Respiratory Syncytial Virus: Pathology, therapeutic drugs and prophylaxis

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(1)Immunology Letters 162 (2014) 237–247. Contents lists available at ScienceDirect. Immunology Letters journal homepage: www.elsevier.com/locate/immlet. Review. Respiratory Syncytial Virus: Pathology, therapeutic drugs and prophylaxis Roberto S. Gomez a,c , Isabelle Guisle-Marsollier c , Karen Bohmwald a , Susan M. Bueno a,c , Alexis M. Kalergis a,b,c,∗ a. Millennium Institute of Immunology and Immunotherapy, Departamento de Genética Molecular y Microbiología, Facultad de Ciencias Biológicas, Chile Departamento de Reumatología, Facultad de Medicina, Pontificia Universidad Católica de Chile, Chile c INSERM U1064, Nantes, France b. a r t i c l e. i n f o. Article history: Received 20 June 2014 Received in revised form 21 August 2014 Accepted 8 September 2014 Available online 28 September 2014 Keywords: Immunity Prophylaxis Human Respiratory Syncytial Virus Infection Therapeutics Vaccine. a b s t r a c t Human Respiratory Syncytial Virus (hRSV) is the leading cause of lower respiratory tract diseases, affecting particularly newborns and young children. This virus is able to modulate the immune response, generating a pro-inflammatory environment in the airways that causes obstruction and pulmonary alterations in the infected host. To date, no vaccines are available for human use and the first vaccine that reached clinical trials produced an enhanced hRSV-associated pathology 50 years ago, resulting in the death of two children. Currently, only two therapeutic approaches have been used to treat hRSV infection in high risk children: 1. Palivizumab, a humanized antibody against the F glycoprotein that reduces to half the number of hospitalized cases and 2. Ribavirin, which fails to have a significant therapeutic effect. A major caveat for these approaches is their high economical cost, which highlights the need of new and affordable therapeutic or prophylactic tools to treat or prevents hRSV infection. Accordingly, several efforts are in progress to understand the hRSV-associated pathology and to characterize the immune response elicited by this virus. Currently, preclinical and clinical trials are being conducted to evaluate safety and efficacy of several drugs and vaccines, which have shown promising results. In this article, we discuss the most important advances in the development of drugs and vaccines, which could eventually lead to better strategies to treat or prevent the detrimental inflammation triggered by hRSV infection. © 2014 Elsevier B.V. All rights reserved.. 1. Introduction Human Respiratory Syncytial Virus (hRSV) is the major cause of bronchiolitis and lower tract illness, affecting nearly 70% of infants before the age of one year and approximately 100% of children by age of two [1–3]. Approximately 30 million of children younger than five years old suffer from acute lower respiratory infection due to hRSV, out of which 10% require hospitalization [4,5]. HRSV causes approximately 200,000 deaths per year, most of the cases take place in developing countries and include mainly children younger than 5 years of age [4]. This virus causes wide complications to premature born patients as well as infants suffering of congenital heart disease and immune deficiency [6,7]. Further,. ∗ Corresponding author at: Dr. Alexis Kalergis Millennium Institute of Immunology and Immunotherapy, Departamento de Genética Molecular y Microbiología, Facultad de Ciencias Biológicas, Pontificia Universidad Católica de Chile, Alameda #340, Santiago E-8331010, Chile. Tel.: +56 2 686 2842; fax: +56 2 222 5515. E-mail addresses: akalergis@bio.puc.cl, akalergis@icloud.com (A.M. Kalergis).. 2% of young children infected with this virus and hospitalized due severe bronchiolitis show symptoms of central nervous system (CNS) alterations, such as seizures, central apnea and encephalopathy, among others [4,8–12]. Recently, an increase in the number of cases of encephalopathy associated to hRSV infection has been described [12]. Further, a recent study has shown that hRSV can be detected in the CNS of infected animals 30 days after infection causing significantly reduced performance in learning and behavioral tests [13]. Therefore, it is a high priority worldwide to generate either a vaccine against hRSV to prevent the respiratory disease or new therapeutic drugs to treat severe infection and reduce the potential long-term effects caused by infection with this virus. Up to date, therapeutic treatments for hRSV infection have consisted of antiviral molecules. One of the most successful treatments has been a humanized monoclonal antibody against hRSV F glycoprotein known as Palivizumab [14,15]. This neutralizing antibody was shown to be able to reduce hRSV-associated hospitalization rates by 55%, as compared to placebo [16,17]. However, monthly antibody re-administration is required to prevent viral dissemination and this passive immunization is not effective for. http://dx.doi.org/10.1016/j.imlet.2014.09.006 0165-2478/© 2014 Elsevier B.V. All rights reserved.. Downloaded from ClinicalKey.com at University of Chile Catholic ALERTA May 16, 2016. For personal use only. No other uses without permission. Copyright ©2016. Elsevier Inc. All rights reserved..

