Brad Gilbertson

Antiviral Activity of the Long Chain Pentraxin PTX3 against Influenza Viruses

Proteins of the innate immune system can act as natural inhibitors of influenza virus, limiting growth and spread of the virus in the early stages of infection before the induction of adaptive immune responses. In this study, we identify the long pentraxin PTX3 as a potent innate inhibitor of influenza viruses both in vitro and in vivo. Human and murine PTX3 bound to influenza virus and mediated a range of antiviral activities, including inhibition of hemagglutination, neutralization of virus infectivity and inhibition of viral neuraminidase. Antiviral activity was associated with binding of the viral hemagglutinin glycoprotein to sialylated ligands present on PTX3. Using a mouse model we found PTX3 to be rapidly induced following influenza infection and that PTX3−/− mice were more susceptible than wild-type mice to infection by PTX3-sensitive virus strains. Therapeutic treatment of mice with human PTX3 promoted survival and reduced viral load in the lungs following infection with PTX3-sensitive, but not PTX3-resistant, influenza viruses. Together, these studies describe a novel antiviral role for PTX3 in early host defense against influenza infections both in vitro and in vivo and describe the therapeutic potential of PTX3 in ameliorating disease during influenza infection.

Neuraminidase-Inhibiting Antibody Is a Correlate of Cross-Protection against Lethal H5N1 Influenza Virus in Ferrets Immunized with Seasonal Influenza Vaccine

ABSTRACT In preparing for the threat of a pandemic of avian H5N1 influenza virus, we need to consider the significant delay (4 to 6 months) necessary to produce a strain-matched vaccine. As some degree of cross-reactivity between seasonal influenza vaccines and H5N1 virus has been reported, this was further explored in the ferret model to determine the targets of protective immunity. Ferrets were vaccinated with two intramuscular inoculations of trivalent inactivated split influenza vaccine or subcomponent vaccines, with and without adjuvant, and later challenged with a lethal dose of A/Vietnam/1203/2004 (H5N1) influenza virus. We confirmed that vaccination with seasonal influenza vaccine afforded partial protection against lethal H5N1 challenge and showed that use of either AlPO4 or Iscomatrix adjuvant with the vaccine resulted in complete protection against disease and death. The protection was due exclusively to the H1N1 vaccine component, and although the hemagglutinin contributed to protection, the dominant protective response was targeted toward the neuraminidase (NA) and correlated with sialic acid cleavage-inhibiting antibody titers. Purified heterologous NA formulated with Iscomatrix adjuvant was also protective. These results suggest that adjuvanted seasonal trivalent vaccine could be used as an interim measure to decrease morbidity and mortality from H5N1 prior to the availability of a specific vaccine. The data also highlight that an inducer of cross-protective immunity is the NA, a protein whose levels are not normally monitored in vaccines and whose capacity to induce immunity in recipients is not normally assessed.

Influenza-induced inflammation drives pneumococcal otitis media.

ABSTRACT Influenza A virus (IAV) predisposes individuals to secondary infections with the bacterium Streptococcus pneumoniae (the pneumococcus). Infections may manifest as pneumonia, sepsis, meningitis, or otitis media (OM). It remains controversial as to whether secondary pneumococcal disease is due to the induction of an aberrant immune response or IAV-induced immunosuppression. Moreover, as the majority of studies have been performed in the context of pneumococcal pneumonia, it remains unclear how far these findings can be extrapolated to other pneumococcal disease phenotypes such as OM. Here, we used an infant mouse model, human middle ear epithelial cells, and a series of reverse-engineered influenza viruses to investigate how IAV promotes bacterial OM. Our data suggest that the influenza virus HA facilitates disease by inducing a proinflammatory response in the middle ear cavity in a replication-dependent manner. Importantly, our findings suggest that it is the inflammatory response to IAV infection that mediates pneumococcal replication. This study thus provides the first evidence that inflammation drives pneumococcal replication in the middle ear cavity, which may have important implications for the treatment of pneumococcal OM.

