Sensing of immature particles produced by dengue virus infected cells induces an antiviral response by plasmacytoid dendritic cells.

Dengue virus (DENV) is the leading cause of mosquito-borne viral illness and death in humans. Like many viruses, DENV has evolved potent mechanisms that abolish the antiviral response within infected cells. Nevertheless, several in vivo studies have demonstrated a key role of the innate immune response in controlling DENV infection and disease progression. Here, we report that sensing of DENV infected cells by plasmacytoid dendritic cells (pDCs) triggers a robust TLR7-dependent production of IFNα, concomitant with additional antiviral responses, including inflammatory cytokine secretion and pDC maturation. We demonstrate that unlike the efficient cell-free transmission of viral infectivity, pDC activation depends on cell-to-cell contact, a feature observed for various cell types and primary cells infected by DENV, as well as West Nile virus, another member of the Flavivirus genus. We show that the sensing of DENV infected cells by pDCs requires viral envelope protein-dependent secretion and transmission of viral RNA. Consistently with the cell-to-cell sensing-dependent pDC activation, we found that DENV structural components are clustered at the interface between pDCs and infected cells. The actin cytoskeleton is pivotal for both this clustering at the contacts and pDC activation, suggesting that this structural network likely contributes to the transmission of viral components to the pDCs. Due to an evolutionarily conserved suboptimal cleavage of the precursor membrane protein (prM), DENV infected cells release uncleaved prM containing-immature particles, which are deficient for membrane fusion function. We demonstrate that cells releasing immature particles trigger pDC IFN response more potently than cells producing fusion-competent mature virus. Altogether, our results imply that immature particles, as a carrier to endolysosome-localized TLR7 sensor, may contribute to regulate the progression of dengue disease by eliciting a strong innate response.

Human Influenza A Virus-Specific CD8+ T-Cell Response Is Long-lived.

Animal and human studies have demonstrated the importance of influenza A virus (IAV)-specific CD8(+) cytotoxic T lymphocytes (CTLs) in heterosubtypic cross-protective immunity. Using peripheral blood mononuclear cells obtained intermittently from healthy HLA-typed blood donors between 1999 and 2012, we were able to demonstrate that IAV-specific CTLs are long-lived. Intercurrent IAV infections transiently increase the frequency of functionally distinct subsets of IAV-specific CTLs, in particular effector and effector memory T cells.

Influenza B virus-specific CD8+ T-lymphocytes strongly cross-react with viruses of the opposing influenza B lineage.

Influenza B viruses fall in two antigenically distinct lineages (B/Victoria/2/1987 and B/Yamagata/16/1988 lineage) that co-circulate with influenza A viruses of the H3N2 and H1N1 subtypes during seasonal epidemics. Infections with influenza B viruses contribute considerably to morbidity and mortality in the human population. Influenza B virus neutralizing antibodies, elicited by natural infections or vaccination, poorly cross-react with viruses of the opposing influenza B lineage. Therefore, there is an increased interest in identifying other correlates of protection which could aid the development of broadly protective vaccines. blast analysis revealed high sequence identity of all viral proteins. With two online epitope prediction algorithms, putative conserved epitopes relevant for study subjects used in the present study were predicted. The cross-reactivity of influenza B virus-specific polyclonal CD8+ cytotoxic T-lymphocyte (CTL) populations obtained from HLA-typed healthy study subjects, with intra-lineage drift variants and viruses of the opposing lineage, was determined by assessing their in vitro IFN-γ response and lytic activity. Here, we show for the first time, to the best of our knowledge, that CTLs directed to viruses of the B/Victoria/2/1987 lineage cross-react with viruses of the B/Yamagata/16/1988 lineage and vice versa.

Human cytotoxic T lymphocytes directed to seasonal influenza A viruses cross-react with the newly emerging H7N9 virus.