(2) 238. R.S. Gomez et al. / Immunology Letters 162 (2014) 237–247. all young children [18,19]. Further, due to its high costs, in most countries Palivizumab is prescribed only for high-risk children. Only few years after the virus was identified, a formalininactivated hRSV preparation (FI-hRSV) was used in a field trial during the mid-1960s as an initial attempt to vaccinate against the virus. Unfortunately, after natural infection with hRSV, children that were immunized with this formulation showed an exacerbated pulmonary disease and suffered more severe symptoms than unvaccinated children [20]. Data explaining the failure of FI-hRSV as a vaccine were obtained after decades of research and suggested that immunization with this formalin-inactivated hRSV vaccine promoted an allergic-like response in the lungs [21,22]. In animal models of hRSV infection, such as cotton rats, immunization with inactivated virus followed by challenge with infective hRSV also induces eosinophil and neutrophil infiltration to the airways [23,24]. Further, deposition of immune complexes and complement activation is another characteristic observed in both animal models and infected patients [25], a feature that seems to be exacerbated by immunization with formalin-inactivated hRSV. This is due to the establishment of unbalanced Th1 –Th2 polarized responses, which are characterized by the secretion of pro-inflammatory cytokines that drive excessive infiltration of eosinophils and neutrophils into the lungs [26]. It is likely that modification of viral epitopes by formalin and the development of low affinity antibodies against modified hRSV antigens could have contributed to the damaging immune response triggered by this early vaccine approach [24]. After the above-mentioned FI-hRSV vaccine failed, several new strategies aiming at inducing protective immunity against hRSV infection have arisen during the past 50 years. However, to date no efficient and affordable products are available for public health systems to counteract the disease. In this review we will discuss the most recent knowledge about the pathology caused by hRSV and the experimental drugs and vaccine developed up to date as an attempt to prevent or treat hRSV infection in humans.. 2. Molecular characteristics of hRSV hRSV was first isolated at the end of the 50s and its name is due to its property to generate syncytia on infected cells in culture [27–29]. HRSV belongs to the Mononegavirales order, Paramixoviridae family and pneumovirus genus. Other members of this family are measles (MeV), mumps (MuV) and human metapneumovirus (hMPV), among others [30]. HRSV consists of an enveloped nonsegmented, negative-sensed and single-stranded RNA genome of 15.2 kb, which has 10 genes that are transcribed in 10 different monocistronic messenger RNAs. However, the M2 mRNA is translated into two different proteins, namely M2-1 and M2-2, obtained by the termination-dependent re-initiation mechanism [31]. Thus, the hRSV genomes encodes for 11 proteins [32,33], out of which eight are structural proteins present both in infected cells and in the virions [32,34,35]. The disulfide-bonded glycoprotein (F) and the large glycoprotein (G) are envelope proteins that constitute the major antigenic determinants of the virus and induce protective antibodies [32,34,35]. While the G glycoprotein mediates viral attachment, the F glycoprotein mediates viral penetration and syncytium formation. The small hydrophobic protein (SH), also at the virus envelope, was recently postulated to be a viroporin-like ion channel, interfering with the normal function of the infected cell [36]. The matrix protein (M) is found underneath the envelope, which is a non-glycosylated protein involved in the assembly of the viral particle [32,34,37]. The nucleoprotein (N), the phosphoprotein (P), the transcription factor M2-1 and the large RNA-dependent RNA-polymerase (L) are found in an inner nucleocapsid that wraps the genomic RNA [38]. The NS1 and NS2 are non-structural proteins that are found only in infected cells but not in virions [39].. Transmission of hRSV takes place through inhalation of infectious droplets or through their direct contact with ocular mucosa [40,41]. The principal cellular targets of this virus are epithelial cells in the respiratory tract, where the virus replicates to produce new infectious viral particles [42]. Once the virus reaches the airways the infection starts with the viral attachment to the host cell, mediated by the G glycoprotein and then the interaction of the F glycoprotein with their specific receptor nucleolin in lung epithelial cells [43–45]. This process triggers the fusion of the viral envelope to the cell plasma membrane and the entry into cell cytoplasm [26,45]. Subsequently, viral nucleocapsid is released into the cellular cytoplasm, where transcription and replication of the viral genome begins [32,45]. The first step of hRSV replication is the synthesis of a complementary, polycistronics ssRNA (+) antigenome. This molecule serves as a template for the synthesis of new fulllength ssRNA (−) genomes. These two processes are modulated by the M2-2 protein [34,35].. 3. Respiratory pathology caused by hRSV infection The clinical manifestations of the pathology caused by hRSV are common to other respiratory viral infections. The first symptoms of infection are rhinitis, cough, fever and nasal congestion [32,35,46]. The severity of the illness is due both to host and viral factors. Prematurity, low birth weight, cardiopulmonary disease, bronchopulmonary dysplasia and immunodeficiency are the most common host risk factors that account for severe bronchiolitis caused by hRSV [47,48]. Infants under one year of age manifest less-specific symptoms, such as periodic breathing, apnea and feeding difficulties [2]. Young infants that have been hospitalized and required mechanic ventilation are in higher risk to developing asthma later during childhood and even in adulthood [49]. Also, it has been suggested that asthma is a factor that predisposes to severe symptoms after hRSV infection [48]. Clinical studies have indicated that hRSV pathology is mediated by inflammatory cells due to the induction of an exacerbated Th2 cellular response [2,49], which impairs viral clearance and reduces cytotoxic T cells activity [48,50–54]. In agreement with this notion, healthy hRSV-infected patients commonly show increased airway obstruction, including alveolar sparing, in contrast to immunocompromised individuals. These latter groups includes a higher frequency of patients with progressive pneumonia, with cellular and fluid infiltration on alveoli, but show reduced wheezing [48,55,56]. Moreover, an association has been described between hRSV hospitalization and later development of asthma [57]. A recent study has shown that repeated hRSV infection in early life can impair maternally transferred tolerance by hampering regulatory T cells (Tregs) function conferring a Th2 -like phenotype to these cells. It is thought that these effects can lead to the loss of tolerance to allergens, which could explain as to how hRSV infection can promote the subsequent asthma development [58]. Although the main sign of hRSV pathology is associated to airway inflammation, recently it has been described that neurological symptoms affect approximately 2% of patients hospitalized with severe bronchiolitis. These symptoms include apnea, seizures, lethargy and encephalopathy [10,11,59,60]. Further, dissemination of hRSV to the brain has been supported by the presence of hRSV proteins and mRNA in several regions of the brain, including cortex and hippocampus [10,13]. The mechanism involved in the infection of the CNS remains unknown, however infection of these regions could result from infected leukocytes going across the blood–brain barrier (BBB) and carrying viral particles. In rodents, learning and behavior disabilities have been evidenced and supported by neurophysiological experiments, showing impaired synaptic plasticity 30 days or more after hRSV infection [13]. Recently an increase in the. Downloaded from ClinicalKey.com at University of Chile Catholic ALERTA May 16, 2016. For personal use only. No other uses without permission. Copyright ©2016. Elsevier Inc. All rights reserved..

