Heme Oxygenase-1 Modulates Human Respiratory Syncytial Virus Replication and Lung Pathogenesis during Infection.

Human respiratory syncytial virus (hRSV) is the leading cause of severe lower respiratory tract infections in children. The development of novel prophylactic and therapeutic antiviral drugs against hRSV is imperative to control the burden of disease in the susceptible population. In this study, we examined the effects of inducing the activity of the host enzyme heme oxygenase-1 (HO-1) on hRSV replication and pathogenesis on lung inflammation induced by this virus. Our results show that after hRSV infection, HO-1 induction with metalloporphyrin cobalt protoporphyrin IX significantly reduces the loss of body weight due to hRSV-induced disease. Further, HO-1 induction also decreased viral replication and lung inflammation, as evidenced by a reduced neutrophil infiltration into the airways, with diminished cytokine and chemokine production and reduced T cell function. Concomitantly, upon cobalt protoporphyrin IX treatment, there is a significant upregulation in the production of IFN-α/ß mRNAs in the lungs. Furthermore, similar antiviral and protective effects occur by inducing the expression of human HO-1 in MHC class II+ cells in transgenic mice. Finally, in vitro data suggest that HO-1 induction can modulate the susceptibility of cells, especially the airway epithelial cells, to hRSV infection.

Replication of Human Norovirus RNA in Mammalian Cells Reveals Lack of Interferon Response.

UNLABELLED: Human noroviruses (HuNoVs), named after the prototype strain Norwalk virus (NV), are a leading cause of acute gastroenteritis outbreaks worldwide. Studies on the related murine norovirus (MNV) have demonstrated the importance of an interferon (IFN) response in host control of virus replication, but this remains unclear for HuNoVs. Despite the lack of an efficient cell culture infection system, transfection of stool-isolated NV RNA into mammalian cells leads to viral RNA replication and virus production. Using this system, we show here that NV RNA replication is sensitive to type I (α/ß) and III (interleukin-29 [IL-29]) IFN treatment. However, in cells capable of a strong IFN response to Sendai virus (SeV) and poly(I·C), NV RNA replicates efficiently and generates double-stranded RNA without inducing a detectable IFN response. Replication of HuNoV genogroup GII.3 strain U201 RNA, generated from a reverse genetics system, also does not induce an IFN response. Consistent with a lack of IFN induction, NV RNA replication is enhanced neither by neutralization of type I/III IFNs through neutralizing antibodies or the soluble IFN decoy receptor B18R nor by short hairpin RNA (shRNA) knockdown of mitochondrial antiviral signaling protein (MAVS) or interferon regulatory factor 3 (IRF3) in the IFN induction pathways. In contrast to other positive-strand RNA viruses that block IFN induction by targeting MAVS for degradation, MAVS is not degraded in NV RNA-replicating cells, and an SeV-induced IFN response is not blocked. Together, these results indicate that HuNoV RNA replication in mammalian cells does not induce an IFN response, suggesting that the epithelial IFN response may play a limited role in host restriction of HuNoV replication. IMPORTANCE: Human noroviruses (HuNoVs) are a leading cause of epidemic gastroenteritis worldwide. Due to lack of an efficient cell culture system and robust small-animal model, little is known about the innate host defense to these viruses. Studies on murine norovirus (MNV) have shown the importance of an interferon (IFN) response in host control of MNV replication, but this remains unclear for HuNoVs. Here, we investigated the IFN response to HuNoV RNA replication in mammalian cells using Norwalk virus stool RNA transfection, a reverse genetics system, IFN neutralization reagents, and shRNA knockdown methods. Our results show that HuNoV RNA replication in mammalian epithelial cells does not induce an IFN response, nor can it be enhanced by blocking the IFN response. These results suggest a limited role of the epithelial IFN response in host control of HuNoV RNA replication, providing important insights into our understanding of the host defense to HuNoVs that differs from that to MNV.

Human metapneumovirus infection activates the TSLP pathway that drives excessive pulmonary inflammation and viral replication in mice.