Bystander Activation of CD8+ T Lymphocytes during Experimental Mycobacterial Infection

ABSTRACT Infection of C57BL/6 mice with Mycobacterium avium leads to the activation of both CD4+ and CD8+ gamma interferon (IFN-γ)-producing T cells, although the CD8+ cells play no role in protection against infection. Using transfer of different lines of transgenic T cells with T-cell receptors (TCRs) which recognize irrelevant antigens, we show here that transferred CD8+ T cells from two of the three lines were activated to the same degree as the host cells, suggesting that the majority of the IFN-γ-producing CD8+ T cells of the host represented bystander activation. The third line, specific for the male HY antigen, showed no activation. Activation required the participation of the CD28 coreceptor on T cells and was unaffected by the removal of CD44hi (memory phenotype) T cells. The transferred CD8+ T cells proliferated in vivo, although this was not essential for IFN-γ production. Taken together, these data are highly reminiscent of homeostatic proliferation of TCR transgenic T cells upon transfer to lymphopenic hosts, and suggest low-affinity stimulation through the TCR, possibly by self peptides. The findings are discussed in relation to homeostatic proliferation and their significance in the possible induction of autoimmune disease.

A Mouse Model for the Study of Contact-Dependent Transmission of Influenza A Virus and the Factors That Govern Transmissibility

ABSTRACT Influenza A virus transmission by direct contact is not well characterized. Here, we describe a mouse model for investigation of factors regulating contact-dependent transmission. Strains within the H3N2 but not H1N1 subtype of influenza virus were transmissible, and reverse-engineered viruses representing hybrids of these subtypes showed that the viral hemagglutinin is a determinant of the transmissible phenotype. Transmission to contact mice occurred within the first 6 to 54 h after cohousing with directly infected index mice, and the proportion of contacts infected within this period was reduced if the index mice had been preinfected with a heterologous subtype virus. A threshold level of virus present in the saliva of the index mice was identified, above which the likelihood of transmission was greatly increased. There was no correlation with transmission and viral loads in the nose or lung. This model could be useful for preclinical evaluation of antiviral and vaccine efficacy in combating contact-dependent transmission of influenza.

Serum amyloid P is a sialylated glycoprotein inhibitor of influenza A viruses.

Members of the pentraxin family, including PTX3 and serum amyloid P component (SAP), have been reported to play a role in innate host defence against a range of microbial pathogens, yet little is known regarding their antiviral activities. In this study, we demonstrate that human SAP binds to human influenza A virus (IAV) strains and mediates a range of antiviral activities, including inhibition of IAV-induced hemagglutination (HA), neutralization of virus infectivity and inhibition of the enzymatic activity of the viral neuraminidase (NA). Characterization of the anti-IAV activity of SAP after periodate or bacterial sialidase treatment demonstrated that α(2,6)-linked sialic acid residues on the glycosidic moiety of SAP are critical for recognition by the HA of susceptible IAV strains. Other proteins of the innate immune system, namely human surfactant protein A and porcine surfactant protein D, have been reported to express sialylated glycans which facilitate inhibition of particular IAV strains, yet the specific viral determinants for recognition of these inhibitors have not been defined. Herein, we have selected virus mutants in the presence of human SAP and identified specific residues in the receptor-binding pocket of the viral HA which are critical for recognition and therefore susceptibility to the antiviral activities of SAP. Given the widespread expression of α(2,6)-linked sialic acid in the human respiratory tract, we propose that SAP may act as an effective receptor mimic to limit IAV infection of airway epithelial cells.

The Source of the PB1 Gene in Influenza Vaccine Reassortants Selectively Alters the Hemagglutinin Content of the Resulting Seed Virus

ABSTRACT The yields of egg-grown influenza vaccines are maximized by the production of a seed strain using a reassortment of the seasonal influenza virus isolate with a highly egg-adapted strain. The seed virus is selected based on high yields of viral hemagglutinin (HA) and expression of the surface antigens from the seasonal isolate. The remaining proteins are usually derived from the high-growth parent. However, a retrospective analysis of vaccine seeds revealed that the seasonal PB1 gene was selected in more than 50% of reassortment events. Using the model seasonal H3N2 virus A/Udorn/307/72 (Udorn) virus and the high-growth A/Puerto Rico/8/34 (PR8) virus, we assessed the influence of the source of the PB1 gene on virus growth and vaccine yield. Classical reassortment of these two strains led to the selection of viruses that predominantly had the Udorn PB1 gene. The presence of Udorn PB1 in the seed virus, however, did not result in higher yields of virus or HA compared to the yields in the corresponding seed virus with PR8 PB1. The 8-fold-fewer virions produced with the seed virus containing the Udorn PB1 were somewhat compensated for by a 4-fold increase in HA per virion. A higher HA/nucleoprotein (NP) ratio was found in past vaccine preparations when the seasonal PB1 was present, also indicative of a higher HA density in these vaccine viruses. As the HA viral RNA (vRNA) and mRNA levels in infected cells were similar, we propose that PB1 selectively alters the translation of viral mRNA. This study helps to explain the variability of vaccine seeds with respect to HA yield.