(3) R.S. Gomez et al. / Immunology Letters 162 (2014) 237–247. number of encephalopathies has been reported, highlighting the importance of developing a cost/effective hRSV prophylaxis [9,61].. 4. HRSV and host immune response HRSV infection triggers the secretion of several proinflammatory molecules that initiate the immune response against this virus (Fig. 1) [45]. The immune response due to the viral infection is characterized by an exacerbated inflammation and weak T cell immunity [26,45,62]. The most commonly secreted molecules by epithelial cells in response to hRSV infection are IL-6, IL-8/CXCL8, IL-10, TNF-␣, RANTES/CCL5, MCP-1/CCL2, MIP-1␣/CCL3, MIP-1␤/CCL4, MIP-2/CXCL2, IP-10/CXCL10 and eotaxin-1/CCL11 [63–68]. This initial response contributes significantly to the inflammation, promoting the migration of immune cells, such as eosinophils, neutrophils and monocytes into the lungs, which are in turn induced to produce additional proinflammatory molecules [62,67]. These inflammatory elements fail to achieve viral clearance and contribute to the persistence of the virus in the airways, leading to tissue damage [62,63,67,69]. Inflammatory molecules that are secreted by the infected epithelium promote the recruitment of cells from the innate immune response, such as macrophages and dendritic cells (DCs) [26,62]. Although these cells are not permissive for viral replication, they can be infected by hRSV [70,71]. The infection of these phagocytic cells results in the production of additional inflammatory cytokines, such as IL-6, IL-10, CCL5, CXCL8, CCL3, CCL4 [72,73]. Myeloid peroxidase (MPO) is another protein produced in significant high amounts by these cells after hRSV infection. Macrophages do not play a relevant role in the development of the adaptive immune response against hRSV, since a depletion of these cells does not prevent the recruitment of CD4+ and CD8+ T cells after infection [74]. However, a deficiency in alveolar macrophage function and numbers caused an increased of airway obstruction and viral loads in murine models [75]. These observations suggest that macrophages could significantly contribute to the innate immune response and the severity of hRSV bronchiolitis. Furthermore, other studies have shown that the immature state of macrophages in neonates is deleterious to viral clearance, since administration of IFN-␥ in neonatal BALB/c mice promotes the differentiation of immature macrophages into activated cells, restoring their ability for intracellular killing, pro-inflammatory cytokine secretion and for initiating the adaptive immune response [76]. During the adaptive immune response, hRSV-specific IgG1 production by B cells provides weak protection against the virus in animal models [77], probably because these antibodies lead to a Th2 polarized immune response. Neonates younger than 3 months old show a less diversified B cell repertoire against hRSV, which provides an explanation for the severity of disease in young infants and the lack of protection mediated by virus-specific B cells [78]. Nevertheless, a strong IgA memory response can be induced in BALB/c mice challenged with hRSV, showing a potential approach to be considered for producing protective immunity [79]. Another described effect of hRSV infection in B cells is the overexpression of activation markers, such as MHC class II and CD86, independently of TLR4 signaling [80,81]. On the other hand, DCs infection is thought to be a crucial step during hRSV immunopathogenesis that accounts for an altered activation of the adaptive immune response. It has been described that hRSV is able to subvert the capacity of DCs to activate naïve T cells [70,82]. Further, inhibition of T cell activation is not due to soluble molecules produced by hRSV-infected DCs, but on the contrary, it seems to require cell-cell contact between T cells and infected DCs [82]. These data suggest that inhibition of CD4+ T cell activation. 239. by hRSV-infected DCs is contact-dependent and that the assembly immunological synapses between these cells may be impaired by hRSV [82]. DCs are fundamental for the development of an effective adaptive immune response and their activation leads to the proliferation and differentiation of effector and memory T and B cells [83–87]. Several pathogenic viruses have evolved molecular strategies to impair the function of DCs and modulate host adaptive immunity to survive and disseminate to other hosts [88]. HRSV is one of such viruses that impairs the function of DCs and promotes a Th2 immune response [89–91]. After hRSV infection, T cells failed to up-regulate the production of granzyme B, perforin and IFN-␥ and to down modulate TCR surface expression [92,93]. Further, administration of IL-2 in vivo restored T cell effector activity and memory function, corroborating that T cells can be targeted by hRSV virulence mechanisms during infection [90]. HRSV also induces a strong Th2 response in the host and promotes the recruitment of pro-inflammatory cells, such as eosinophils, neutrophils and monocytes, as well as the production of IL-4, IL-5 and IL-13. This combination of immune cells and cytokines dampens CD8+ T cell immunity and prevents virus clearance [91,93]. In summary, all these studies suggest that a balanced Th1 /Th2 immune response is required to reduce hRSV replication and lung pathology. Such concepts have been strengthened by data obtained from studies of vaccination and subsequent hRSV challenge in animal models [94–99]. Reinfection with hRSV is one of the main features of the pathology caused by this virus and correlates directly with a weak immunity induced after infection [100]. As mentioned above, the polarization an adaptive immune response toward inflammation is determined by the nature of antibodies generated during hRSV infection [90,92,93]. Despite that a memory response is generated against hRSV, it is not long-term, fails to clear the virus and allows repeated reinfection