Human metapneumovirus (hMPV) is a leading cause of acute respiratory tract infections in children and the elderly. The mechanism by which this virus triggers an inflammatory response still remains unknown. Here, we evaluated whether the thymic stromal lymphopoietin (TSLP) pathway contributes to lung inflammation upon hMPV infection. We found that hMPV infection promotes TSLP expression both in human airway epithelial cells and in the mouse lung. hMPV infection induced lung infiltration of OX40L(+) CD11b(+) DCs. Mice lacking the TSLP receptor deficient mice (tslpr(-/-) ) showed reduced lung inflammation and hMPV replication. These mice displayed a decreased number of neutrophils as well a reduction in levels of thymus and activation-regulated chemokine/CCL17, IL-5, IL-13, and TNF-α in the airways upon hMPV infection. Furthermore, a higher frequency of CD4(+) and CD8(+) T cells was found in tslpr(-/-) mice compared to WT mice, which could contribute to controlling viral spread. Depletion of neutrophils in WT and tslpr(-/-) mice decreased inflammation and hMPV replication. Remarkably, blockage of TSLP or OX40L with specific Abs reduced lung inflammation and viral replication following hMPV challenge in mice. Altogether, these results suggest that activation of the TSLP pathway is pivotal in the development of pulmonary pathology and pulmonary hMPV replication.

New insights on the viral and host factors contributing to the airway pathogenesis caused by the respiratory syncytial virus.

The respiratory syncytial virus (RSV) is the most prevalent etiological agent of lower respiratory tract infections and the first cause of hospitalization in infants due to respiratory disease worldwide. However, efforts to develop safe and effective vaccines and antivirals have been challenged by an incomplete understanding of the RSV pathogenesis and the host immune response to RSV infection in the airways. Here, we discuss recent advances in understanding the interaction between RSV and the epithelium to induce pathogenesis in the airways, such as the role of the RSV NS2 protein in the airway epithelium, as well as the events involved in the RSV entry process. In addition, we summarize the cellular factors produced by airway epithelial cells (AECs) in response to RSV infection that lead to the activation of innate and adaptive immune responses, inducing lung inflammation and disease. Further, we discuss the possible contribution of a recently identified cytokine, thymic stromal lymphopoitein (TSLP), in the lung immunopathology caused by RSV.

Understanding Lung Immunopathology Caused by the Human Metapneumovirus: Implications for Rational Vaccine Design.

Acute respiratory tract infections (ARTIs) are the major cause of child mortality worldwide. The human metapneumovirus (hMPV) is one of the leading causes of child hospitalizations due to pneumonia. The adaptive immune response generated by the host against hMPV is usually inefficient at protecting from reinfections, which is repeat throughout life, from childhood to old age. Despite considerable research efforts, to date there are no licensed vaccines to prevent respiratory disease caused by hMPV infection. In this article we review current vaccine strategies tested in animal models and the implication of such studies in understanding the different immune cell populations that contribute to hMPV clearance and the prevention and resolution of lung inflammation upon exposure to the virus.

Elevated IL-3 and IL-12p40 levels in the lower airway of infants with RSV-induced bronchiolitis correlate with recurrent wheezing.

Respiratory Syncytial Virus (RSV) is the first cause of hospitalization due to bronchiolitis in infants. RSV bronchiolitis has been linked to asthma and recurrent wheezing, however the mechanisms behind this association have not been elucidated. Here, we evaluated the cytokine and chemokine profiles in the airways in infants with RSV bronchiolitis. Nasopharyngeal Aspirates (NPA) and Bronchoalveolar Lavage Fluids (BALF) from infants hospitalized due to RSV bronchiolitis and healthy controls were analyzed for cytokine and chemokine production. We observed elevated levels of Th2 cytokines (IL-3, IL-4, IL-10 and IL-13), pro-inflammatory cytokines and chemokines (IL-1ß, IL-6, TNF-ß, MCP-1/CCL2, MIP-1α/CCL3 and IL-8/CXCL8) in BALF from infants with RSV bronchiolitis, as compared to controls. We found a direct correlation of IL-3 and IL-12p40 levels with the development of recurrent wheezing later in life. These results suggest that IL-3 and IL-12p40 could be considered as molecular predictors for recurrent wheezing due to RSV infection.

