Triple tandem trimer immunogens for HIV-1 and influenza nucleic acid-based vaccines.

Recombinant native-like HIV-1 envelope glycoprotein (Env) trimers are used in candidate vaccines aimed at inducing broadly neutralizing antibodies. While state-of-the-art SOSIP or single-chain Env designs can be expressed as native-like trimers, undesired monomers, dimers and malformed trimers that elicit non-neutralizing antibodies are also formed, implying that these designs could benefit from further modifications for gene-based vaccination approaches. Here, we describe the triple tandem trimer (TTT) design, in which three Env protomers are genetically linked in a single open reading frame and express as native-like trimers. Viral vectored Env TTT induced similar neutralization titers but with a higher proportion of trimer-specific responses. The TTT design was also applied to generate influenza hemagglutinin (HA) trimers without the need for trimerization domains. Additionally, we used TTT to generate well-folded chimeric Env and HA trimers that harbor protomers from three different strains. In summary, the TTT design is a useful platform for the design of HIV-1 Env and influenza HA immunogens for a multitude of vaccination strategies.

Vaccine-induced neutralizing antibody responses to seasonal influenza virus H1N1 strains are not enhanced during subsequent pandemic H1N1 infection.

The first exposure to influenza is presumed to shape the B-cell antibody repertoire, leading to preferential enhancement of the initially formed responses during subsequent exposure to viral variants. Here, we investigated whether this principle remains applicable when there are large genetic and antigenic differences between primary and secondary influenza virus antigens. Because humans usually have a complex history of influenza virus exposure, we conducted this investigation in influenza-naive cynomolgus macaques. Two groups of six macaques were immunized four times with influenza virus-like particles (VLPs) displaying either one (monovalent) or five (pentavalent) different hemagglutinin (HA) antigens derived from seasonal H1N1 (H1N1) strains. Four weeks after the final immunization, animals were challenged with pandemic H1N1 (H1N1pdm09). Although immunization resulted in robust virus-neutralizing responses to all VLP-based vaccine strains, there were no cross-neutralization responses to H1N1pdm09, and all animals became infected. No reductions in viral load in the nose or throat were detected in either vaccine group. After infection, strong virus-neutralizing responses to H1N1pdm09 were induced. However, there were no increases in virus-neutralizing titers against four of the five H1N1 vaccine strains; and only a mild increase was observed in virus-neutralizing titer against the influenza A/Texas/36/91 vaccine strain. After H1N1pdm09 infection, both vaccine groups showed higher virus-neutralizing titers against two H1N1 strains of intermediate antigenic distance between the H1N1 vaccine strains and H1N1pdm09, compared with the naive control group. Furthermore, both vaccine groups had higher HA-stem antibodies early after infection than the control group. In conclusion, immunization with VLPs displaying HA from antigenically distinct H1N1 variants increased the breadth of the immune response during subsequent H1N1pdm09 challenge, although this phenomenon was limited to intermediate antigenic variants.

Primary antibody response after influenza virus infection is first dominated by low-mutated HA-stem antibodies followed by higher-mutated HA-head antibodies.

Several studies have shown that the first encounter with influenza virus shapes the immune response to future infections or vaccinations. However, a detailed analysis of the primary antibody response is lacking as this is difficult to study in humans. It is therefore not known what the frequency and dynamics of the strain-specific hemagglutinin (HA) head- and stem-directed antibody responses are directly after primary influenza virus infection. Here, sera of twelve H1N1pdm2009 influenza virus-infected cynomolgus macaques were evaluated for HA-head and HA-stem domain antibody responses. We observed an early induction of HA-stem antibody responses, which was already decreased by day 56. In contrast, responses against the HA-head domain were low early after infection and increased at later timepoint. The HA-specific B cell repertoires in each animal showed diverse VH-gene usage with preferred VH-gene and JH-gene family usage for HA-head or HA-stem B cells but a highly diverse allelic variation within the VH-usage. HA-head B cells had shorter CDRH3s and higher VH-gene somatic hyper mutation levels relative to HA-stem B cells. In conclusion, our data suggest that HA-stem antibodies are the first to react to the infection while HA-head antibodies show a delayed response, but a greater propensity to enter the germinal center and undergo affinity maturation.

A public antibody class recognizes an S2 epitope exposed on open conformations of SARS-CoV-2 spike.