episodes throughout life, especially in young children [35,100,101]. Along these lines, 43% and 73% of adults naturally infected by hRSV and exposed 2–26 months later with the same virus strain (no antigenic change) showed more than three or two reinfections, respectively [100]. However, individual with high titer of neutralizing anti-hRSV antibodies have reduced possibilities of reinfection. Because hRSV infects approximately 70% of children by age of one, it is important to understand how the immune system of the host can contribute to generating protective immunity against infection. However, newborns have reduced efficiencies at triggering immune response as compared to older individuals. Consistent with this notion is the observation that a group of children between 4 and 8 months of life can show 8- to 10-fold lower neutralizing antibodies than do older children (9–21 months) after primary hRSV infection [102]. These differences could be explained by the lack of maturity of the immune responses shown by young children, characterized by biased innate, humoral and cellular response [103,104]. In fact, hRSV-specific B cells from very young infants display reduced somatic mutations, which explains the poor antibody response shown by these children [78]. This insufficient immune variability in neonates is an important point to considerer in the development of vaccines against hRSV, as the vaccination can promote certain type of immunity in young children that could be not the best against the virus [103,104]. Because regulatory T cells were found to be enriched in hRSVinfected lungs [105], the first thought was that this virus was exploiting these cells to down modulate the cytotoxic capacity of effector T cells [106]. However, recent studies in mice depleted from regulatory T cells with an anti-CD25 antibody showed that regulatory T cells play a beneficial role for the host, because they are able to regulate lung inflammation by down modulating the secretion of pro-inflammatory cytokines and promote an efficient adaptive. Downloaded from ClinicalKey.com at University of Chile Catholic ALERTA May 16, 2016. For personal use only. No other uses without permission. Copyright ©2016. Elsevier Inc. All rights reserved..

(4) 240. R.S. Gomez et al. / Immunology Letters 162 (2014) 237–247. Fig. 1. Innate and Adaptive response against Respiratory Syncytial Virus. (1) After hRSV infects the airways, lung epithelial cells start to secrete inflammatory signals, such different cytokines and chemokines. (2) Cytokines and Chemokines are able to attack and activate neutrophils, monocytes and eosinophils, generating the recruitments of these cells into the airways. (3) Alveolar macrophages play an important role decreasing the viral loads in the lungs. (4) In the border between innate and adaptive response are DCs, that are infected by the virus and are able to migrate to the lymph nodes, where impair the T cell activation. (5) hRSV infection triggers an inefficient T cells response for virus clearance, promoting a Th2 response and decreasing the effectiveness to kill infected cells, due to down-regulation of important proteins such as Granzyme B and Perforin. (6) B cells, after hRSV infection, generate non-protective antibodies, which normally are IgG1 that promote a Th2 response and non-neutralizating antibodies. (7) Tregs play an important role promoting the functionality of hRSV-specific CD8+ T cells.. immune response [107]. Consistently, another model for regulatory T cells depletion (DEREG mice), showed that the absence of these cells decreased viral loads after infection but increased lung infiltration with inflammatory cells, such as macrophages, neutrophil and eosinophils. These data suggest a protective role for regulatory T cells during acute hRSV pathology, which seem to be mediated by Granzyme B [108]. Absence of regulatory T cells led to an increase in the frequency and activation of T cells, especially of those expressing IL-13 and GATA-3, two major markers of Th2 polarized responses [109]. Furthermore, the frequency of circulating regulatory T cells decrease in hRSV-infected children, probably due to the migration of these cells to the lungs or the draining lymph nodes [110]. Due to the protective capacity of regulatory T cells during hRSV pathology, a vaccine capable of promoting the recruitment of these cells to the lungs to suppress the T cell inflammatory response triggered by the virus. Consistently with this notion, immunization with FI-hRSV vaccine causes a decrease on regulatory T cells in the airways. Lack of protection by the vaccine could be due to the inability to sustain a strong regulatory T cell response in the lungs, which seems to be required to prevent the damage caused by hRSV to the host [111]. 5. Development of therapeutic drugs and vaccine against hRSV Although several drugs have been shown to reduce hRSV infection, currently only Rivabirin is allowed for human use. However the use of this molecule remains controversial due to concerns relative to cost/efficacy issues, as well as potential side effects [112,113]. Drugs have been developed to target several steps of the infectious cycle of virus, such as entry, replication and transcription (Fig. 2). These approaches can be significant as they can directly impair the viral infective cycle, without compromising the host immune response. These features are particularly important for the treatment of newborns due to the impaired capacity to trigger an efficient immune response against hRSV. Based on this. reason, drugs that can directly inhibit virus spreading are likely to become high potential prophylactic candidates. A considerable number of drugs have been tested, which targeted either viral entry events, hRSV assembly and function [114–119]. For the targeting of viral entry, several strategies have been explored: molecules that neutralize the fusion of viral protein with the host by blocking interaction