents DENV infection (107–109).

Antibody-enhanced DENV infection also is believed to contribute to the evasion of the innate immune response through a mechanism termed "intrinsic ADE" (110). A monocytic cell line, THP-1, showed decreased production of inflammatory cytokines and mediators, such as IL-12, IFN-γ, TNFα, and nitric oxide radicals when they were infected with DENV in the presence but not in the absence of enhancing antibodies (111). Similarly, ADE infection suppressed TLR-mediated signals and the secretion of IFN-β, but increased the production of anti-inflammatory cytokines, such as IL-10, compared to DENV infection without antibodies (112). These effects depended on FcγR binding because antibodies blocking FcγRI or FcγRIIA restored IFN-β production (112). These findings were unexpected because activating FcγR signals should induce expression of ISGs and thus block DENV replication. Recent studies showed that the leukocyte immunoglobulin-like receptor-B1 binds DENV-antibody aggregates and blocks activating FcγR signals, which enabled DENV to evade the early antiviral response during ADE (113). These results suggest that antibody-mediated DENV entry also triggers intracellular signals that suppress innate responses in infected cells to increase viral production. This "intrinsic ADE" is complemented by "extrinsic ADE," which refers to the enhanced FcγR-mediated binding and uptake of DENV described above.

## **PRIMING AND EVASION OF ADAPTIVE IMMUNE RESPONSES**

Dendritic cells link innate and adaptive immune responses by integrating innate signals from PAMPs with pathogen-derived antigens to induce antigen-specific T-cell and B-cell responses. DCs achieve this by (a) taking up and processing antigen and presenting antigen-derived peptides on MHC I to CD8<sup>+</sup> T-cells or on MHC II to CD4<sup>+</sup> T-cells; (b) expressing co-stimulatory molecules that activate T-cells; and (c) secreting chemokines and cytokines that attract T-cells and modulate the priming T-cell effector functions.

Because DC survival is required for optimal T-cell activation, DENV-induced apoptosis of DCs could antagonize the priming of immune responses. Bulk culture studies observed increased survival of moDCs (50, 114) and monocytes (115) after exposure to DENV. However, intracellular staining studies revealed a higher fraction of Annexin V<sup>+</sup> apoptotic cells in those co-staining for DENV E protein (116). These results suggest that DENV induces apoptosis in infected cells, but increases survival in non-infected bystander cells. The impact of cell survival on the number of antigen-presenting cells and priming of DENV-specific adaptive immune responses warrants further study.

Pathogen recognition leads to DC maturation, which is characterized by increased expression of MHC II and co-stimulatory markers required for efficient priming of T-cell responses (117). After exposure to DENV, non-infected bystander moDCs upregulate MHC I and II molecules, as well as co-stimulatory molecules CD80 (B7-1), CD83, and CD86 (B7-2), although DENV blocks activation and maturation of infected moDCs within the same cultures (59, 116) (**Figure 2A**). Intracellular staining for DENV proteins revealed a block in activation of DENV-infected moDCs that was not observed in bulk culture (50, 59). Similarly, non-infected bystander monocytes, moDCs and cDCs, expressed higher levels of CD80 and CD86 than DENV-infected cells in the dermis of intradermally infected *Ifnar*−/<sup>−</sup> mice (53). In addition, DCs produce cytokines and chemokines to modulate T-cell responses. DENV-exposed moDCs (50, 59, 116) or cDCs (61), produce IL-6, IL-10, TNFα, and IFN-α. Furthermore, DENV-exposed moDCs secrete CXCL9, CXCL10, and CXCL11 (118) that bind the chemokine receptor CXCR3 and could attract effector and memory T-cells. However, it remains unclear whether it is the DENV-infected or non-infected DCs that produce these inflammatory mediators. Together, these data suggest that DENV blocks activation in infected DCs, which may decrease the priming of CD4<sup>+</sup> or CD8<sup>+</sup> T-cells, whereas non-infected bystander cells still can become activated.

