Suryaprakash Sambhara

Cutting Edge: Impaired Toll-Like Receptor Expression and Function in Aging

Toll-like receptors (TLR) are pattern recognition receptors that recognize conserved molecular patterns on microbes and link innate and adaptive immune systems. We investigated whether the enhanced susceptibility to bacterial, yeast, and viral infections and poor adaptive immune responses in aging are a result of diminished expression and function of TLRs. We examined the expression and function of all murine TLRs on macrophages from young and aged mice. Both splenic and activated peritoneal macrophages from aged mice expressed significantly lower levels of all TLRs. Furthermore, macrophages from aged mice secreted significantly lower levels of IL-6 and TNF-α when stimulated with known ligands for TLR1 and 2, 2 and 6,TLR3, TLR4, TLR5, and TLR9 when compared with those from young mice. These results support the concept that increased susceptibility to infections and poor adaptive immune responses in aging may be due to the decline in TLR expression and function.

Role of Host Cytokine Responses in the Pathogenesis of Avian H5N1 Influenza Viruses in Mice

ABSTRACT Highly pathogenic avian H5N1 influenza viruses are now widespread in poultry in Asia and have recently spread to some African and European countries. Interspecies transmission of these viruses to humans poses a major threat to public health. To better understand the basis of pathogenesis of H5N1 viruses, we have investigated the role of proinflammatory cytokines in transgenic mice deficient in interleukin-6 (IL-6), macrophage inflammatory protein 1 alpha (MIP-1α), IL-1 receptor (IL-1R), or tumor necrosis factor receptor 1 (TNFR1) by the use of two avian influenza A viruses isolated from humans, A/Hong Kong/483/97 (HK/483) and A/Hong Kong/486/97 (HK/486), which exhibit high and low lethality in mice, respectively. The course of disease and the extent of virus replication and spread in IL-6- and MIP-1α-deficient mice were not different from those observed in wild-type mice during acute infection with 1,000 50% mouse infective doses of either H5N1 virus. However, with HK/486 virus, IL-1R-deficient mice exhibited heightened morbidity and mortality due to infection, whereas no such differences were observed with the more virulent HK/483 virus. Furthermore, TNFR1-deficient mice exhibited significantly reduced morbidity following challenge with either H5N1 virus but no difference in viral replication and spread or ultimate disease outcome compared with wild-type mice. These results suggest that TNF-α may contribute to morbidity during H5N1 influenza virus infection, while IL-1 may be important for effective virus clearance in nonlethal H5N1 disease.

Innate immunity in aging: impact on macrophage function

Innate and adaptive immune functions decline with age, leading to increased susceptibility to infectious diseases and cancer, and reduced responses to preventive vaccination in the elderly population. Macrophages function as ‘pathogen sensors’ and play an important role in the initiation of inflammatory responses, elimination of pathogens, manipulation of the adaptive immune response and reparation of damaged tissue. In this paper, we review the literature addressing the impact of aging on the macrophage population.

Development of adenoviral-vector-based pandemic influenza vaccine against antigenically distinct human H5N1 strains in mice

INTRODUCTION Avian H5N1 influenza viruses currently circulating in southeast Asia could potentially cause the next pandemic. However, currently licensed human vaccines are subtype-specific and do not protect against these H5N1 viruses. We aimed to develop an influenza vaccine and assessed its immunogenicity and efficacy to confer protection in BALB/c mice. METHODS We developed an egg-independent strategy to combat the avian influenza virus, because the virus is highly lethal to chickens and the maintenance of a constant supply of embryonated eggs would be difficult in a pandemic. We used a replication-incompetent, human adenoviral-vector-based, haemagglutinin subtype 5 influenza vaccine (HAd-H5HA), which induces both humoral and cell-mediated immune responses against avian H5N1 influenza viruses isolated from people. FINDINGS Immunisation of mice with HAd-H5HA provided effective protection from H5N1 disease, death, and primary viral replication (p<0.0001) against antigenically distinct strains of H5N1 influenza viruses. Unlike the recombinant H5HA vaccine, which is based on a traditional subunit vaccine approach, HAd-H5HA vaccine induced a three-fold to eight-fold increase in HA-518-epitope-specific interferon-gamma-secreting CD8 T cells (p=0.01). INTERPRETATION Our findings highlight the potential of an Ad-vector-based delivery system, which is both egg-independent and adjuvant-independent and offers stockpiling options for the development of a pandemic influenza vaccine.

Critical Role of an Antiviral Stress Granule Containing RIG-I and PKR in Viral Detection and Innate Immunity

Retinoic acid inducible gene I (RIG-I)-like receptors (RLRs) function as cytoplasmic sensors for viral RNA to initiate antiviral responses including type I interferon (IFN) production. It has been unclear how RIG-I encounters and senses viral RNA. To address this issue, we examined intracellular localization of RIG-I in response to viral infection using newly generated anti-RIG-I antibody. Immunohistochemical analysis revealed that RLRs localized in virus-induced granules containing stress granule (SG) markers together with viral RNA and antiviral proteins. Because of similarity in morphology and components, we termed these aggregates antiviral stress granules (avSGs). Influenza A virus (IAV) deficient in non-structural protein 1 (NS1) efficiently generated avSGs as well as IFN, however IAV encoding NS1 produced little. Inhibition of avSGs formation by removal of either the SG component or double-stranded RNA (dsRNA)-dependent protein kinase (PKR) resulted in diminished IFN production and concomitant enhancement of viral replication. Furthermore, we observed that transfection of dsRNA resulted in IFN production in an avSGs-dependent manner. These results strongly suggest that the avSG is the locus for non-self RNA sensing and the orchestration of multiple proteins is critical in the triggering of antiviral responses.