between host cell receptors and viral proteins and drugs that destroy the conformation of the viral F glycoprotein [119]. The fact that none of these molecules has reached clinical testing in humans highlights the complexity of designing drugs that efficiently target hRSV fusion with host cells [114,115,117,118]. An efficient combination of inhibitory drugs could provide a more powerful method for treating the pathology associated to hRSV, although this strategy has not been evaluated yet. “De novo” pyrimidine biosynthesis pathway has been identified as a potential target for new hRSV antiviral drugs. Molecules targeting this pathway, such as leflunomide, have shown promising results by reducing inflammation in the lungs in vivo [116]. The development of immunization strategies started in the 60s with the FI-hRSV vaccine, which not only failed to confer immunity against hRSV, but exacerbated detrimental inflammation in vaccinated children [25]. Several attempts have been made at generating an efficient and safe vaccine formulation that induces protective hRSV immunity and keeps the host from suffering the enhanced inflammatory damage caused by the virus in airways (Table 1). One of the most successful and common strategies used for developing vaccines is the attenuation of microbe virulence, which reduces pathogenicity to a minimum but retains most of the microbe antigenicity and immunogenicity. Among the approaches for pathogen attenuation, replication restriction by temperature has been widely evaluated for hRSV [120,121]. A recently strategy to inactivate the virus and induce immunity consisted on the use of nanoemulsions that are capable to disrupting the viral envelope [122]. This formulation prevented enhanced disease due to hRSV infection and promoted virus-specific humoral and cellular responses. High levels of IgG and IgA, with absence of IgE and Th1 /Th17. Downloaded from ClinicalKey.com at University of Chile Catholic ALERTA May 16, 2016. For personal use only. No other uses without permission. Copyright ©2016. Elsevier Inc. All rights reserved..

(5) R.S. Gomez et al. / Immunology Letters 162 (2014) 237–247. 241. Table 1 Strategies used to generate RSV vaccines. Vaccine model. Vaccine name. Administration route. Model evaluated. Evaluation time. Effects after vaccination. Evaluation of enhanced diseasea. Reference. Atennuated virus. cpts-248/404. Intranasal. Human. No information. Not evaluated. [153]. rA2cp248/404/ 1030SH (MEDI-559). Intranasal. Human. No information. Upper respiratory tract congestion Generation of neutralizing antibodies. Non-enhanced disease after natural infection with hRSVb. [121,154]. Bacterium-like particle (BLP-F). Intranasal. Balb/c mice Cotton rats. up to 4 weeks after last immunization. Increase on IgG2a and IgA antibodies Reduction of viral titer after challenge. [126]. AdF.RGD. Intramuscular. Balb/c mice. 4–5 weeks after immunization. Increase on hRSV antibody titer Reduction of viral titer after challenge. PR8/NA-F85–93. Intranasal. Balb/c mice. 10 days. Virus like particle (VLP-F and VLP-G) F-TriAdj. Intramuscular. Balb/c mice. Not evaluated. [133]. Intranasal or Intramuscular. Balb/c mice Cotton rats. 3 weeks after last immunization 3 weeks until 1 year after last immunization. Intramuscular. Cotton rats Human. 4 weeks after last immunization. Low sign of alveolitis Reduction of lung viral titer High virus neutralization titers No sign of inflammatory response in the lungs. [134–136]. F-Nanoparticles. F + Alum. Intramuscular. Balb/c mice. 2 weeks after last immunization. Increase on hRSV specific CTL cells Reduction of viral titer after challenge Increase in serum and lung IgG Low lung viral titer Increase in lung neutralizing antibodies and IgA titer in the lungs Generation of memory T cells and hRSV-specific T cells Increase in neutralizing antibodies Low lung viral titer Generation of palivizumab competing antibodies High levels of hRSV-specific antibodies in young and old mice Reduction on Th2 cytokines production. High virus neutralization titers Reduction of lung viral titer No sign of pulmonary pathology No sign of inflammatory response Reduction of lung viral titer No sign of pulmonary pathology Not evaluated. Not evaluated. [139]. Target: N protein. BCG-N. Subdermal. Balb/c mice. 3 weeks after immunization. Th1 response polarization on T cells. Not evaluated. [99,147]. Target: G protein. rhRSV and G-hRSV. Intranasal. Cotton rats. 21 weeks after immunization. High serum hRSV antibody titers Low lung viral titer. [148]. GNanoparticles. Subcutaneous. Balb/c mice. 3 weeks until 6 weeks after last immunization. High serum antibody titer and neutralizing antibodies Minor airway inflammation and low viral titer. No sign of inflammatory response in the lungs Not evaluated. Target: M2 protein. TriVax. Intravenous o Intraperitonal. Balb/c mice. 6 days until 7 weeks after last immunization. Induction of hRSV-specific CTLs Reduced viral loads Generation of memory CD8T cells. Not evaluated. [152]. Virosomes. RSV-P3CSK4. Intramuscular. Balb/c mice Cotton rats. 2 weeks until 3 weeks after last immunization. Intramuscular or Intransal. Balb/c mice Cotton rats. 2 weeks. No signs of aveolitis and fewer or no mast cells and macrophages in lungs No sign of perivascular and peribronchial infiltration or alveolitis. [156]. RSV-MPLA. High serum hRSV-antibodies titer and neutralizing antibodies Polarization to Th1 response by T cells Increase on IgG and IgA antibodies Low lung viral titer Polarization to Th1 response by T cells. Target: F protein. a b. Enhanced disease is only considered when the vaccine was evaluated in the Cotton Rats model. Only case where enhanced disease was evaluated in human model.. Downloaded from ClinicalKey.com at University of Chile Catholic ALERTA May 16, 2016. For personal use only. No other uses without permission. Copyright ©2016. Elsevier Inc. All rights reserved.. [131]. [132]. [137,138]. [150]. [157,158].