New insights into the pathogenesis of SARS-CoV-2 during and after the COVID-19 pandemic.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the respiratory distress condition known as COVID-19. This disease broadly affects several physiological systems, including the gastrointestinal, renal, and central nervous (CNS) systems, significantly influencing the patient's overall quality of life. Additionally, numerous risk factors have been suggested, including gender, body weight, age, metabolic status, renal health, preexisting cardiomyopathies, and inflammatory conditions. Despite advances in understanding the genome and pathophysiological ramifications of COVID-19, its precise origins remain elusive. SARS-CoV-2 interacts with a receptor-binding domain within angiotensin-converting enzyme 2 (ACE2). This receptor is expressed in various organs of different species, including humans, with different abundance. Although COVID-19 has multiorgan manifestations, the main pathologies occur in the lung, including pulmonary fibrosis, respiratory failure, pulmonary embolism, and secondary bacterial pneumonia. In the post-COVID-19 period, different sequelae may occur, which may have various causes, including the direct action of the virus, alteration of the immune response, and metabolic alterations during infection, among others. Recognizing the serious adverse health effects associated with COVID-19, it becomes imperative to comprehensively elucidate and discuss the existing evidence surrounding this viral infection, including those related to the pathophysiological effects of the disease and the subsequent consequences. This review aims to contribute to a comprehensive understanding of the impact of COVID-19 and its long-term effects on human health.

Lack of norovirus replication and histo-blood group antigen expression in 3-dimensional intestinal epithelial cells.

Noroviruses (NoVs) are a leading cause of gastroenteritis worldwide. An in vitro model for NoV replication remains elusive, making study of the virus difficult. A previous study, which used a 3-dimensional (3-D) intestinal model derived from INT-407 cells reported NoV replication and extensive cytopathic effects (CPE). Using the same 3-D model, but with highly purified Norwalk virus (NV), we attempted to replicate this study. Our results showed no evidence of NV replication by real-time PCR of viral RNA or by immunocytochemical detection of viral structural and nonstructural proteins. Immunocytochemical analysis of the 3-D cultures also showed no detectable presence of histo-blood group antigens that participate in NV binding and host tropism. To determine the potential cause of CPE observed in the previous study, we exposed 3-D cultures to lipopolysaccharide concentrations consistent with contaminated stool samples and observed morphologic features similar to CPE. We conclude that the 3-D INT-407 model does not support NV replication.

Non-capsular based immunization approaches to prevent Streptococcus pneumoniae infection.

Streptococcus pneumoniae is a Gram-positive bacterium and the leading cause of bacterial pneumonia in children and the elderly worldwide. Currently, two types of licensed vaccines are available to prevent the disease caused by this pathogen: the 23-valent pneumococcal polysaccharide-based vaccine and the 7-, 10, 13, 15 and 20-valent pneumococcal conjugate vaccine. However, these vaccines, composed of the principal capsular polysaccharide of leading serotypes of this bacterium, have some problems, such as high production costs and serotype-dependent effectiveness. These drawbacks have stimulated research initiatives into non-capsular-based vaccines in search of a universal vaccine against S. pneumoniae. In the last decades, several research groups have been developing various new vaccines against this bacterium based on recombinant proteins, live attenuated bacterium, inactivated whole-cell vaccines, and other newer platforms. Here, we review and discuss the status of non-capsular vaccines against S. pneumoniae and the future of these alternatives in a post-pandemic scenario.

Human Norovirus Proteins: Implications in the Replicative Cycle, Pathogenesis, and the Host Immune Response.

Human noroviruses (HuNoVs) are the cause of more than 95% of epidemic non-bacterial gastroenteritis worldwide, with some lethal cases. These viral agents affect people of all ages. However, young children and older adults are the highest-risk groups, being affected with the greatest rate of hospitalizations and morbidity cases. HuNoV structural proteins, especially VP1, have been studied extensively. In contrast, the functions of the non-structural proteins of the virus have been undescribed in depth. Studies on HuNoV non-structural proteins have mostly been made by expressing them individually in in vitro cultures, providing insights of their functions and the role that they play in HuNoV replication and pathogenesis. This review examines exhaustively the functions of both HuNoV structural and non-structural proteins and their possible role within the viral replicative cycle and the pathogenesis of the virus. It also highlights recent findings regarding the host's innate and adaptive immune responses against HuNoV, which are of great relevance for diagnostics and vaccine development so as to prevent infections caused by these fastidious viruses.

