Tropism and neutralisation studies on bat influenza H17N10

The diversity of subtypes within Influenza A recently expanded with identification of H17N10 and H18N11 from bats. To study the tropism and zoonotic potential of these viruses, we successfully produced lentiviral pseudotypes bearing haemagglutinin H17 and neuraminidase N10. We investigated a range of cell lines from different species for their susceptibility to infection by these pseudotypes and show that a number of human haematopoietic cancer cell lines and the canine kidney MDCK II (but not MDCK I) cells are susceptible. Using microarrays and qRT-PCR we show that the dog leukocyte antigen DLA-DRA mRNA is over expressed in late passaged parental MDCK and commercial MDCK II cells, compared to early passaged parental MDCK and MDCK I cells, respectively. The human orthologue HLA-DRA encodes the alpha subunit of the MHC class II HLA-DR antigen-binding heterodimer. Small interfering RNA- or neutralizing antibody-targeting HLA-DRA, drastically reduced the susceptibility of Raji B cells to H17-PV. Conversely, over expression of HLA-DRA and its paralogue HLA-DRB1 on the surface of unsusceptible HEK293T/17 cells conferred susceptibility to H17-PV. The identification of HLA-DR as an H17N10 entry mediator will contribute to understanding the tropism of the virus and help to elucidate its zoonotic transmission. We also show that H17 pseudotypes can be efficiently neutralised by the broadly-neutralizing HA2 stalk monoclonal antibodies CR9114 and FI6. The lentiviral pseudotype system is a useful research tool, amenable for investigation of bat influenza tropism, restriction and pandemic preparedness, without safety issues of producing a replication-competent virus, to which the human population is naïve

Dramatic potentiation of the antiviral activity of HIV antibodies by cholesterol conjugation

The broadly neutralizing antibodies HIV 2F5 and 4E10, which bind to overlapping epitopes in the membrane- proximal external region (MPER) of the fusion protein gp41, have been proposed to use a two-step mechanism for neutralization: first, they bind and pre-concentrate at the viral membrane through their long, hydrophobic CDRH3 loops and second, they form a high-affinity complex with the protein epitope. Accordingly, mutagenesis of the CDRH3 can abolish their neutralizing activity, with no change in the affinity for the peptide epitope. We show here that we can mimic this mechanism by conjugating a cholesterol group outside of the paratope of an antibody. Cholesterol- conjugated antibodies bind to lipid-raft domains on the membrane and because of this enrichment, they show increased antiviral potency. In particular we find that cholesterol conjugation: (i) rescues the antiviral activity of CDRH3- mutated 2F5, (ii) increases the antiviral activity of WT 2F5, (iii) potentiates the non-membrane binding HIV antibody D5 10-100 fold (depending on the virus strain), and (iv) increases synergy between 2F5 and D5. Conjugation can be made at several positions, including variable and constant domains. Cholesterol conjugation therefore appears a general strategy to boost potency of antiviral antibodies and, since membrane affinity is engineered outside of the antibody paratope, it can complement affinity maturation strategies

Cholesterol conjugation potentiates the antiviral activity of an HIV immunoadhesin

Immunoadhesins are engineered proteins combining the constant domain (Fc) of an antibody with a ligand-binding (adhesion) domain. They have significant potential as therapeutic agents, because they maintain the favourable pharmacokinetics of antibodies with an expanded repertoire of ligand-binding domains: proteins, peptides, or small molecules. We have recently reported that the addition of a cholesterol group to two HIV antibodies can dramatically improve their antiviral potency. Cholesterol, which can be conjugated at various positions in the antibody, including the constant (Fc) domain, endows the conjugate with affinity for the membrane lipid rafts, thus increasing its concentration at the site where viral entry occurs. Here, we extend this strategy to an HIV immunoadhesin, combining a cholesterol-conjugated Fc domain with the peptide fusion inhibitor C41. The immunoadhesin C41-Fc-chol displayed high affinity for Human Embryonic Kidney (HEK) 293 cells, and when tested on a panel of HIV-1 strains, it was considerably more potent than the unconjugated C41-Fc construct. Potentiation of antiviral activity was comparable to what was previously observed for the cholesterol-conjugated HIV antibodies. Given the key role of cholesterol in lipid raft formation and viral fusion, we expect that the same strategy should be broadly applicable to enveloped viruses, for many of which it is already known the sequence of a peptide fusion inhibitor similar to C41. Moreover, the sequence of heptad repeat-derived fusion inhibitors can often be predicted from genomic information alone, opening a path to immunoadhesins against emerging viruses