In February 2013, zoonotic transmission of a novel influenza A virus of the H7N9 subtype was reported in China. Although at present no sustained human-to-human transmission has been reported, a pandemic outbreak of this H7N9 virus is feared. Since neutralizing antibodies to the hemagglutinin (HA) globular head domain of the virus are virtually absent in the human population, there is interest in identifying other correlates of protection, such as cross-reactive CD8(+) T cells (cytotoxic T lymphocytes [CTLs]) elicited during seasonal influenza A virus infections. These virus-specific CD8(+) T cells are known to recognize conserved internal proteins of influenza A viruses predominantly, but it is unknown to what extent they cross-react with the newly emerging H7N9 virus. Here, we assessed the cross-reactivity of seasonal H3N2 and H1N1 and pandemic H1N1 influenza A virus-specific polyclonal CD8(+) T cells, obtained from HLA-typed study subjects, with the novel H7N9 virus. The cross-reactivity of CD8(+) T cells to H7N9 variants of known influenza A virus epitopes and H7N9 virus-infected cells was determined by their gamma interferon (IFN-γ) response and lytic activity. It was concluded that, apart from recognition of individual H7N9 variant epitopes, CD8(+) T cells to seasonal influenza viruses display considerable cross-reactivity with the novel H7N9 virus. The presence of these cross-reactive CD8(+) T cells may afford some protection against infection with the new virus.

Infection of the upper respiratory tract with seasonal influenza A(H3N2) virus induces protective immunity in ferrets against infection with A(H1N1)pdm09 virus after intranasal, but not intratracheal, inoculation.

The clinical symptoms caused by infection with influenza A virus vary widely and depend on the strain causing the infection, the dose and route of inoculation, and the presence of preexisting immunity. In most cases, seasonal influenza A viruses cause relatively mild upper respiratory tract disease, while sometimes patients develop an acute severe pneumonia. Heterosubtypic immunity induced by previous infections with influenza A viruses may dampen the development of clinical symptoms caused by infection with influenza A viruses of another subtype, as is the case during influenza pandemics. Here we show that ferrets acquire protective immunity after infection of the upper respiratory tract with a seasonal influenza A(H3N2) virus against subsequent infection with influenza A(H1N1)pdm09 virus inoculated by the intranasal route. However, protective heterosubtypic immunity was afforded locally, since the prior infection with the A(H3N2) virus did not provide protection against the development of pneumonia induced after intratracheal inoculation with the A(H1N1)pdm09 virus. Interestingly, some of these animals developed more severe disease than that observed in naïve control animals. These findings are of interest in light of the development of so-called universal influenza vaccines that aim at the induction of cross-reactive T cell responses.

Human T-cells directed to seasonal influenza A virus cross-react with 2009 pandemic influenza A (H1N1) and swine-origin triple-reassortant H3N2 influenza viruses.

Virus-specific CD8(+) T-cells contribute to protective immunity against influenza A virus (IAV) infections. As the majority of these cells are directed to conserved viral proteins, they may afford protection against IAVs of various subtypes. The present study assessed the cross-reactivity of human CD8(+) T-lymphocytes, induced by infection with seasonal A (H1N1) or A (H3N2) influenza virus, with 2009 pandemic influenza A (H1N1) virus [A(H1N1)pdm09] and swine-origin triple-reassortant A (H3N2) [A(H3N2)v] viruses that are currently causing an increasing number of human cases in the USA. It was demonstrated that CD8(+) T-cells induced after seasonal IAV infections exerted lytic activity and produced gamma interferon upon in vitro restimulation with A(H1N1)pdm09 and A(H3N2)v influenza A viruses. Furthermore, CD8(+) T-cells directed to A(H1N1)pdm09 virus displayed a high degree of cross-reactivity with A(H3N2)v viruses. It was concluded that cross-reacting T-cells had the potential to afford protective immunity against A(H1N1)pdm09 viruses during the pandemic and offer some degree of protection against infection with A(H3N2)v viruses.

Cross-protective immunity against influenza pH1N1 2009 viruses induced by seasonal influenza A (H3N2) virus is mediated by virus-specific T-cells.