Mixed lymphocyte reactions (MLR) are a functional read-out for DC–T-cell interactions, in which DCs from MHC mismatched donors activate allogeneic T-cells. DENV infection decreased the capacity of moDCs (116, 119) as well as DCs isolated from human skin explants (65) to stimulate proliferation of DENV-naïve CD4<sup>+</sup> T-cells in MLRs *in vitro*, suggesting that DENV-infected DCs are less capable of activating CD4<sup>+</sup> T-cells (**Figure 2B**). Others have reported that DENV-infected cultures of moDCs can prime CD4<sup>+</sup> T-cells, but with decreased T-cell effector functions, such as secretion of IFN-γ or TNFα (109). The impaired ability of DENVinfected moDCs to produce IFN-α and IFN-β may explain the decreased ability to prime T-cell responses (120). Although these surrogate MLR assays are interesting, little information exists as to how DCs prime DENV-specific T-cells. Non-infected moDCs that were pulsed with DENV E protein efficiently activated CD4<sup>+</sup> or CD8<sup>+</sup> CD45RO<sup>+</sup> memory T-cells from DENV-immune but not from naïve individuals to produce IFN-γ (121). More study

bystander DCs efficiently prime adaptive T-cell responses, whereas DENV-infected DCs prime naïve T-cells less efficiently. **(C)** Activated T-cells display CD40L on their surface or secrete CD40L that acts on DENV-infected DCs and can restore DC maturation and function. Purple denotes DENV infection, while orange denotes inflammation/cell activation.

is needed to determine the capacity of DCs to prime DENVspecific naïve T-cells or to reactivate memory T-cells during reexposure. Rapid progress should be possible given the publication of large numbers of HLA-restricted immunodominant DENV antigens (29, 31).

Also, activated T-cells can support the maturation of DCs. An initial study showed that co-culture of moDCs with a CD40Ltransfected cell line restored the ability of DENV-infected moDCs to induce MLR responses (114) (**Figure 2C**). Subsequent work demonstrated that co-culture of activated T-cells with DENVinfected moDCs rescued the otherwise suppressed MLR response, and the activation of non-infected bystander moDCs depended on TNFα and IFN-α/β (118). Consequently, stimulation of the adaptive response to DENV infection requires signals from both cells in DC–T-cell interactions, likely via cell-to-cell contact. In clinical studies, gene expression analysis of acute dengue patients revealed that DHF cases expressed lower levels of genes linked to antigen processing, presentation, and T-cell activation compared to DF patients (122). Thus, impaired antigen presentation and functionality of DENV-infected DCs may reflect a viral immune escape strategy to dampen T-cell responses and impact disease severity.

To date, mostly *in vitro*-generated human moDCs have been used to study the role of DCs in inducing innate or adaptive immune responses during DENV infection. Nevertheless, diverse subsets of cDCs in lymphoid or non-lymphoid tissues execute specialized functions in presenting antigen and inducing CD4<sup>+</sup> or CD8<sup>+</sup> T-cell responses (123, 124). Expanding previous findings using human cDC subsets directly isolated from blood or tissues will be important to characterize the full spectrum of immune responses to DENV infection.

## **CONCLUDING REMARKS**

Dengue virus infects the same cells (DCs, monocytes, and MΦs) that are essential for inducing and maintaining optimal innate and adaptive immune responses. This tropism of DENV appears to impair DC function, which may undermine the priming of DENV-specific memory responses. Is it possible to block DENV replication in DCs to reduce viral load and restore DC function, which could impact the generation of neutralizing or potentially harmful memory B- and T-cell responses? Can we as a field harness the knowledge gained about DC biology during DENV infection to prevent human disease? Can DC function be optimized in the context of live-attenuated DENV vaccines to stimulate protective immunity?

Significant progress has been made on characterizing DENV infection and activation of DCs, as well as protective or enhancing T-cell and B-cell responses. Nevertheless, early events after DENV transmission in the skin require further study, including a greater understanding of early virus replication, local immune responses at the site of transmission (i.e., the skin), and immunomodulatory effects of mosquito saliva. Further effort should focus on how responses of DCs impact disease outcome in an acute infection and prime immunological memory responses that will affect dengue pathogenesis and disease severity in secondary DENV infections. A collaborative effort using a multi-disciplinary approach among experts in dengue virology, medicine, vector biology, and immunology is called for to reach these goals.

## **ACKNOWLEDGMENTS**

This work was supported by funds from the National Institute of Allergy and Infectious Diseases, National Institutes of Health [grant R01-AI085607 (Eva Harris) and R01-AI077955 (Michael S. Diamond)], and the German Research Foundation,*Deutsche Forschungsgemeinschaft* [research fellowship SCHM 3011/1-1 (Michael A. Schmid)]. The authors declare no competing financial interests.

