Multiplex evaluation of influenza neutralizing antibodies with potential applicability to in-field serological studies

The increased number of outbreaks of H5 and H7 LPAI and HPAI viruses in poultry has major public and animal health implications. The continuous rapid evolution of these subtypes and the emergence of new variants influence the ability to undertake effective surveillance. Retroviral pseudotypes bearing influenza haemagglutinin (HA) and neuraminidase (NA) envelope glycoproteins represent a flexible platform for sensitive, readily standardized influenza serological assays. We describe a multiplex assay for the study of neutralizing antibodies that are directed against both influenza H5 and H7 HA. This assay permits the measurement of neutralizing antibody responses against two antigenically distinct HAs in the same serum/plasma sample thus increasing the amount and quality of serological data that can be acquired from valuable sera. Sera obtained from chickens vaccinated with a monovalent H5N2 vaccine, chickens vaccinated with a bivalent H7N1/H5N9 vaccine, or turkeys naturally infected with an H7N3 virus were evaluated in this assay and the results correlated strongly with data obtained by HI assay. We show that pseudotypes are highly stable under basic cold-chain storage conditions and following multiple rounds of freeze-thaw. We propose that this robust assay may have practical utility for in-field sero-surveillance and vaccine studies in resource-limited regions worldwide

Bat and pig IFN-induced transmembrane protein 3 restrict cell entry by influenza virus and lyssaviruses

IFN-induced transmembrane protein 3 (IFITM3) is a restriction factor that blocks cytosolic entry of numerous viruses that utilize acidic endosomal entry pathways. In humans and mice, IFITM3 limits influenza-induced morbidity and mortality. Although many IFITM3-sensitive viruses are zoonotic, whether IFITMs function as antiviral restriction factors in mammalian species other than humans and mice is unknown. Here, IFITM3 orthologues in the microbat (Myotis myotis) and pig (Sus scrofa domesticus) were identified using rapid amplification of cDNA ends. Amino acid residues known to be important for IFITM3 function were conserved in the pig and microbat orthologues. Ectopically expressed pig and microbat IFITM3 co-localized with transferrin (early endosomes) and CD63 (late endosomes/multivesicular bodies). Pig and microbat IFITM3 restricted cell entry mediated by multiple influenza haemagglutinin subtypes and lyssavirus glycoproteins. Expression of pig or microbat IFITM3 in A549 cells reduced influenza virus yields and nucleoprotein expression. Conversely, small interfering RNA knockdown of IFITM3 in pig NPTr cells and primary microbat cells enhanced virus replication, demonstrating that these genes are functional in their species of origin at endogenous levels. In summary, we showed that IFITMs function as potent broad-spectrum antiviral effectors in two mammals – pigs and bats – identified as major reservoirs for emerging viruses

Chicken interferon-inducible transmembrane protein 3 restricts influenza viruses and lyssaviruses in vitro.

Interferon-inducible transmembrane protein 3 (IFITM3) is an effector protein of the innate immune system. It confers potent, cell-intrinsic resistance to infection by diverse enveloped viruses both in vitro and in vivo, including influenza viruses, West Nile virus, and dengue virus. IFITM3 prevents cytosolic entry of these viruses by blocking complete virus envelope fusion with cell endosome membranes. Although the IFITM locus, which includes IFITM1, -2, -3, and -5, is present in mammalian species, this locus has not been unambiguously identified or functionally characterized in avian species. Here, we show that the IFITM locus exists in chickens and is syntenic with the IFITM locus in mammals. The chicken IFITM3 protein restricts cell infection by influenza A viruses and lyssaviruses to a similar level as its human orthologue. Furthermore, we show that chicken IFITM3 is functional in chicken cells and that knockdown of constitutive expression in chicken fibroblasts results in enhanced infection by influenza A virus. Chicken IFITM2 and -3 are constitutively expressed in all tissues examined, whereas IFITM1 is only expressed in the bursa of Fabricius, gastrointestinal tract, cecal tonsil, and trachea. Despite being highly divergent at the amino acid level, IFITM3 proteins of birds and mammals can restrict replication of viruses that are able to infect different host species, suggesting IFITM proteins may provide a crucial barrier for zoonotic infections