(6) 242. R.S. Gomez et al. / Immunology Letters 162 (2014) 237–247. Fig. 2. Therapeutic drugs against hRSV infection target 3 main pathways: virus entry, replication and transcription. Virus entry is inhibited by drugs preventing virus fusion either by direct interaction with viral F glycoprotein and G (RFI 641; JNJ-2408068; Anti CX3C; Palivizumab) or by destroying the conformation of fusion proteins (BMS433771). Virus replication and transcription is inhibited either by drugs targeting L and N protein (YM-53403; RSV-604), by drug inhibiting pyrimidine synthesis pathway (leflunomide) or by siRNA repressing N, P or L viral genes.. T cell responses were observed after immunization with this vaccine [122]. 6. The use of hRSV-F glycoprotein as target for drug and vaccine design Recently, fragment antibodies called “Nanobodies” have been evaluated as treatment against hRSV infection [123,124]. Thus, heavy chain against F-hRSV glycoprotein has been evaluated as an efficient hRSV neutralizing tool in HEp-2 cells. The neutralizing capacity of this molecule was about 4000-fold higher as compared to whole classical anti-F antibodies [125]. In vivo, nanobodies were found to be efficient at preventing infection and excessive airway inflammation [123]. These nanobodies are thought to prevent the structural conformation change of F-hRSV glycoprotein, which is required for the fusion with the host cell surface [123]. One recent approach for vaccine design has been the use of Bacteria-Like Particles (BLPs) that carry hRSV-derived antigens [126]. Lactococcuslactis was used as a vector expressing a recombinant F glycoprotein stabilized in its pre-fusion conformation form [126]. Interestingly, immunization with this formulation led to an increased humoral response evidenced as high IgG and IgA nasal titers, reduced viral loads on Balb/c infected mice and no sign of interstitial pneumonia nor alveolitis [126]. Further, cotton rats vaccinated with this formulations showed no signs of enhanced inflammatory lung disease and low viral titers after hRSV challenge [126].. Another approach explored for vaccine development is the use of viral vectors, such as adenoviruses [127–130], which similarly to attenuated bacteria, can express viral proteins and confer immune protection against hRSV. A recent study with adenovirus expressing F glycoprotein showed strong humoral and cellular responses after vaccination/exposure to hRSV. Further, vaccination with this adenovirus-based vaccine led to an increae of IFN-␥secreting CD8+ T cells, with a reduction both in viral loads and CD4+ T cells secreting IL-4. Consistently, the adenovirus-based vaccine caused no signs vaccine-enhanced pulmonary disease, nor infiltration of inflammatory cells into the lungs of hRSV-challenged Balb/c mice [131], as observed for the FI-hRSV vaccine [20]. This study also showed an increase in the regulatory T cells after vaccination/challenge with the adenoviral-based vaccine. The expression of a truncated form of the F glycoprotein (without the transmembrane domain) by these vectors also led to a reduction on the pathology promoting a Th1 immune response, after immunization with recombinant vaccine and challenge with the virus [131]. It was recently shown that a chimeric influenza live virus carrying an hRSV CD8+ T cell epitope F (85–93) in the neuraminidase stalk prevented high viral loads in the lungs after hRSV infection [132]. This effect is associated with an efficient cellular response against hRSV, by the generation of F85–93 -specific CTLs, and to low replication levels of hRSV [132]. However, in this study an additional influenza-specific antibody was required to counteract influenza-enhanced disease, which raised the issue of controlling of the influenza-caused disease as an additional parameter for. Downloaded from ClinicalKey.com at University of Chile Catholic ALERTA May 16, 2016. For personal use only. No other uses without permission. Copyright ©2016. Elsevier Inc. All rights reserved..

