Neutralizing antibody activity against 21 SARS-CoV-2 variants in older adults vaccinated with BNT162b2.

SARS-CoV-2 variants may threaten the effectiveness of vaccines and antivirals to mitigate serious COVID-19 disease. This is of most concern in clinically vulnerable groups such as older adults. We analysed 72 sera samples from 37 individuals, aged 70-89 years, vaccinated with two doses of BNT162b2 (Pfizer-BioNTech) 3 weeks apart, for neutralizing antibody responses to wildtype SARS-CoV-2. Between 3 and 20 weeks after the second vaccine dose, neutralizing antibody titres fell 4.9-fold to a median titre of 21.3 (neutralization dose 80%), with 21.6% of individuals having no detectable neutralizing antibodies at the later time point. Next, we examined neutralization of 21 distinct SARS-CoV-2 variant spike proteins with these sera, and confirmed substantial antigenic escape, especially for the Omicron (B.1.1.529, BA.1/BA.2), Beta (B.1.351), Delta (B.1.617.2), Theta (P.3), C.1.2 and B.1.638 spike variants. By combining pseudotype neutralization with specific receptor-binding domain (RBD) enzyme-linked immunosorbent assays, we showed that changes to position 484 in the spike RBD were mainly responsible for SARS-CoV-2 neutralizing antibody escape. Nineteen sera from the same individuals boosted with a third dose of BNT162b2 contained higher neutralizing antibody titres, providing cross-protection against Omicron BA.1 and BA.2. Despite SARS-CoV-2 immunity waning over time in older adults, booster vaccines can elicit broad neutralizing antibodies against a large number of SARS-CoV-2 variants in this clinically vulnerable cohort.

Information-Driven Docking for TCR-pMHC Complex Prediction.

T cell receptor (TCR) recognition of peptides presented by major histocompatibility complex (MHC) molecules is a fundamental process in the adaptive immune system. An understanding of this recognition process at the molecular level is crucial for TCR based therapeutics and vaccine design. The broad nature of TCR diversity and cross-reactivity presents a challenge for traditional structural resolution. Computational modelling of TCR-pMHC complexes offers an efficient alternative. This study compares the ability of four general-purpose docking platforms (ClusPro, LightDock, ZDOCK and HADDOCK) to make use of varying levels of binding interface information for accurate TCR-pMHC modelling. Each platform was tested on an expanded benchmark set of 44 TCR-pMHC docking cases. In general, HADDOCK is shown to be the best performer. Docking strategy guidance is provided to obtain the best models for each platform for future research. The TCR-pMHC docking cases used in this study can be downloaded from https://github.com/innate2adaptive/ExpandedBenchmark.

The furin cleavage site in the SARS-CoV-2 spike protein is required for transmission in ferrets.

SARS-CoV-2 entry requires sequential cleavage of the spike glycoprotein at the S1/S2 and the S2' cleavage sites to mediate membrane fusion. SARS-CoV-2 has a polybasic insertion (PRRAR) at the S1/S2 cleavage site that can be cleaved by furin. Using lentiviral pseudotypes and a cell-culture-adapted SARS-CoV-2 virus with an S1/S2 deletion, we show that the polybasic insertion endows SARS-CoV-2 with a selective advantage in lung cells and primary human airway epithelial cells, but impairs replication in Vero E6, a cell line used for passaging SARS-CoV-2. Using engineered spike variants and live virus competition assays and by measuring growth kinetics, we find that the selective advantage in lung and primary human airway epithelial cells depends on the expression of the cell surface protease TMPRSS2, which enables endosome-independent virus entry by a route that avoids antiviral IFITM proteins. SARS-CoV-2 virus lacking the S1/S2 furin cleavage site was shed to lower titres from infected ferrets and was not transmitted to cohoused sentinel animals, unlike wild-type virus. Analysis of 100,000 SARS-CoV-2 sequences derived from patients and 24 human postmortem tissues showed low frequencies of naturally occurring mutants that harbour deletions at the polybasic site. Taken together, our findings reveal that the furin cleavage site is an important determinant of SARS-CoV-2 transmission.

Disrupting HIV-1 capsid formation causes cGAS sensing of viral DNA.

Detection of viral DNA by cyclic GMP-AMP synthase (cGAS) is a first line of defence leading to the production of type I interferon (IFN). As HIV-1 replication is not a strong inducer of IFN, we hypothesised that an intact capsid physically cloaks viral DNA from cGAS. To test this, we generated defective viral particles by treatment with HIV-1 protease inhibitors or by genetic manipulation of gag. These viruses had defective Gag cleavage, reduced infectivity and diminished capacity to saturate TRIM5α. Importantly, unlike wild-type HIV-1, infection with cleavage defective HIV-1 triggered an IFN response in THP-1 cells that was dependent on viral DNA and cGAS. An IFN response was also observed in primary human macrophages infected with cleavage defective viruses. Infection in the presence of the capsid destabilising small molecule PF-74 also induced a cGAS-dependent IFN response. These data demonstrate a protective role for capsid and suggest that antiviral activity of capsid- and protease-targeting antivirals may benefit from enhanced innate and adaptive immunity in vivo.

