More Than Just Gene Therapy Vectors: Lentiviral Vector Pseudotypes for Serological Investigation

Serological assays detecting neutralising antibodies are important for determining the immune responses following infection or vaccination and are also often considered a correlate of protection. The target of neutralising antibodies is usually located in the Envelope protein on the viral surface, which mediates cell entry. As such, presentation of the Envelope protein on a lentiviral particle represents a convenient alternative to handling of a potentially high containment virus or for those viruses with no established cell culture system. The flexibility, relative safety and, in most cases, ease of production of lentiviral pseudotypes, have led to their use in serological assays for many applications such as the evaluation of candidate vaccines, screening and characterization of anti-viral therapeutics, and sero-surveillance. Above all, the speed of production of the lentiviral pseudotypes, once the envelope sequence is published, makes them important tools in the response to viral outbreaks, as shown during the COVID-19 pandemic in 2020. In this review, we provide an overview of the landscape of the serological applications of pseudotyped lentiviral vectors, with a brief discussion on their production and batch quality analysis. Finally, we evaluate their role as surrogates for the real virus and possible alternatives.

Entomological Surveillance for Zika and Dengue Virus in Aedes Mosquitoes: Implications for Vector Control in Thailand

Entomological surveillance for arthropod-borne viruses is vital for monitoring vector-borne diseases and informing vector control programs. In this study, we conducted entomological surveillance in Zika virus endemic areas. In Thailand, it is standard protocol to perform mosquito control within 24 h of a reported dengue case. Aedes females were collected within 72 h of case reports from villages with recent Zika–human cases in Kamphaeng Phet Province, Thailand in 2017 and 2018. Mosquitoes were bisected into head-thorax and abdomen and then screened for Zika (ZIKV) and dengue (DENV) viruses using real-time RT-PCR. ZIKV RNA was detected in three samples from two female Ae. aegypti (1.4%). A partial envelope sequence analysis revealed that the ZIKV sequences were the Asian lineage identical to sequences from ZIKV-infected cases reported in Thailand during 2016 and 2017. Dengue virus-1 (DENV-1) and dengue virus-4 (DENV-4) were found in four Ae. aegypti females (2.8%), and partial capsid sequences were nearly identical with DENV-1 and DENV-4 from Thai human cases reported in 2017. Findings in the current study demonstrate the importance of entomological surveillance programs to public health mosquito-borne disease prevention measures and control.

Characterization of envelope sequence of HIV virus in children infected with HIV in Vietnam

Background: HIV is characterized by high levels of genetic variability, including increased numbers of heterogeneous sequences of the envelope region. Therefore, studying genetic variability of HIV in relation to viral replication might facilitate prognosis of disease progression. Methods: The study was designed as cross-sectional; data and samples of participants collected and analyzed env genes were obtained from 23 children enrolled by Vietnam National Children’s Hospital. Results: Substantial mutations in the C2 region were found in patients with high levels of viral replication while changes in the C3 region were mostly found in patients with low viral load. In the V1 region, we found profound amino acid modifications in patients with low HIV viral loads in contrast to the V2 sequence, where we identified single point mutations in patients with increased HIV viral load. The V3 region was relatively homogeneous, while profound deletions in the V4 region were detected in patients with increased viral replication. Conclusion: Our results suggest that genetic variations in different regions of the HIV envelope sequence, including both conserved C2 and C3 and variable V1/V2 and V4 regions, might be involved in increased viral infectivity and replication capacity. Such knowledge might help improve prediction of HIV progress and treatment in patients.

THETA: a new genotypic approach for predicting HIV-1 CRF02-AG coreceptor usage

Motivation The circulating recombinant form of HIV-1 CRF02-AG is the most frequent non-B subtype in Europe. Anti-HIV therapy and pathophysiological studies on the impact of HIV-1 tropism require genotypic determination of HIV-1 tropism for non-B subtypes. But genotypic approaches based on analysis of the V3 envelope region perform poorly when used to determine the tropism of CRF02-AG. We, therefore, designed an algorithm based on information from the gp120 and gp41 ectodomain that better predicts the tropism of HIV-1 subtype CRF02-AG. Results We used a bio-statistical method to identify the genotypic determinants of CRF02-AG coreceptor use. Toulouse HIV Extended Tropism Algorithm (THETA), based on a Least Absolute Shrinkage and Selection Operator method, uses HIV envelope sequence from phenotypically characterized clones. Prediction of R5X4/X4 viruses was 86% sensitive and that of R5 viruses was 89% specific with our model. The overall accuracy of THETA was 88%, making it sufficiently reliable for predicting the tropism of subtype CRF02-AG sequences. Availability and implementation Binaries are freely available for download at https://github.com/viro-tls/THETA. It was implemented in Matlab and supported on MS Windows platform. The sequence data used in this work are available from GenBank under the accession numbers MK618182-MK618417.

From predicting to analyzing HIV-1 resistance to broadly neutralizing antibodies

Treatment with broadly neutralizing antibodies (bNAbs) has recently proven effective against HIV-1 infections in humanized mice, non-human primates, and humans. For optimal treatment, susceptibility of the patient's viral strains to a particular bNAb has to be ensured. Since no computational approaches are so far available, susceptibility can only be tested in expensive and time-consuming neutralization experiments. Here, we present well-performing computational models (AUC up to 0.84) that can predict HIV-1 resistance to bNAbs given the envelope sequence of the virus. Having learnt important binding sites of the bNAbs from the envelope sequence, the models are also biologically meaningful and useful for epitope recognition. Additional to the prediction result, we provide a motif logo that displays the contribution of the pivotal residues of the test sequence to the prediction. As our prediction models are based on non-linear kernels, we introduce a new visualization technique to improve the model interpretability. Moreover, we confirmed previous experimental findings that there is a trend towards antibody resistance for the subtype B population of the virus. While previous experiments considered rather small and selected cohorts, we were able to show a similar trend for the global HIV-1 population comprising all major subtypes by predicting the neutralization sensitivity for around 36,000 HIV-1 sequences - a scale-up which is very difficult to achieve in an experimental setting.

From predicting to analyzing HIV-1 resistance to broadly neutralizing antibodies

Treatment with broadly neutralizing antibodies (bNAbs) has recently proven effective against HIV-1 infections in humanized mice, non-human primates, and humans. For optimal treatment, susceptibility of the patient's viral strains to a particular bNAb has to be ensured. Since no computational approaches are so far available, susceptibility can only be tested in expensive and time-consuming neutralization experiments. Here, we present well-performing computational models (AUC up to 0.84) that can predict HIV-1 resistance to bNAbs given the envelope sequence of the virus. Having learnt important binding sites of the bNAbs from the envelope sequence, the models are also biologically meaningful and useful for epitope recognition. Additional to the prediction result, we provide a motif logo that displays the contribution of the pivotal residues of the test sequence to the prediction. As our prediction models are based on non-linear kernels, we introduce a new visualization technique to improve the model interpretability. Moreover, we confirmed previous experimental findings that there is a trend towards antibody resistance for the subtype B population of the virus. While previous experiments considered rather small and selected cohorts, we were able to show a similar trend for the global HIV-1 population comprising all major subtypes by predicting the neutralization sensitivity for around 36,000 HIV-1 sequences - a scale-up which is very difficult to achieve in an experimental setting.