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OA01.02 B. Heydarchi (1); D. Fong (1); A. Sethi (2); J. Edwards (1); N. Salazar Quiroz (1); T. Aktepe (1); C. Gonelli (1); S. Grimley (1); C. Mackenzie (1); B. [...], Background: A vaccine against HIV would ideally elicit broadly neutralising antibodies (BrNAbs). In HIV+ patients these immunoglobulins (Ig) are rare and often display long third heavy complementarity determining regions (CDRH3) with high levels of somatic hypermutation. The average length of bovine Ig-CDRH3 is notably longer than other species and has potential for raising novel BrNAbs. We previously showed that cows vaccinated with HIV envelope (Env) produced BrNAbs that target the CD4 binding site (CD4bs). Here we used a stabilized trimer gp140 SOSIPv4.1 from the neutralisation resistant ADA subtype-B strain to select a lineage of chimeric human monoclonal BrNAbs containing bovine V-regions with ultra-long CDRH3. Methods: To produce monoclonal BrNAbs (mAbs), bovine memory B-cells were isolated from cows vaccinated with AD8 gp140 (clade B), AD8 SOSIP gp140v4.1 or KNH1 SOSIP gp140 and BG505 SOSIP664 gp140. The IgG+, SOSIP gp140 v4.1 AD8+ cells were FACS-sorted and variable heavy (VH) and light (VL) genes were amplified and cloned into human constant region expression vectors. The mAbs were expressed and characterised for Env binding and neutralisation activity. Results: Among many Env binding mAbs, fourteen showed heterologous neutralisation with CDRH3 length of 12 to 61 residues. Six mAbs with shorter-length CDRH3 showed modest neutralisation against tier 1 clade B viruses. Another six mAbs with ultra-long CDRH3 could neutralise tier 1 and tier 2 of clade B and clade C as well as tier 2 of clade A HIV virus. But three common-lineage mAbs isolated from a KNH1/BG505 SOSIP vaccinated cow were highly potent (IC50 ~ 0.001 [micro]g/ml) in neutralising the global panel of twelve HIV pseuodoviruses in the TZM-bl assay. Binding and neutralisation of Env mutant pseudoviruses confirmed these three potent bovine mAbs bound the CD4 binding site and required contact residues in C3, C4 and C5 of gp120. These potent mAbs strongly out-competed VRC01 and other elite human BrNAbs for binding the CD4bs, yet were not polyreactive with human antigens. Antigen binding activity of these bovine V-region chimeric mAbs was fully retained after re-engineering cellular-immune functionality into the human IgG Fc region. Conclusions: BrNAbs with novel structured ultra-long CDRH3 from Env vaccinated cows give new insights into conserved gp120-CD4bs epitopes.

Human immunodeficiency virus type 1 (HIV-1) vaccination of cows has elicited broadly neutralizing antibodies (bNAbs). In this study, monoclonal antibodies (mAbs) are isolated from a clade A (KNH1144 and BG505) vaccinated cow using a heterologous clade B antigen (AD8). CD4 binding site (CD4bs) bNAb (MEL-1872) is more potent than a majority of CD4bs bNAbs isolated so far. MEL-1872 mAb with CDRH3 of 57 amino acids shows more potency (geometric mean half-maximal inhibitory concentration [IC 50 ]: 0.009 μg/mL; breadth: 66%) than VRC01 against clade B viruses (29-fold) and than CHO1-31 against tested clade A viruses (21-fold). It also shows more breadth and potency than NC-Cow1, the only other reported anti-HIV-1 bovine bNAb, which has 60% breadth with geometric mean IC 50 of 0.090 μg/mL in this study. Using successive different stable-structured SOSIP trimers in bovines can elicit bNAbs focusing on epitopes ubiquitous across subtypes. Furthermore, the cross-clade selection strategy also results in ultra-potent bNAbs., Competing Interests: Declaration of interests B.H. and D.F.J.P. are inventors on a corresponding patent from University of Melbourne: HIV-1 antibodies PCT/AU2021/050593. The other authors declare no competing interests., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

In recent years, bats have been identified as a natural reservoir for a diverse range of viruses. Nelson Bay orthoreovirus (NBV) was first isolated from the heart blood of a fruit bat (Pteropus poliocephalus) in 1968. While the pathogenesis of NBV remains unknown, other related members of this group have caused acute respiratory disease in humans. Thus the potential for NBV to impact human health appears plausible. Here, to increase our knowledge of NBV, we examined the replication and infectivity of NBV using different mammalian cell lines derived from bat, human, mouse and monkey. All cell lines supported the replication of NBV; however, L929 cells showed a greater than 2 log reduction in virus titre compared with the other cell lines. Furthermore, NBV did not induce major cytopathic effects in the L929 cells, as was observed in other cell lines. Interestingly, the related Pteropine orthoreoviruses, Pulau virus (PulV) and Melaka virus (MelV) were able to replicate to high titres in L929 cells but infection resulted in reduced cytopathic effect. Our study demonstrates a unique virus-host interaction between NBV and L929 cells, where cells effectively control viral infection/replication and limit the formation of syncytia. By elucidating the molecular mechanisms that control this unique relationship, important insights will be made into the biology of this fusogenic virus.

Background: Bats are the suspected natural reservoir hosts for a number of new and emerging zoonotic viruses including Nipah virus, Hendra virus, severe acute respiratory syndrome coronavirus and Ebola virus. Since the discovery of SARS-like coronaviruses in Chinese horseshoe bats, attempts to isolate a SL-CoV from bats have failed and attempts to isolate other bat-borne viruses in various mammalian cell lines have been similarly unsuccessful. New stable bat cell lines are needed to help with these investigations and as tools to assist in the study of bat immunology and virus-host interactions., Methodology/findings: Black flying foxes (Pteropus alecto) were captured from the wild and transported live to the laboratory for primary cell culture preparation using a variety of different methods and culture media. Primary cells were successfully cultured from 20 different organs. Cell immortalisation can occur spontaneously, however we used a retroviral system to immortalise cells via the transfer and stable production of the Simian virus 40 Large T antigen and the human telomerase reverse transcriptase protein. Initial infection experiments with both cloned and uncloned cell lines using Hendra and Nipah viruses demonstrated varying degrees of infection efficiency between the different cell lines, although it was possible to infect cells in all tissue types., Conclusions/significance: The approaches developed and optimised in this study should be applicable to bats of other species. We are in the process of generating further cell lines from a number of different bat species using the methodology established in this study.

We present evidence of linkage between markers on chromosome 12 and asthma using the BETA program for nonparametric single- and multipoint linkage analysis. We have used quantitative scores as our phenotypic variables, combining data into indices for asthma and atopy and maximizing heritability. The largest single-locus LODs were achieved for asthma: D12S342 and asthma score (LOD 2.255), D12S324 and asthma affection (LOD 2.214), and D12S366 and wheeze (LOD 3.307). The region of interest identified using multipoint analysis, with a maximum LOD of 2.29, centers around D12S97 at location 173.5 cM with a standard error of 6.5 for the asthma score and close agreement for asthma affection and wheeze. Such evidence merits further investigation of this area in an attempt to define the region with greater precision with a view to identifying candidate genes. We hope that the methods presented will encourage researchers to use phenotypic information in a way that encourages meta-analysis., (Copyright 1998 Academic Press.)