Infection of Mouse Macrophages by Seasonal Influenza Viruses Can Be Restricted at the Level of Virus Entry and at a Late Stage in the Virus Life Cycle.

UNLABELLED: Airway epithelial cells are susceptible to infection with seasonal influenza A viruses (IAV), resulting in productive virus replication and release. Macrophages (MΦ) are also permissive to IAV infection; however, virus replication is abortive. Currently, it is unclear how productive infection of MΦ is impaired or the extent to which seasonal IAV replicate in MΦ. Herein, we compared mouse MΦ and epithelial cells for their ability to support genomic replication and transcription, synthesis of viral proteins, assembly of virions, and release of infectious progeny following exposure to genetically defined IAV. We confirm that seasonal IAV differ in their ability to utilize cell surface receptors for infectious entry and that this represents one level of virus restriction. Following virus entry, we demonstrate synthesis of all eight segments of genomic viral RNA (vRNA) and mRNA, as well as seven distinct IAV proteins, in IAV-infected mouse MΦ. Although newly synthesized hemagglutinin (HA) and neuraminidase (NA) glycoproteins are incorporated into the plasma membrane and expressed at the cell surface, electron microscopy confirmed that virus assembly was defective in IAV-infected MΦ, defining a second level of restriction late in the virus life cycle. IMPORTANCE: Seasonal influenza A viruses (IAV) and highly pathogenic avian influenza viruses (HPAI) infect macrophages, but only HPAI replicate productively in these cells. Herein, we demonstrate that impaired virus uptake into macrophages represents one level of restriction limiting infection by seasonal IAV. Following uptake, seasonal IAV do not complete productive replication in macrophages, representing a second level of restriction. Using murine macrophages, we demonstrate that productive infection is blocked late in the virus life cycle, such that virus assembly is defective and newly synthesized virions are not released. These studies represent an important step toward identifying host-encoded factors that block replication of seasonal IAV, but not HPAI, in macrophages.

Influenza virus PB1 and neuraminidase gene segments can cosegregate during vaccine reassortment driven by interactions in the PB1 coding region.

UNLABELLED: 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.

The source of the PB1 gene in influenza vaccine reassortants selectively alters the hemagglutinin content of the resulting seed virus.

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.

Abortive replication of influenza virus in mouse dendritic cells.

The interaction between influenza virus and dendritic cells (DCs) remains poorly defined and controversial. Here we show that influenza virus replication in mouse bone marrow-derived DCs is abortive, despite viral genome transcription and replication occurring for each gene segment and viral hemagglutinin and nucleoprotein, at least, being produced. Electron microscopy reveals that virus assembly, rather than release of virus from the cell surface, is defective.

Cold adaptation generates mutations associated with the growth of influenza B vaccine viruses.

Seasonal inactivated influenza vaccines are usually trivalent or quadrivalent and are prepared from accredited seed viruses. Yields of influenza A seed viruses can be enhanced by gene reassortment with high-yielding donor strains, but similar approaches for influenza B seed viruses have been largely unsuccessful. For vaccine manufacture influenza B seed viruses are usually adapted for high-growth by serial passage. Influenza B antigen yields so obtained are often unpredictable and selection of influenza B seed viruses by this method can be a rate-limiting step in seasonal influenza vaccine manufacture. We recently have shown that selection of stable cold-adapted mutants from seasonal epidemic influenza B viruses is associated with improved growth. In this study, specific mutations were identified that were responsible for growth enhancement as a consequence of adaptation to growth at lower temperatures. Molecular analysis revealed that the following mutations in the HA, NP and NA genes are required for enhanced viral growth: G156/N160 in the HA, E253, G375 in the NP and T146 in the NA genes. These results demonstrate that the growth of seasonal influenza B viruses can be optimized or improved significantly by specific gene modifications.

Cold adaptation improves the growth of seasonal influenza B vaccine viruses.

Gene reassortment has proved useful in improving yields of influenza A antigens of egg-based inactivated vaccines, but similar approaches have been difficult with influenza B antigens. Current regulations for influenza vaccine seed viruses limit the number of egg passages and as a result resultant yields from influenza B vaccine seed viruses are frequently inconsistent. Therefore, reliable approaches to enhance yields of influenza B vaccine seed viruses are required for efficient vaccine manufacture. In the present study three stable cold-adapted (ca) mutants, caF, caM and caB derived from seasonal epidemic strains, B/Florida/4/2006, B/Malaysia/2506/2004 and B/Brisbane/60/2008 were prepared, which produced high hemagglutinin antigen yields and also increased viral yields of reassortants possessing the desired 6:2 gene constellation. The results demonstrate that consistent improvements in yields of influenza B viruses can be obtained by cold adaptation following extended passage. Taken together, the three ca viruses were shown to have potential as donor viruses for the preparation of high-yielding influenza B vaccine viruses by reassortment.

Targeting lymphocyte Peyer's patch adhesion molecule-1: a relay approach to gut immunization.

Targeting vaccines to dendritic cells (DCs) can enhance responses to weak vaccine antigens. Although there are molecules that are relatively specific for the various DC subsets, there are none that are both region-specific and DC-specific. This has provided some limitation to targeting regional DC populations. We proposed that these limits could be overcome by targeting antigens not to the DC subsets directly but to cells that persistently seek out and closely interact with DCs, namely lymphocytes. To investigate this hypothesis, we targeted antigens to a unique population of gut-homing lymphocytes and then looked at the induction of immune responses at this site. Using an anti-LPAM-1 (Lymphocyte Peyer's patch adhesion molecule-1; alpha(4)beta(7) integrin) monoclonal antibody (mAb) as a model antigen, we found that targeting gut-homing lymphocytes could significantly elevate the gut mucosal IgA response. Moreover, such a strategy greatly elevated the systemic IgG as well as IgA response. We found that LPAM-1-targeting enhanced the localization of antigen to both the systemic and mucosal lymphoid compartments where both IgA and IgG responses were induced. We also found that any parenteral route of delivery sufficed. Overall, targeting unique populations of lymphocytes may provide a strategy for ferrying antigen to sites that such lymphocytes home to.