## **REFERENCES**

infection in mice, whereas T and B cell-dependent immunity is less critical. *J Virol* (2004) **78**:2701–10. doi:10.1128/JVI.78.6.2701-2710.2004

124. Haniffa M, Collin M, Ginhoux F. Identification of human tissue crosspresenting dendritic cells: a new target for cancer vaccines. *Oncoimmunology* (2013) **2**(3):e23140. doi:10.4161/onci.23140

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Received: 09 October 2014; paper pending published: 05 November 2014; accepted: 04 December 2014; published online: 17 December 2014.*

*Citation: Schmid MA, Diamond MS and Harris E (2014) Dendritic cells in dengue virus infection: targets of virus replication and mediators of immunity. Front. Immunol. 5:647. doi: 10.3389/fimmu.2014.00647*

*This article was submitted to Microbial Immunology, a section of the journal Frontiers in Immunology.*

*Copyright © 2014 Schmid, Diamond and Harris. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

## Utility, limitations, and future of non-human primates for dengue research and vaccine development

## **Carlos A. Sariol 1,2 and Laura J.White<sup>3</sup>\***

<sup>1</sup> Department of Microbiology and Medical Zoology, Caribbean Primate Research Center, University of Puerto Rico-Medical Sciences Campus, San Juan, PR, USA

<sup>2</sup> Department of Internal Medicine, Caribbean Primate Research Center, University of Puerto Rico-Medical Sciences Campus, San Juan, PR, USA

<sup>3</sup> Global Vaccine Incorporation, Research Triangle Park, NC, USA

#### **Edited by:**

Scott B. Halstead, International Vaccine Institute, South Korea

#### **Reviewed by:**

George W. Anderson, Lawrence Livermore National Laboratory, USA Leah Corinne Katzelnick, University of Cambridge, UK

Subhash Vasudevan, Duke-NUS Graduate Medical School, Singapore

#### **\*Correspondence:**

Laura J. White, Global Vaccines Incorporation, PO Box 14827, 801-11 Capitola Dr., Durham, Research Triangle Park, NC 27713-4827, USA e-mail<EMAIL_ADDRESS>
Dengue is considered the most important emerging, human arboviruses, with worldwide distribution in the tropics. Unfortunately, there are no licensed dengue vaccines available or specific anti-viral drugs.The development of a dengue vaccine faces unique challenges.The four serotypes co-circulate in endemic areas, and pre-existing immunity to one serotype does not protect against infection with other serotypes, and actually may enhance severity of disease. One foremost constraint to test the efficacy of a dengue vaccine is the lack of an animal model that adequately recapitulates the clinical manifestations of a dengue infection in humans. In spite of this limitation, non-human primates (NHP) are considered the best available animal model to evaluate dengue vaccine candidates due to their genetic relatedness to humans and their ability to develop a viremia upon infection and a robust immune response similar to that in humans. Therefore, most dengue vaccines candidates are tested in primates before going into clinical trials. In this article, we present a comprehensive review of published studies on dengue vaccine evaluations using the NHP model, and discuss critical parameters affecting the usefulness of the model. In the light of recent clinical data, we assess the ability of the NHP model to predict immunological parameters of vaccine performances in humans and discuss parameters that should be further examined as potential correlates of protection. Finally, we propose some guidelines toward a more standardized use of the model to maximize its usefulness and to better compare the performance of vaccine candidates from different research groups.

**Keywords: dengue, macaques, non-human primates, vaccine, genetic, neutralizing antibodies, cell-mediated immunity, animal model**

## **CURRENT STATE OF THE NHP MODEL TO EVALUATE DENGUE VACCINE CANDIDATES, USEFULNESS AND LIMITATIONS**

Non-human primates (NHP) have been used to model a number of human infections and diseases, based on their genetic, physiological, and immune similarities with humans. In the case of dengue, NHP are the only natural vertebrate hosts besides humans susceptible to infection with the four serotypes of dengue viruses (DENV1–4). A number of species of NHP are infected in the wild by sylvatic DENV strains and experimentally by human clinical isolates without the need for virus adaptation [reviewed in Ref. (1, 2)]. A major limitation of this model for the study of dengue pathogenesis and vaccine efficacy evaluations is that the outcome of infection is subclinical in most instances (3), and does not recapitulate the symptoms seen in patients with dengue fever (DF) and dengue hemorrhagic fever and shock syndrome (DHF/DSS). Nevertheless, important similarities make it the best available model to evaluate vaccine immunogenicity and protective efficacy. Virus replication in NHP results in a peripheral viremia, with similar onset and duration to that reported in humans, although lower in magnitude. The induction of a robust neutralizing antibody (NAb) response and cellular immune responses also parallels the human immune response to dengue infection.

Two comprehensive reviews have been published recently on NHP as a potential model for dengue pathogenesis (2) and NHP infected with dengue in natural settings (1). Therefore, those topics will not be included here.