Influenza A (H1N1) viruses of swine origin were introduced into the human population in 2009 and caused a pandemic. The disease burden in the elderly was relatively low, which was attributed to the presence of cross-reacting serum antibodies in this age group, which were raised against seasonal influenza A (H1N1) viruses that circulated before 1957. It has also been described how infection with heterosubtypic influenza viruses can induce some degree of protection against infection by a novel strain of influenza virus. Here, we assess the extent of protective immunity against infection with the 2009 influenza A (H1N1) pandemic influenza virus that is afforded by infection with a seasonal influenza A (H3N2) virus in mice. Mice that experienced a primary A (H3N2) influenza virus infection displayed reduced weight loss after challenge infection and cleared the 2009 influenza A (H1N1) virus infection more rapidly. To elucidate the correlates of protection of this heterosubtypic immunity to pandemic H1N1 virus infection, adoptive transfer experiments were carried out by using selected post-infection lymphocyte populations. Virus-specific CD8(+) T-cells in concert with CD4(+) T-cells were responsible for the observed protection. These findings may not only provide an explanation for epidemiological differences in the incidence of severe pandemic H1N1 infections, they also indicate that the induction of cross-reactive virus-specific CD8(+) and CD4(+) T-cell responses may be a suitable approach for the development of universal influenza vaccines.

Induction of virus-specific cytotoxic T lymphocytes as a basis for the development of broadly protective influenza vaccines.

There is considerable interest in the development of broadly protective influenza vaccines because of the continuous emergence of antigenic drift variants of seasonal influenza viruses and the threat posed by the emergence of antigenically distinct pandemic influenza viruses. It has been recognized more than three decades ago that influenza A virus-specific cytotoxic T lymphocytes recognize epitopes located in the relatively conserved proteins like the nucleoprotein and that they cross-react with various subtypes of influenza A viruses. This implies that these CD8+ T lymphocytes may contribute to protective heterosubtypic immunity induced by antecedent influenza A virus infections. In the present paper, we review the evidence for the role of virus-specific CD8+ T lymphocytes in protective immunity against influenza virus infections and discuss vaccination strategies that aim at the induction of cross-reactive virus-specific T-cell responses.

Vaccination with whole inactivated virus vaccine affects the induction of heterosubtypic immunity against influenza virus A/H5N1 and immunodominance of virus-specific CD8+ T-cell responses in mice.

It was recently shown that the use of an experimental subunit vaccine protected mice against infection with a human A/H3N2 influenza virus, but consequently affected the induction of heterosubtypic immunity to a highly pathogenic A/H5N1 influenza virus, which was otherwise induced by the A/H3N2 infection. As whole inactivated virus (WIV) vaccines are widely used to protect against seasonal influenza and also contain inner viral proteins such as the nucleoprotein (NP), the potential of a WIV vaccine to induce protective immunity against infection was tested with a homologous A/H3N2 (A/Hong Kong/2/68) and a heterosubtypic A/H5N1 influenza virus (A/Indonesia/5/05). As expected, the vaccine afforded protection against infection with the A/H3N2 virus only. In addition, it was demonstrated that the use of WIV vaccine for protection against A/H3N2 infection affected the induction of heterosubtypic immunity that was otherwise afforded by A/H3N2 influenza virus infection. The reduction in protective immunity correlated with changes in the immunodominance patterns of the CD8(+) T-cell responses directed to the epitopes located in the acid polymerase subunit of the viral RNA polymerase (PA(224-233)) and the NP (NP(366-374)). In unvaccinated mice that experienced infection with the A/H3N2 influenza virus, the magnitude of the CD8(+) T-cell response to both peptides was similar on secondary infection with A/H5N1 influenza virus. In contrast, prior vaccination with WIV affected the immunodominance pattern and skewed the response after infection with influenza virus A/Indonesia/5/05 towards a dominant NP(366-374)-specific response. These findings may have implications for vaccination strategies aimed at the induction of protective immunity to seasonal and/or pandemic influenza.

Enhanced Antiviral Activity of Human Surfactant Protein D by Site-Specific Engineering of the Carbohydrate Recognition Domain.

