Genetic Characterization of H3N2 Influenza Viruses Isolated from Pigs in North America, 1977–1999: Evidence for Wholly Human and Reassortant Virus Genotypes

Since 1998, H3N2 viruses have caused epizootics of respiratory disease in pigs throughout the major swine production regions of the U.S. These outbreaks are remarkable because swine influenza in North America had previously been caused almost exclusively by H1N1 viruses. We sequenced the full-length protein coding regions of all eight RNA segments from four H3N2 viruses that we isolated from pigs in the Midwestern U.S. between March 1998 and March 1999, as well as from H3N2 viruses recovered from a piglet in Canada in January 1997 and from a pig in Colorado in 1977. Phylogenetic analyses demonstrated that the 1977 Colorado and 1997 Ontario isolates are wholly human influenza viruses. However, the viruses isolated since 1998 from pigs in the Midwestern U.S. are reassortant viruses containing hemagglutinin, neuraminidase and PB1 polymerase genes from human influenza viruses, matrix, non-structural and nucleoprotein genes from classical swine viruses, and PA and PB2 polymerase genes from avian viruses. The HA proteins of the Midwestern reassortant swine viruses can be differentiated from those of the 1995 lineage of human H3 viruses by 12 amino acid mutations in HA1. In contrast, the Sw:ONT:97 virus, which did not spread from pig-to-pig, lacks 11 of these changes

Restricted Infectivity of a Human-Lineage H3N2 Influenza A Virus in Pigs Is Hemagglutinin and Neuraminidase Gene Dependent

Influenza A viruses cause pandemics at sporadic intervals. Pandemic viruses can potentially be introduced into the human population through in toto transfer of an avian influenza virus or through reassortment between avian and human strains. Pigs are believed to play a central role in the creation of pandemic viruses through reassortment because of their susceptibility to infection with both avian and human influenza viruses. However, we recently found that a human-lineage H3N2 influenza virus was highly restricted in its ability to infect pigs after intranasal inoculation. We hypothesized that this restricted infectivity phenotype was controlled by the hemagglutinin (HA) and neuraminidase (NA). To test this, we infected pigs with reverse genetics-created HA plus NA reassortant viruses. Specifically, introduction of the HA and NA genes of a contemporary H3N2 swine virus into the genetic background of the wholly human virus resulted in a significant increase in virus shedding and pathogenicity. These data indicate that the HA/NA can play important roles in controlling human influenza virus infectivity in pigs. The results further support the premise that a barrier exists to human influenza virus infection in pigs, which may limit the role of pigs in pandemic virus creation through reassortment of human and avian influenza viruses

Nfatc2 and Tob1 have non-overlapping function in T cell negative regulation and tumorigenesis.

Nfatc2 and Tob1 are intrinsic negative regulators of T cell activation. Nfatc2-deficient and Tob1-deficient T cells show reduced thresholds of activation; however, whether these factors have independent or overlapping roles in negative regulation of T cell responses has not been previously examined. Here, we show that Nfatc2 knockout (KO) but not Tob1 KO mice have age-associated accumulation of persistently activated T cells in vivo and expansion of the CD44+ memory cell compartment and age-associated lymphocytic infiltrates in visceral organs, without significant changes in numbers of CD4+CD25+Foxp3+ regulatory T cells (Treg). In vitro, CD4+CD25- "conventional" T cells (Tconvs) from both KO strains showed greater proliferation than wild type (WT) Tconvs. However, while Tregs from Nfatc2 KO mice retained normal suppressive function, Tregs from Tob1 KOs had enhanced suppressive activity. Nfatc2 KO Tconvs expanded somewhat more rapidly than WT Tconvs under conditions of homeostatic proliferation, but their accelerated growth capacity was negated, at least acutely, in a lymphoreplete environment. Finally, Nfatc2 KO mice developed a previously uncharacterized increase in B-cell malignancies, which was not accelerated by the absence of Tob1. The data thus support the prevailing hypothesis that Nfatc2 and Tob1 are non-redundant regulators of lymphocyte homeostasis

An anti-gdnf family receptor alpha 1 (gfra1 antibody–drug conjugate for the treatment of hormone receptor–positive breast cancer

Luminal A (hormone receptor-positive) breast cancer constitutes 70% of total breast cancer patients. In an attempt to develop a targeted therapeutic for this cancer indication, we have identified and characterized Glial cell line–Derived Neurotrophic Factor (GDNF) Family Receptor Alpha 1 (GFRA1) antibody–drug conjugates (ADC) using a cleavable valine-citrulline-MMAE (vcMMAE) linker-payload. RNAseq and IHC analysis confirmed the abundant expression of GFRA1 in luminal A breast cancer tissues, whereas minimal or no expression was observed in most normal tissues. Anti–GFRA-vcMMAE ADC internalized to the lysosomes and exhibited target-dependent killing of GFRA1-expressing cells both in vitro and in vivo. The ADCs using humanized anti-GFRA1 antibodies displayed robust therapeutic activity in clinically relevant cell line–derived (MCF7 and KPL-1) tumor xenograft models. The lead anti-GFRA1 ADC cross-reacts with rodent and cynomolgus monkey GFRA1 antigen and showed optimal pharmacokinetic properties in both species. These properties subsequently enabled a target-dependent toxicity study in rats. Anti-GFRA1 ADC is well tolerated in rats, as seen with other vcMMAE linker–payload based ADCs. Overall, these data suggest that anti–GFRA1-vcMMAE ADC may provide a targeted therapeutic opportunity for luminal A breast cancer patients. Mol Cancer Ther; 17(3); 638–49. 2017 AACR

<i>Nfatc2</i> KO mice, but not <i>Tob1</i> KO mice show age-related accumulation of memory T cells.

<p>(A) Spleen and lymph node cells were isolated from age-matched WT, <i>Nfatc2</i> KO, and <i>Tob1</i> KO mice, and cells from each genotype and for each organ were pooled for experiments. Expression of CD44 and CD62L was measured by conventional cell surface staining in WT, <i>Nfatc2</i> KO, and <i>Tob1</i> KO T cells immediately after isolation from spleens or lymph nodes. Panels are representative two-dimensional contour plots showing CD44 and CD62L staining from young (top) and old (bottom) mice gated on CD3 T cells from representative mice as indicated. Similar data for young and old mice were obtained in 9 and 4 experiments using WT cells, 6 and 4 experiments using <i>Nfatc2</i> KO cells, and 4 and 4 experiments using <i>Tob1</i> KO cells, respectively. Means ± SD are provided in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0100629#pone-0100629-t001" target="_blank">Table 1</a>.</p

<i>Nfatc2</i> deficiency leads to enhanced proliferation in a lymphodepleted environment of homeostatic expansion, but does not provide improved survival fitness in a competitive lymphoreplete environment.

<p>CD4⁺CD25⁻ Tconvs were prepared as described from WT and from <i>Nfatc2</i> KO mice. Sorted cells (100,000) were adoptively transferred into the tail vein of two B6.SCID (top) or two B6.CD45.1 (bottom) mice. Recipients were sacrificed 15 days later and total cell numbers recovered from spleens (SP) and lymph nodes (LN) were enumerated using a CellDyn 3500 hematology analyzer. The percent of donor cells in each recipient was then calculated based on the percent of CD4⁺CD45.2⁺ cells present in each preparation, and is represented by a symbol in the graphs. Lines indicate the mean for each recipient group.</p