(7) R.S. Gomez et al. / Immunology Letters 162 (2014) 237–247. clinical studies. Previous studies have used influenza M1 matrix protein of Virus-Like Particle (VLP) bound to hRSV F or G glycoproteins and showed better results without the problems of using the an infectious influenza virus [133]. This study showed that while VLP-G significantly reduced viral replication on the lungs, as compared to VLP-F, both approached induced high amounts of hRSV-specific IgG2a and neutralizing antibodies [133]. A truncated F glycoprotein has been used as vaccine coupled with three different adjuvants (F-TriAdj) [134,135]. In these studies, adjuvant-coupled proteins showed no enhanced disease effect, produced efficient and specific humoral response, characterized by high titers of neutralizing antibodies and production of IgA in the lungs. Furthermore, this vaccine formulation induced T cells immunity, consisting of memory T cells and high frequencies of effector hRSV-specific CD8+ T cells that conferred long term protection (up to one year) to infection [136]. Another strategy that has been tested is the production of F-alum nanoparticles in Sf9 insect cells, which have shown promising results after immunization. In cotton rats, this subunit vaccine had no side effects, produce an increase in the amount of neutralizing antibodies and a decrease in the viral loads in lungs after hRSV challenge. These positive responses were even more significant when this formulation was accompanied with Alum as adjuvant. In fact immunization with this formulation prevents enhanced disease after challenge with hRSV, as measured by lung pathology [137]. These nanoparticles were tested in phase I clinical trial. Adult subjects between 18 and 49 years of age generated neutralizing antibodies and Palivizumablike antibodies without adverse effect when vaccinated with this “F rosette” nanoparticle [138]. Thus, this vaccine is a good candidate for further clinical evaluation. Another study showed an interesting protective role of F-adjuvant conjugated in senescent mice, reducing viral titers and increasing virus-specific antibody titers [139]. The alum formulation was able to increase IFN-␥ in both young and aged mice with no increase of IL-5 and CCL11 in aged mice. Interestingly, although the F glycoprotein by itself failed to prevent hRSV-induced lung pathology both in young and aged mice the Falum formulation significantly decreased pathology in aged but not in young mice [139]. 7. Strategies to target the hRSV-N protein One of the most efficient experimental drugs against hRSV is RSV-604, which targets the N nucleoprotein, interfering with the replicative cycle of the virus [140]. This molecule is currently being tested in phase II trials, which have shown promising results despite the tendency of hRSV to acquire mutations during long exposure to the drug [141]. siRNA targeting methods against N, P and L, interfering with replication cycle was found to reduce viral titers in the lungs [142–145]. For instance when administrated in adults humans before and after hRSV challenge, siRNA targeting N gene was found to reduce infection in 40% of the patients [145]. This study showed the potential promising therapeutic effect of siRNA in humans and highlighted the necessity of testing this drug as a real therapy in infants already manifesting signs of the disease. The use of attenuated bacterial vectors in the development of vaccine has been a widely used strategy. One of the most famous examples is related to the discovery of the Bacillus Calmette-Guerin: the attenuated form of tuberculosis in bovine (Mycobacterium bovis, also known as Bacille Calmette-Guerin BCG) was found to give an efficient protection in human against tuberculosis. Later, BCG has been used as a powerful immunogenic recombinant vector against other diseases [146,147]. Concordant to this idea, the use of recombinant BCG for hRSV N nucleoprotein was found to induce an efficient protection against innate and adaptive events of the pathology, decreasing cellular infiltration. 243. and viral loads in the airways [99,148]. In fact, inflammatory events observed in mice after rBCG-N vaccination and hRSV challenge were comparable to those of uninfected animals suggesting that the effector/memory Th1 cell mediated response was fully protective [148]. 8. Vaccine development based on hRSV-G glycoprotein Studies on hRSV-G glycoprotein have been focused on recombinant viruses, which could be an efficient strategy for reducing hRSV pathology. Two recombinant hRSV particles (rhRSV and GhRSV) were found to be protective against hRSV challenge in vivo with no enhanced disease phenomenon, described by low pulmonary inflammation. Replication level of these 2 particles was either null or low; leading to the conclusion that G would play an important role in hRSV replication [149]. Unfortunately, protection against viral replication and inflammation in the lungs after challenge lasted only 147 and 75 days, respectively, after vaccination [149]. In 2009, a study showed that antibodies to the CX3C motif of the G glycoprotein reduced viral titers and cellular infiltration when administered during the first few days after infection [150]. Based on this result, other studies have used the immunogenic part of G glycoprotein [150,151]. As described, in these studies the use of this motif in nanoparticles formulation increased hRSV-specific humoral response in immunized animals, decreased viral titers and lung pathology in infected mice and promoted CD8+ -specific T cells and Th1 response over Th2 response in BAL fluids [151]. Interestingly, the use of this motif of G glycoprotein in FI-hRSV formulation helped to decrease the negative effects of this vaccine showing less pathology in vaccinated animals [152]. 9. Peptides-based vaccines against h-RSV M2 protein TriVax is a combination of M2 CD8+ immunodominant peptide combined with poly I:C and anti-CD40 co-stimulatory antibody [153]. Immunization with this formulation increased the production of M2-specific CD8+ T cells that have CTL activity [153]. Vaccination promoted the secretion of IFN-␥, IL-2 and TNF-␣ by those CD8+ T cells and reduced the pathology-associated effects caused by a hRSV strain that generates the production of mucin in the airways. This vaccine approach was able to reduce mucin expression and interstitial inflammation in the lungs after hRSV challenge. Interestingly this vaccine candidate was able to promote memory CD8+ T cells. However, these memory T cells failed to produce the same level of antiviral cytokines as effector CD8+ T cells produced by the vaccination [153]. 10. Alternative strategies to generate hRSV vaccines An attenuated viral particle, cpts-248/404, was found to be a good candidate among the low-temperature attenuated particles. Infants vaccinated with this particle did not manifest lower respiratory tract infection, but the upper airway was found affected after immunization [120,154]. Later, a derived attenuated particle from cpt-248/404 mutated for SH and L protein named rA2cp248/404/1030SH has been found to generate an efficient IgG and IgA specific response against hRSV in 44% of the 1–2 month old infants [155]. A further derivate particle of rA2cp248/404/1030SH, called MEDI-559, successfully maintained virus attenuation in the respiratory tract of cotton rat [121]. Surprisingly, in recent studies, one viral particle has been found to revert its attenuated temperature properties by a deleterious mutation (1313), resulting in a strong replication capacity at low temperature and in attenuated replication properties at 37 ◦ C [156]. In the same study, this promising viral particle was then. Downloaded from ClinicalKey.com at University of Chile Catholic ALERTA May 16, 2016. For personal use only. No other uses without permission. Copyright ©2016. Elsevier Inc. All rights reserved..