Innate immunity is critical in the early containment of influenza A virus (IAV) infection and surfactant protein D (SP-D) plays a crucial role in innate defense against IAV in the lungs. Multivalent lectin-mediated interactions of SP-D with IAVs result in viral aggregation, reduced epithelial infection, and enhanced IAV clearance by phagocytic cells. Previous studies showed that porcine SP-D (pSP-D) exhibits distinct antiviral activity against IAV as compared to human SP-D (hSP-D), mainly due to key residues in the lectin domain of pSP-D that contribute to its profound neutralizing activity. These observations provided the basis for the design of a full-length recombinant mutant form of hSP-D, designated as "improved SP-D" (iSP-D). Inspired by pSP-D, the lectin domain of iSP-D has 5 amino acids replaced (Asp324Asn, Asp330Asn, Val251Glu, Lys287Gln, Glu289Lys) and 3 amino acids inserted (326Gly-Ser-Ser). Characterization of iSP-D revealed no major differences in protein assembly and saccharide binding selectivity as compared to hSP-D. However, hemagglutination inhibition measurements showed that iSP-D expressed strongly enhanced activity compared to hSP-D against 31 different IAV strains tested, including (pandemic) IAVs that were resistant for neutralization by hSP-D. Furthermore, iSP-D showed increased viral aggregation and enhanced protection of MDCK cells against infection by IAV. Importantly, prophylactic or therapeutic application of iSP-D decreased weight loss and reduced viral lung titers in a murine model of IAV infection using a clinical isolate of H1N1pdm09 virus. These studies demonstrate the potential of iSP-D as a novel human-based antiviral inhalation drug that may provide immediate protection against or recovery from respiratory (pandemic) IAV infections in humans.

Assessment of the antiviral properties of recombinant surfactant protein D against influenza B virus in vitro.

The armamentarium of antiviral drugs against influenza viruses is limited. Furthermore, influenza viruses emerge that are resistant to existing antiviral drugs like the M2 and NA inhibitors. Therefore, there is an urgent need for the development of novel classes of antiviral drugs. Here we investigated the antiviral properties of recombinant porcine surfactant protein D (RpSP-D), an innate defense molecule with lectin properties, against influenza B viruses. We have previously shown that porcine SP-D has more potent neutralizing activity against influenza A viruses than human SP-D. Here we show that RpSP-D neutralizes influenza B viruses efficiently and inhibited the binding of these viruses to epithelial cells of the human trachea.

Recombinant porcine surfactant protein D inhibits influenza A virus replication ex vivo.

Influenza is a major burden to public health. Due to high mutation rates and selection pressure, mutant viruses emerge which are resistant to currently used antiviral drugs. Therefore, there is a need for the development of novel classes of antiviral drugs that suffer less from the emergence of resistant viruses. Antiviral drugs based on collectin-like surfactant protein D (SP-D) may fulfil these requirements. Especially porcine SP-D displays strong antiviral activity to influenza A viruses. In the present study the antiviral activity of recombinant porcine SP-D was investigated in ex vivo cultures of respiratory tract tissue infected with human influenza A virus of the H3N2 subtype. Porcine SP-D has antiviral activity in these test systems. It is suggested that porcine SP-D may be used as a venue to develop a novel class of antiviral drugs.

Pulmonary surfactant protein D in first-line innate defence against influenza A virus infections.

Influenza A viruses (IAV) cause respiratory tract infections annually associated with excess mortality and morbidity. Nonspecific, innate immune mechanisms play a key role in protection against viral invasion at early stages of infection. A soluble protein present in mucosal secretions of the lung, surfactant protein D (SP-D), is an important component of this initial barrier that helps to prevent and limit IAV infections of the respiratory epithelium. This collagenous C-type lectin binds IAVs and thereby inhibits attachment and entry of the virus but also contributes to enhanced clearance of SP-D-opsonized virus via interactions with phagocytic cells. In addition, SP-D modulates the inflammatory response and helps to maintain a balance between effective neutralization/killing of IAV, and protection against alveolar damage resulting from IAV-induced excessive inflammatory responses. The mechanisms of interaction between SP-D and IAV not only depend on the structure and binding properties of SP-D but also on strain-specific features of IAV, and both issues will be discussed. SP-D from pigs exhibits distinct anti-IAV properties and is discussed in more detail. Finally, the potential of SP-D as a prophylactic and/or therapeutic antiviral agent to protect humans against infections by IAV is discussed.