Host Components Contributing to Respiratory Syncytial Virus Pathogenesis.

Respiratory syncytial virus (RSV) is the most prevalent viral etiological agent of acute respiratory tract infection. Although RSV affects people of all ages, the disease is more severe in infants and causes significant morbidity and hospitalization in young children and in the elderly. Host factors, including an immature immune system in infants, low lymphocyte levels in patients under 5 years old, and low levels of RSV-specific neutralizing antibodies in the blood of adults over 65 years of age, can explain the high susceptibility to RSV infection in these populations. Other host factors that correlate with severe RSV disease include high concentrations of proinflammatory cytokines such as interleukins (IL)-6, IL-8, tumor necrosis factor (TNF)-α, and thymic stromal lymphopoitein (TSLP), which are produced in the respiratory tract of RSV-infected individuals, accompanied by a strong neutrophil response. In addition, data from studies of RSV infections in humans and in animal models revealed that this virus suppresses adaptive immune responses that could eliminate it from the respiratory tract. Here, we examine host factors that contribute to RSV pathogenesis based on an exhaustive review of in vitro infection in humans and in animal models to provide insights into the design of vaccines and therapeutic tools that could prevent diseases caused by RSV.

BCG-Induced Cross-Protection and Development of Trained Immunity: Implication for Vaccine Design.

The Bacillus Calmette-Guérin (BCG) is a live attenuated tuberculosis vaccine that has the ability to induce non-specific cross-protection against pathogens that might be unrelated to the target disease. Vaccination with BCG reduces mortality in newborns and induces an improved innate immune response against microorganisms other than Mycobacterium tuberculosis, such as Candida albicans and Staphylococcus aureus. Innate immune cells, including monocytes and natural killer (NK) cells, contribute to this non-specific immune protection in a way that is independent of memory T or B cells. This phenomenon associated with a memory-like response in innate immune cells is known as "trained immunity." Epigenetic reprogramming through histone modification in the regulatory elements of particular genes has been reported as one of the mechanisms associated with the induction of trained immunity in both, humans and mice. Indeed, it has been shown that BCG vaccination induces changes in the methylation pattern of histones associated with specific genes in circulating monocytes leading to a "trained" state. Importantly, these modifications can lead to the expression and/or repression of genes that are related to increased protection against secondary infections after vaccination, with improved pathogen recognition and faster inflammatory responses. In this review, we discuss BCG-induced cross-protection and acquisition of trained immunity and potential heterologous effects of recombinant BCG vaccines.

Presence of wild-type and attenuated Salmonella enterica strains in brain tissues following inoculation of mice by different routes.

Salmonella enterica serovar Typhi and Typhimurium vaccine candidates elicit significant immune responses in mice by intranasal (i.n.) immunization. Because of the proximity of the cribriform plate of the ethmoid bone, we were concerned that Salmonella bacteria delivered i.n. might access the brain. Accordingly, wild-type and attenuated (by single and double mutations) strains of S. enterica serovars Typhimurium and Typhi were recovered at low numbers initially from the olfactory lobe and then from the brain for 3 to 4 days after i.n. immunization. This was independent of invA gene function. Although the presence of bacteria in blood 1 to 3 h after i.n. inoculation was sometimes observed, this was infrequent compared to the frequency of bacteria detected in brain tissues. In confirmation of recent observations by Wickham et al. (M. E. Wickham, N. F. Brown, J. Provias, B. B. Finlay, and B. K. Coombes, BMC Infect. Dis. 7:65, 2007) that oral inoculation with wild-type S. enterica serovar Typhimurium strains lead to bacteria in blood with subsequent colonization of brain tissues with neurological symptoms of disease, we found similar results by using the i.n. and intraperitoneal (i.p.) routes of inoculation for wild-type but not for attenuated strains of S. enterica serovar Typhimurium. In contrast, a highly modified attenuated S. enterica serovar Typhimurium strain was not present in brain tissues when administered at higher doses by the oral, i.n., and i.p. routes than the wild-type strain even though the presence of bacteria in blood was detectable 1 to 3 h after inoculation by each of the three routes. Our results indicate that i.n. and possibly even oral delivery of live Salmonella vaccines may be unsafe although it is possible to reduce this risk by appropriate genetic modifications.