Challenges for vaccination in the elderly

The increased susceptibility of the elderly to infection presents a major challenge to public health services. An aging immune system is well documented as the cause of increased infection rates in elderly people. Such immunosenescence is multi-factorial and incompletely understood. Immunosenescent changes include malfunctioning of innate immune system cellular receptors; involution of the thymus, with consequent reduction of the naïve T cell population; alteration of the T cell population composition; modified phenotypes of individual T cells; and replicative senescence of memory cells expressing naïve markers. Unfortunately, immunosenescence also renders vaccination less effective in the elderly. It is therefore important that the vaccines used against common but preventable diseases, such as influenza, are specifically enhanced to overcome the reduced immune responsiveness of this vulnerable population.

Early Control of H5N1 Influenza Virus Replication by the Type I Interferon Response in Mice

ABSTRACT Widespread distribution of highly pathogenic avian H5N1 influenza viruses in domesticated and wild birds continues to pose a threat to public health, as interspecies transmission of virus has resulted in increasing numbers of human disease cases. Although the pathogenic mechanism(s) of H5N1 influenza viruses has not been fully elucidated, it has been suggested that the ability to evade host innate responses, such as the type I interferon response, may contribute to the virulence of these viruses in mammals. We investigated the role that type I interferons (alpha/beta interferon [IFN-α/β]) might play in H5N1 pathogenicity in vivo, by comparing the kinetics and outcomes of H5N1 virus infection in IFN-α/β receptor (IFN-α/βR)-deficient and SvEv129 wild-type mice using two avian influenza A viruses isolated from humans, A/Hong Kong/483/97 (HK/483) and A/Hong Kong/486/97 (HK/486), which exhibit high and low lethality in mice, respectively. IFN-α/βR-deficient mice experienced significantly more weight loss and more rapid time to death than did wild-type mice. HK/486 virus caused a systemic infection similar to that with HK/483 virus in IFN-α/βR-deficient mice, suggesting a role for IFN-α/β in controlling the systemic spread of this H5N1 virus. HK/483 virus replicated more efficiently than HK/486 virus both in vivo and in vitro. However, replication of both viruses was significantly reduced following pretreatment with IFN-α/β. These results suggest a role for the IFN-α/β response in the control of H5N1 virus replication both in vivo and in vitro, and as such it may provide some degree of protection to the host in the early stages of infection.

Immunity to influenza: the challenges of protecting an aging population.

Influenz a viruses cause annual epidemics and occasional pandemics of acute respiratory disease. Improved vaccines that can overcome the decline in immune function with aging and/or can induce broader immunity to novel pandemic strains are a high priority. To design improved vaccines for the elderly, we need to better understand the effects of age on both innate and adaptive immunity. In a murine model, we have determined that defects in antigen-presenting cell (APC) expression of pattern-recognition molecules, costimulatory molecules, and cytokine production may play an important role in the reduced clonal expansion of T cells in aging. The use of immunomodulators such as adjuvants may overcome some of the defects of aging immunity and may also be, useful in the development of improved vaccines for avian influenza A subtypes that pose a pandemic threat. Several novel strategies including the use of ISCOM-formulated vaccines, mucosal delivery, or DNA vaccination provided cross-subtype protection that could provide an important component of immunity in the event of a pandemic.

A Broadly Protective Vaccine against Globally Dispersed Clade 1 and Clade 2 H5N1 Influenza Viruses

Development of effective and immunogenic vaccines against highly pathogenic avian influenza H5N1 viruses with the potential to cause a pandemic is a public health priority. The global demand for a vaccine cannot be met in the event of an influenza pandemic because of the limited capacity to manufacture egg-derived vaccines as well as potential problems with the availability of embryonated eggs. Thus, there is an urgent need to develop alternative, egg-independent vaccines. We developed an adenoviral vector-based vaccine that contains hemagglutinin protein from clade 1 and clade 2 viruses, as well as conserved nucleoprotein, to broaden the vaccine coverage against H5N1 viruses.

Immunosenescence and Influenza Vaccine Efficacy

A number of protective immune functions decline with age along with physiological and anatomical changes, contributing to the increased susceptibility of older adults to infectious diseases and suboptimal protective immune responses to vaccination. Influenza vaccination is the most cost-effective strategy to prevent complications from influenza viral infections; however, the immunogenicity and effectiveness of currently licensed vaccines in the United States is about 30–50% in preventing complications arising from influenza and preventing death from all causes during winter months in older adults. Hence, it is crucial to understand the molecular mechanisms that lead to immune dysfunction as a function of age so that appropriate strategies can be developed to enhance the disease resistance and immunogenicity of preventive vaccines, including influenza vaccines, for the older adult population.