(8) 244. R.S. Gomez et al. / Immunology Letters 162 (2014) 237–247. combined with another one, carrying attenuating properties mutation in NS2 gene. Interestingly, the viral particle obtained generated another spontaneous compensating mutation (I1314T) that could be efficiently stabilized by a subsequent substitution mutation. The evaluation of this stabilized particle NS2/1313/1314L in juvenile chimpanzee led to the conclusion that this particle could be a good candidate for further vaccination studies in children. This study shows also one important event to consider in hRSV infection, the fact that the virus in its attenuated form or not, are able to generate compensating mutations. Virosomes particles, which are the combination of viral envelope and lipopeptides formulated to be recognized by TLR2 and TLR4 (P3CSK4 and MPLA respectively) showed also promising results. Both formulations efficiently decreased viral titer and increased IgG specific antibodies. The formulation with P3CSK4 adjuvant generated a Th1 /Th2 balanced response, contrarily to the use of monophosphoril A (MPLA) that generated a polarized Th1 response, consisting of high levels of IFN-␥ and low levels of IL5 [157–159]. In cotton rat model, MPLA-adjuvanted virosomes increases IgA response, neutralizing antibodies, with no sign of enhanced disease. This study show that TLR4 ligand pathway could be an interesting strategy related subunit vaccine. 11. Immune protection induced by maternal immunization As newborns are the population most affected by severe complications after hRSV infection, maternal immunization during pregnancy is also an important point to consider. Maternal antibodies are passively transferred to babies by placenta and breast milk [160,161]. Only few studies related to maternal antibody transfer are published in the hRSV vaccine field [162]. A recent study using FI-hRSV particles in animal models showed increased antibody titers and less severe disease in pups [163]. The contribution of maternal antibodies against hRSV remains controversial. While some studies support the idea that high levels of maternal antibodies transferred to children can be beneficial against hRSV infection [164,165], other studies have suggested that these antibodies fail to protect against hRSV infection and can generate undesirable effect, such as airway hypersensitivity, reinfection and recurrent wheezing [166–168]. Nevertheless, high titers of hRSV antibodies in children were found to be detrimental to the production of self-made antibodies [169]. A recent commentary about maternal transfers points out that important parameters, as the seasonality of hRSV, the pregnancy state, vaccine type and infants immune system immaturity, can also influence the effectiveness of the vaccination [170]. 12. Concluding remarks The knowledge about hRSV is wide and a significant numbers of products have been tested against the disease. Some of these products have therapeutic promising anti-viral effect against the disease and others are already candidate prophylactic vaccine in newborns. Several drugs shown promising effects against the pathology in vivo in animal models, but also many of these drugs probably will not reach clinical studies. Thus a significant fraction of the work still remains to be completed, such as evaluating carefully any side effects of these drugs in children in a controlled manner, because newborns be deficient at promoting good immunity, these drugs could be very helpful for controlling hRSV infection and preventing subsequent pathology. The use of bacterial vectors seems to be a good way to advance in the field of hRSV vaccines, but the adverse secondary effect of these vectors must be evaluated and an important attention must be paid when these prototype vaccines will be evaluated in clinical trials.. Both, F and G hRSV glycoproteins are good candidates in the use of subunit vaccine since they produce significant anti-hRSV titers and reduce hRSV-associated pathology. Adjuvants also seems to play a key protective role and more specifically the ones promoting TLR engagement, raising the hypothesis that functional antigen presenting cell engagement would be important (Table 1). Finally, the complexity of hRSV-induced pathology makes the development of safe treatment and prophylaxis for newborns a challenge for vaccine candidates. As a consequence, special care as to be taken in the secondary effects attributed to “De novo” synthetized products. Combination of hRSV epitopes with products that promotes humoral and cellular response, such as TLR ligands, seems to be one of the best strategies to generate effective, safety and long-term protection against the virus. Acknowledgements This study was supported by the following grants: FONDO NACIONAL DE CIENCIA Y TECNOLOGIA DE CHILE (FONDECYT numbers 1140010, 1110604, 1100971, 1131012 and 1110397), Millennium Institute of Immunology and Immunotherapy P09/016-F and Grant “Nouvelles Equipes-nouvelles thématiques” from the La Région Pays De La Loire. RSG and KB are supported by the Comisión Nacional de Investigación Científica y Tecnológica (CONICYT). AMK is a Chaire De La Région Pays De La Loire, Chercheur Étranger D’excellence, France. References [1] Domachowske JB, Rosenberg HF. Respiratory syncytial virus infection: immune response, immunopathogenesis, and treatment. Clin Microbiol Rev 1999;12:298–309. [2] Collins PL, Graham BS. 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