Influenza Virus PB1 and Neuraminidase Gene Segments Can Cosegregate during Vaccine Reassortment Driven by Interactions in the PB1 Coding Region

ABSTRACT Egg-grown influenza vaccine yields are maximized by infection with a seed virus produced by “classical reassortment” of a seasonal isolate with a highly egg-adapted strain. Seed viruses are selected based on a high-growth phenotype and the presence of the seasonal hemagglutinin (HA) and neuraminidase (NA) surface antigens. Retrospective analysis of H3N2 vaccine seed viruses indicated that, unlike other internal proteins that were predominantly derived from the high-growth parent A/Puerto Rico/8/34 (PR8), the polymerase subunit PB1 could be derived from either parent depending on the seasonal strain. We have recently shown that A/Udorn/307/72 (Udorn) models a seasonal isolate that yields reassortants bearing the seasonal PB1 gene. This is despite the fact that the reverse genetics-derived virus that includes Udorn PB1 with Udorn HA and NA on a PR8 background has inferior growth compared to the corresponding virus with PR8 PB1. Here we use competitive plasmid transfections to investigate the mechanisms driving selection of a less fit virus and show that the Udorn PB1 gene segment cosegregates with the Udorn NA gene segment. Analysis of chimeric PB1 genes revealed that the coselection of NA and PB1 segments was not directed through the previously identified packaging sequences but through interactions involving the internal coding region of the PB1 gene. This study identifies associations between viral genes that can direct selection in classical reassortment for vaccine production and which may also be of relevance to the gene constellations observed in past antigenic shift events where creation of a pandemic virus has involved reassortment. IMPORTANCE Influenza vaccine must be produced and administered in a timely manner in order to provide protection during the winter season, and poor-growing vaccine seed viruses can compromise this process. To maximize vaccine yields, manufacturers create hybrid influenza viruses with gene segments encoding the surface antigens from a seasonal virus isolate, important for immunity, and others from a virus with high growth properties. This involves coinfection of cells with both parent viruses and selection of dominant progeny bearing the seasonal antigens. We show that this method of creating hybrid viruses does not necessarily select for the best yielding virus because preferential pairing of gene segments when progeny viruses are produced determines the genetic makeup of the hybrids. This not only has implications for how hybrid viruses are selected for vaccine production but also sheds light on what drives and limits hybrid gene combinations that arise in nature, leading to pandemics.

Apoptosis of CD4+ and CD8+ T cells during experimental infection with Mycobacterium avium is controlled by Fas/FasL and Bcl-2-sensitive pathways, respectively.

Both CD4+ and CD8+ T cells from mice infected with Mycobacterium avium suffered a high rate of apoptosis, beginning with the onset of the immune response and culminating in the loss of T cells from the tissues and loss of IFN‐γ production. Fas expression increased over the course of infection on both T cell populations, as did their susceptibility to the induction of apoptosis in vitro by anti‐Fas mAb. Nevertheless, although the rate of apoptosis among CD4+ T cells from infected mice was reduced to normal levels in lpr mice with a defective Fas, CD8+ T cells were unaffected, implying that Fas/FasL interaction was not important in these cells in vivo. Conversely, over‐expression of B‐cell lymphoma‐2 (Bcl‐2), which is known to protect T cells from apoptosis signalled through the TNF receptor or due to the withdrawal of cytokines, totally protected CD8+ T cells from infected mice but had no effect on CD4+. It is of interest that these two contrasting pathways of T‐cell apoptosis operate at the same time during a single infection.

Lipopeptide vaccines illustrate the potential role of subtype‐crossreactive T cells in the control of highly virulent influenza

Background  The best form of protection against influenza is high‐titred virus‐neutralizing antibody specific for the challenge strain. However, this is not always possible to achieve by vaccination due to the need for predicting the emerging virus, whether it be a drift variant of existing human endemic influenza type A subtypes or the next pandemic virus, for incorporation into the vaccine. By activating additional arms of the immune system to provide heterosubtypic immunity, that is immunity active against all viruses of type A influenza regardless of subtype or strain, it should be possible to provide significant benefit in situations where appropriate antibody responses are not achieved. Although current inactivated vaccines are unable to induce heterosubtypic CD8+ T cell immunity, we have shown that lipopeptides are particularly efficient in this regard.