Publisher Correction: Spatial heterogeneity of the T cell receptor repertoire reflects the mutational landscape in lung cancer.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Swine ANP32A Supports Avian Influenza Virus Polymerase.

Avian influenza viruses occasionally infect and adapt to mammals, including humans. Swine are often described as "mixing vessels," being susceptible to both avian- and human-origin viruses, which allows the emergence of novel reassortants, such as the precursor to the 2009 H1N1 pandemic. ANP32 proteins are host factors that act as influenza virus polymerase cofactors. In this study, we describe how swine ANP32A, uniquely among the mammalian ANP32 proteins tested, supports the activity of avian-origin influenza virus polymerases and avian influenza virus replication. We further show that after the swine-origin influenza virus emerged in humans and caused the 2009 pandemic, it evolved polymerase gene mutations that enabled it to more efficiently use human ANP32 proteins. We map the enhanced proviral activity of swine ANP32A to a pair of amino acids, 106 and 156, in the leucine-rich repeat and central domains and show these mutations enhance binding to influenza virus trimeric polymerase. These findings help elucidate the molecular basis for the mixing vessel trait of swine and further our understanding of the evolution and ecology of viruses in this host.IMPORTANCE Avian influenza viruses can jump from wild birds and poultry into mammalian species such as humans or swine, but they only continue to transmit if they accumulate mammalian adapting mutations. Pigs appear uniquely susceptible to both avian and human strains of influenza and are often described as virus "mixing vessels." In this study, we describe how a host factor responsible for regulating virus replication, ANP32A, is different between swine and humans. Swine ANP32A allows a greater range of influenza viruses, specifically those from birds, to replicate. It does this by binding the virus polymerase more tightly than the human version of the protein. This work helps to explain the unique properties of swine as mixing vessels.

Quantitative analysis of the T cell receptor repertoire.

The T cell receptor repertoire provides a window into the cellular adaptive immune response. In the context of cancer, determining the repertoire within a tumor can give important insights into the evolution of the T cell anti-cancer response, and has the potential to identify specific personalized biomarkers for tracking host responses during cancer therapy, including immunotherapy. We describe a protocol for amplifying, sequencing and analyzing T cell receptors which is economical, robust, sensitive and versatile. The key experimental step is the ligation of a single stranded oligonucleotide to the 3' end of the T cell receptor cDNA, which allows easy amplification of all possible rearrangements using only a single set of primers per locus, while simultaneously introducing a unique molecular identifier to label each starting cDNA molecule. After sequencing, this molecular identifier can be used to correct both sequence errors and the effects of differential PCR amplification efficiency, thus producing a more accurate measure of the true T cell receptor frequency within the sample. We describe a detailed protocol describing this method to create libraries of T cell receptors from in vitro T cell cultures, blood or tissue samples. We combine this with a computational pipeline, which incorporates sample multiplexing, T cell receptor annotation and error correction to provide accurate counts of individual T cell receptor sequences within samples. The integrated experimental and computational pipeline should be of value to researchers interested in documenting and understanding the T cell immune response to cancer, and in manipulating it for therapeutic purposes.

Spatial heterogeneity of the T cell receptor repertoire reflects the mutational landscape in lung cancer.

Somatic mutations together with immunoediting drive extensive heterogeneity within non-small-cell lung cancer (NSCLC). Herein we examine heterogeneity of the T cell antigen receptor (TCR) repertoire. The number of TCR sequences selectively expanded in tumors varies within and between tumors and correlates with the number of nonsynonymous mutations. Expanded TCRs can be subdivided into TCRs found in all tumor regions (ubiquitous) and those present in a subset of regions (regional). The number of ubiquitous and regional TCRs correlates with the number of ubiquitous and regional nonsynonymous mutations, respectively. Expanded TCRs form part of clusters of TCRs of similar sequence, suggestive of a spatially constrained antigen-driven process. CD8+ tumor-infiltrating lymphocytes harboring ubiquitous TCRs display a dysfunctional tissue-resident phenotype. Ubiquitous TCRs are preferentially detected in the blood at the time of tumor resection as compared to routine follow-up. These findings highlight a noninvasive method to identify and track relevant tumor-reactive TCRs for use in adoptive T cell immunotherapy.

Affimer proteins are versatile and renewable affinity reagents.

Molecular recognition reagents are key tools for understanding biological processes and are used universally by scientists to study protein expression, localisation and interactions. Antibodies remain the most widely used of such reagents and many show excellent performance, although some are poorly characterised or have stability or batch variability issues, supporting the use of alternative binding proteins as complementary reagents for many applications. Here we report on the use of Affimer proteins as research reagents. We selected 12 diverse molecular targets for Affimer selection to exemplify their use in common molecular and cellular applications including the (a) selection against various target molecules; (b) modulation of protein function in vitro and in vivo; (c) labelling of tumour antigens in mouse models; and (d) use in affinity fluorescence and super-resolution microscopy. This work shows that Affimer proteins, as is the case for other alternative binding scaffolds, represent complementary affinity reagents to antibodies for various molecular and cell biology applications.