Role of Regulatory T Cells in Infection and Vaccination During Early Infancy.

Reducing infant mortality due to infectious diseases is one of the most important public health goals worldwide. Several approaches have been implemented to reach this goal and vaccination has been an effective strategy for reducing infant and newborn mortality. However, the immunological features of neonates and infants represent a significant barrier to the effectiveness of vaccination. Since regulatory T cells (Treg cells) are known to play an active role in contributing to various mechanisms of suppression of the immune cell function. It has been proposed that these immune cells could decrease the immunogenicity of vaccines administered in newborns and infants. In this article, we discuss the various types of Treg cells, along with their suppressing and inhibitory mechanisms, which are used by these cells in the context of infectious and immunization processes in newborns and infants.

Human Metapneumovirus: Mechanisms and Molecular Targets Used by the Virus to Avoid the Immune System.

Human metapneumovirus (hMPV) is a respiratory virus, first reported the year 2001. Since then, it has been described as one of the main etiological agents that causes acute lower respiratory tract infections (ALRTIs), which is characterized by symptoms such as bronchiolitis, wheezing and coughing. Susceptible population to hMPV-infection includes newborn, children, elderly and immunocompromised individuals. This viral agent is a negative-sense, single-stranded RNA enveloped virus, that belongs to the Pneumoviridae family and Metapneumovirus genus. Early reports-previous to 2001-state several cases of respiratory illness without clear identification of the responsible pathogen, which could be related to hMPV. Despite the similarities of hMPV with several other viruses, such as the human respiratory syncytial virus or influenza virus, mechanisms used by hMPV to avoid the host immune system are still unclear. In fact, evidence indicates that hMPV induces a poor innate immune response, thereby affecting the adaptive immunity. Among these mechanisms, is the promotion of an anergic state in T cells, instead of an effective polarization or activation, which could be induced by low levels of cytokine secretion. Further, the evidences support the notion that hMPV interferes with several pattern recognition receptors (PRRs) and cell signaling pathways triggered by interferon-associated genes. However, these mechanisms reported in hMPV are not like the ones reported for hRSV, as the latter has two non-structural proteins that are able to inhibit these pathways. Several reports suggest that viral glycoproteins, such as G and SH, could play immune-modulator roles during infection. In this work, we discuss the state of the art regarding the mechanisms that underlie the poor immunity elicited by hMPV. Importantly, these mechanisms will be compared with those elicited by other common respiratory viruses.

Assessing the Importance of Domestic Vaccine Manufacturing Centers: An Overview of Immunization Programs, Vaccine Manufacture, and Distribution.

Vaccines have significantly reduced the detrimental effects of numerous human infectious diseases worldwide, helped to reduce drastically child mortality rates and even achieved eradication of major pathogens, such as smallpox. These achievements have been possible due to a dedicated effort for vaccine research and development, as well as an effective transfer of these vaccines to public health care systems globally. Either public or private institutions have committed to developing and manufacturing vaccines for local or international population supply. However, current vaccine manufacturers worldwide might not be able to guarantee sufficient vaccine supplies for all nations when epidemics or pandemics events could take place. Currently, different countries produce their own vaccine supplies under Good Manufacturing Practices, which include the USA, Canada, China, India, some nations in Europe and South America, such as Germany, the Netherlands, Italy, France, Argentina, and Brazil, respectively. Here, we discuss some of the vaccine programs and manufacturing capacities, comparing the current models of vaccine management between industrialized and developing countries. Because local vaccine production undoubtedly provides significant benefits for the respective population, the manufacture capacity of these prophylactic products should be included in every country as a matter of national safety.