Retroviral Pseudotypes – From Scientific Tools to Clinical Utility

Retroviral pseudotypes are important research and diagnostic tools for basic and clinical virology studies, facilitating the detailed investigation of individual genes, cellular receptors, antibody responses, serosurveillance and antiviral therapies. Importantly, pseudotypes enable the study of highly pathogenic viruses, without the need for high containment. Their use as gene therapy vectors is widely documented, but other uses, once less well known, are becoming more prominent. The substitution of envelope proteins expressed on the virion surface enables pseudotypes to be employed as surrogates for wildtype viruses in antibody neutralisation or antiviral screening assays and for the study of cell–virus receptor interactions. In addition, they are increasingly being utilised as vaccine immunogens, expressing the antigen either on the particle surface or as a transfer gene for cellular expression. These studies demonstrate the potential for using pseudotypes for both scientific and clinical applications

Current progress with serological assays for exotic emerging/re-emerging viruses

Recent decades have witnessed an unprecedented rise in the outbreak occurrence of infectious and primarily zoonotic viruses. Contributing factors to this phenomenon include heightened global connectivity via air travel and international trade links, as well as man-made environmental alterations such as deforestation and climate change, which all serve to co-localize humans with animal reservoirs and alter the habitat of vector species, thus facilitating transmission of viruses between species. Serological assays are integral to track the epidemiological spread of a virus, evaluate mass vaccination programs by quantifying neutralizing antibody responses raised against antigenic epitopes on the viral surface. However, conventional serological tests are somewhat marred by equipment and reagent costs, the necessity for high containment laboratories for many emerging viruses and inter-laboratory variability amongst other issues. This review details ‘next-generation’ assays aimed at addressing some of the persistent problems with viral serology, focusing on how manipulating the genomes of RNA viruses can produce attenuated or chimeric viruses that can be exploited as surrogate viruses in neutralization assays. Despite the undoubted promise of such novel serological platforms, it must be remembered that these assays have to withstand rigorous validation and standardization measures before they can play an integral role in curtailing the severity of future emerging virus outbreaks

Development of a pseudotype neutralisation assay-based diagnostic kit for in-field vaccine evaluation and serosurveillance for highly pathogenic viruses

Virus neutralisation assays quantitatively detect levels of neutralising antibody response against antigenic surface glycoproteins on many viruses, following vaccination or natural infection. However, high biosafety level requirements and extensive personnel training prevent these tests from broad laboratory application, especially in resource-limited regions. Therefore, development of methods for vaccine evaluation and serosurveillance which can be used in these areas are urgently required. To address these issues lentiviral pseudotype viruses (PVs) have been utilised. PVs are chimeric, replication-deficient particles that mimic the infective mechanisms of their wild-type counterparts. Pseudotype neutralisation assays (PNAs) circumvent the requirement for high biosafety precautions whilst maintaining comparable sensitivity and specificity with existing assays. This study ascertains pseudotype stability through subjection to environmental conditions likely to be encountered in assembly, transport and usage of a PNA-based diagnostic kit. Pseudotypes of clinically-important viruses (e.g. influenza and lyssaviruses) have been used and titres monitored through cumulative freeze-thaw cycles, lyophilisation, and varying temperatures and humidities. Results demonstrated the ability to retain acceptable levels of virus activity following treatments, indicating the potential of PNA-based kits for global distribution and diagnostic application. Such flexible and durable kits could permit accurate in-field vaccine evaluation and serosurveillance for many viruses of endemic and pandemic concern

The evaluation of antibody responses to rabies virus using retroviral pseudotypes and the implications for ‘in-field’ vaccine trials

Using retroviral pseudotype technology we have recently established assays for the characterisation of neutralising antibodies against rabies virus. Neutralisation assays using sera from vaccinated animals and pseudotyped Challenge Virus Standard-11 detected positive and negative samples with 100% specificity and sensitivity, and titres strongly correlated with the Office International des Epizooties FAVN test (R2 = 0.89). We have also studied the cross-neutralising potency of current rabies vaccines against other lyssavirus genotypes. Compared to conventional live virus assays, studying neutralisation with pseudotypes removes the need for high containment facilities and large sample volumes, therefore increasing the number of laboratories that can undertake active surveillance. Further to this we have adapted the assay so that neutralisation tests can be conducted using luciferase, enhanced green fluorescent protein (GFP) or lacZ as the pseudotype reporter gene. These facets combined make our rabies pseudotype assay an attractive option for use in large vaccine campaigns where it would be advantageous to determine serum neutralising titres in situ. The assay will allow a far greater number of laboratories worldwide, who are presently unable to run live virus test, to be more directly involved in front-line vaccine and antiviral therapy trials, especially in resource limited countries where rabies is a major burden. We aim to develop kits for the distribution of these assays that could be selected with a choice of viral pseudotype and reporter gene