Binding of DC-SIGN to the hemagglutinin of influenza A viruses supports virus replication in DC-SIGN expressing cells.

Dendritic cells express lectins receptors, like DC-SIGN, which allow these cells to sense glycans that are present on various bacterial and viral pathogens. Interaction of DC-SIGN with carbohydrate moieties induces maturation of dendritic cells and promotes endocytosis of pathogens which is an important property of these professional antigen presenting cells. Uptake of pathogens by dendritic cells may lead to cross-presentation of antigens or infection of these cells, which ultimately results in activation of virus-specific T cells in draining lymph nodes. Little is known about the interaction of DC-SIGN with influenza A viruses. Here we show that a virus with a non-functional receptor binding site in its hemagglutinin, can replicate in cells expressing DC-SIGN. Also in the absence of sialic acids, which is the receptor for influenza A viruses, these viruses replicate in DC-SIGN expressing cells including human dendritic cells. Furthermore, the efficiency of DC-SIGN mediated infection is dependent on the extent of glycosylation of the viral hemagglutinin.

The number and position of N-linked glycosylation sites in the hemagglutinin determine differential recognition of seasonal and 2009 pandemic H1N1 influenza virus by porcine surfactant protein D.

C-type lectins are important molecules of the innate immune system. These molecules, like surfactant protein D (SP-D) can recognize glycans on pathogens and neutralize these. Also influenza viruses are recognized by SP-D and their susceptibility to neutralization by SP-D is dependent on the number of N-linked glycosylation sites in the hemagglutinin in particular. Porcine SP-D displayed stronger neutralizing activity to human influenza A viruses than to swine influenza A viruses. Although viruses from these species differ with regard to the number of glycosylation sites in the hemagglutinin, the mechanism underlying the differential recognition by porcine SP-D is poorly understood. Here we investigated the molecular basis for the differential recognition of a seasonal H1N1 and a 2009 pandemic H1N1 virus by porcine SP-D. We demonstrated that the number and position of glycosylation sites determine viral susceptibility to the neutralizing activity of porcine SP-D. However, predicting the effect remains difficult as it was shown to be dependent on the strain and the position of the glycosylation sites.

Assessment of the antiviral properties of recombinant porcine SP-D against various influenza A viruses in vitro.

The emergence of influenza viruses resistant to existing classes of antiviral drugs raises concern and there is a need for novel antiviral agents that could be used therapeutically or prophylacticaly. Surfactant protein D (SP-D) belongs to the family of C-type lectins which are important effector molecules of the innate immune system with activity against bacteria and viruses, including influenza viruses. In the present study we evaluated the potential of recombinant porcine SP-D as an antiviral agent against influenza A viruses (IAVs) in vitro. To determine the range of antiviral activity, thirty IAVs of the subtypes H1N1, H3N2 and H5N1 that originated from birds, pigs and humans were selected and tested for their sensitivity to recombinant SP-D. Using these viruses it was shown by hemagglutination inhibition assay, that recombinant porcine SP-D was more potent than recombinant human SP-D and that especially higher order oligomeric forms of SP-D had the strongest antiviral activity. Porcine SP-D was active against a broad range of IAV strains and neutralized a variety of H1N1 and H3N2 IAVs, including 2009 pandemic H1N1 viruses. Using tissue sections of ferret and human trachea, we demonstrated that recombinant porcine SP-D prevented attachment of human seasonal H1N1 and H3N2 virus to receptors on epithelial cells of the upper respiratory tract. It was concluded that recombinant porcine SP-D holds promise as a novel antiviral agent against influenza and further development and evaluation in vivo seems warranted.