Modulation of Host Immunity by Human Respiratory Syncytial Virus Virulence Factors: A Synergic Inhibition of Both Innate and Adaptive Immunity.

The Human Respiratory Syncytial Virus (hRSV) is a major cause of acute lower respiratory tract infections (ARTIs) and high rates of hospitalizations in children and in the elderly worldwide. Symptoms of hRSV infection include bronchiolitis and pneumonia. The lung pathology observed during hRSV infection is due in part to an exacerbated host immune response, characterized by immune cell infiltration to the lungs. HRSV is an enveloped virus, a member of the Pneumoviridae family, with a non-segmented genome and negative polarity-single RNA that contains 10 genes encoding for 11 proteins. These include the Fusion protein (F), the Glycoprotein (G), and the Small Hydrophobic (SH) protein, which are located on the virus surface. In addition, the Nucleoprotein (N), Phosphoprotein (P) large polymerase protein (L) part of the RNA-dependent RNA polymerase complex, the M2-1 protein as a transcription elongation factor, the M2-2 protein as a regulator of viral transcription and (M) protein all of which locate inside the virion. Apart from the structural proteins, the hRSV genome encodes for the non-structural 1 and 2 proteins (NS1 and NS2). HRSV has developed different strategies to evade the host immunity by means of the function of some of these proteins that work as virulence factors to improve the infection in the lung tissue. Also, hRSV NS-1 and NS-2 proteins have been shown to inhibit the activation of the type I interferon response. Furthermore, the hRSV nucleoprotein has been shown to inhibit the immunological synapsis between the dendritic cells and T cells during infection, resulting in an inefficient T cell activation. Here, we discuss the hRSV virulence factors and the host immunological features raised during infection with this virus.

Aberrant T cell immunity triggered by human Respiratory Syncytial Virus and human Metapneumovirus infection.

Human Respiratory syncytial virus (hRSV) and human metapneumovirus (hMPV) are the two major etiological viral agents of lower respiratory tract diseases, affecting mainly infants, young children and the elderly. Although the infection of both viruses trigger an antiviral immune response that mediate viral clearance and disease resolution in immunocompetent individuals, the promotion of long-term immunity appears to be deficient and reinfection are common throughout life. A possible explanation for this phenomenon is that hRSV and hMPV, can induce aberrant T cell responses, which leads to exacerbated lung inflammation and poor T and B cell memory immunity. The modulation of immune response exerted by both viruses include different strategies such as, impairment of immunological synapse mediated by viral proteins or soluble factors, and the induction of pro-inflammatory cytokines by epithelial cells, among others. All these viral strategies contribute to the alteration of the adaptive immunity in order to increase the susceptibility to reinfections. In this review, we discuss current research related to the mechanisms underlying the impairment of T and B cell immune responses induced by hRSV and hMPV infection. In addition, we described the role each virulence factor involved in immune modulation caused by these viruses.

Inflammatory damage on respiratory and nervous systems due to hRSV infection.

The exacerbated inflammatory response elicited by human Respiratory Syncytial Virus (hRSV) in the lungs of infected patients causes a major health burden in the pediatric and elderly population. Since the discovery of hRSV, the exacerbated host immune-inflammatory response triggered by this virus has been extensively studied. In this article, we review the effects on the airways caused by immune cells and cytokines/chemokines secreted during hRSV infection. While molecules such as interferons contribute at controlling viral infection, IL-17 and others produce damage to the hRSV-infected lung. In addition to affecting the airways, hRSV infection can cause significant neurologic abnormalities in the host, such as seizures and encephalopathy. Although the origin of these symptoms remains unclear, studies from patients suffering neurological alteration suggest an involvement of the inflammatory response against hRSV.

Advances in understanding respiratory syncytial virus infection in airway epithelial cells and consequential effects on the immune response.

This article reviews aspects of respiratory syncytial virus (RSV) infection in airway epithelial cells (AECs), including cytopathogenesis, entry, replication and the induction of immune response to the virus, including a new role for thymic stromal lymphopoietin in RSV immunopathology.