Delineating the origins and properties of antibodies elicited by SARS-CoV-2 infection and vaccination is critical for understanding their benefits and potential shortcomings. Therefore, we investigate the SARS-CoV-2 spike (S)-reactive B cell repertoire in unexposed individuals by flow cytometry and single-cell sequencing. We show that ∼82% of SARS-CoV-2 S-reactive B cells harbor a naive phenotype, which represents an unusually high fraction of total human naive B cells (∼0.1%). Approximately 10% of these naive S-reactive B cells share an IGHV1-69/IGKV3-11 B cell receptor pairing, an enrichment of 18-fold compared to the complete naive repertoire. Following SARS-CoV-2 infection, we report an average 37-fold enrichment of IGHV1-69/IGKV3-11 B cell receptor pairing in the S-reactive memory B cells compared to the unselected memory repertoire. This class of B cells targets a previously undefined non-neutralizing epitope on the S2 subunit that becomes exposed on S proteins used in approved vaccines when they transition away from the native pre-fusion state because of instability. These findings can help guide the improvement of SARS-CoV-2 vaccines.

Decreased Passive Immunity to Respiratory Viruses through Human Milk during the COVID-19 Pandemic.

Infants may develop severe viral respiratory tract infections because their immune system is still developing in the first months after birth. Human milk provides passive humoral immunity during the first months of life. During the COVID-19 pandemic, circulation of common respiratory viruses was virtually absent due to the preventative measures resulting in reduced maternal exposure. Therefore, we hypothesized that this might result in lower antibody levels in human milk during the pandemic and, subsequently, decreased protection of infants against viral respiratory tract infections. We assessed antibody levels against respiratory syncytial virus (RSV), Influenza virus, and several seasonal coronaviruses in different periods of the COVID-19 pandemic in serum and human milk using a Luminex assay. IgG levels against RSV, Influenza, HCoV-OC43, HCoV-HKU1, and HCoV-NL63 in human milk were reduced with a factor of 1.7 (P < 0.001), 2.2 (P < 0.01), 2.6 (P < 0.05), 1.4 (P < 0.01), and 2.1 (P < 0.001), respectively, since the introduction of the COVID-19 restrictions. Furthermore, we observed that human milk of mothers that experienced COVID-19 contained increased levels of IgG and IgA binding to other respiratory viruses. Passive immunity via human milk against common respiratory viruses was reduced during the COVID-19 pandemic, which may have consequences for the protection of breastfed infants against respiratory infections. IMPORTANCE Passive immunity derived from antibodies in human milk is important for protecting young infants against invading viruses. During the COVID-19 pandemic, circulation of common respiratory viruses was virtually absent due to preventative measures. In this study, we observed a decrease in human milk antibody levels against common respiratory viruses several months into the COVID-19 pandemic. This waning of antibody levels might partially explain the previously observed surge of hospitalizations of infants, mostly due to RSV, when preventative hygiene measures were lifted. Knowledge of the association between preventative measures, antibody levels in human milk and subsequent passive immunity in infants might help predict infant hospital admissions and thereby enables anticipation to prevent capacity issues. Additionally, it is important in the consideration for strategies for future lockdowns to best prevent possible consequences for vulnerable infants.

Influenza A Virus Hemagglutinin Trimer, Head and Stem Proteins Identify and Quantify Different Hemagglutinin-Specific B Cell Subsets in Humans.

Antibody responses against the influenza A virus hemagglutinin (HA)-protein are studied intensively because they can protect against (re)infection. Previous studies have focused on antibodies targeting the head or stem domains, while other possible specificities are often not taken into account. To study such specificities, we developed a diverse set of HA-domain proteins based on an H1N1pdm2009-like influenza virus strain, including monomeric head and trimeric stem domain, as well as the full HA-trimer. These proteins were used to study the B cell and antibody responses in six healthy human donors. A large proportion of HA-trimer B cells bound exclusively to HA-trimer probe (54-77%), while only 8-18% and 9-23% were able to recognize the stem or head probe, respectively. Monoclonal antibodies (mAbs) were isolated and three of these mAbs, targeting the different domains, were characterized in-depth to confirm the binding profile observed in flow cytometry. The head-directed mAb, targeting an epitope distinct from known head-specific mAbs, showed relatively broad H1N1 neutralization and the stem-directed mAb was able to broadly neutralize diverse H1N1 viruses. Moreover, we identified a trimer-directed mAb that did not compete with known head or stem domain specific mAbs, suggesting that it targets an unknown epitope or conformation of influenza virus' HA. These observations indicate that the described method can characterize the diverse antibody response to HA and might be able to identify HA-specific B cells and antibodies with previously unknown specificities that could be relevant for vaccine design.

Potent neutralizing antibodies from COVID-19 patients define multiple targets of vulnerability.

The rapid spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has had a large impact on global health, travel, and economy. Therefore, preventative and therapeutic measures are urgently needed. Here, we isolated monoclonal antibodies from three convalescent coronavirus disease 2019 (COVID-19) patients using a SARS-CoV-2 stabilized prefusion spike protein. These antibodies had low levels of somatic hypermutation and showed a strong enrichment in VH1-69, VH3-30-3, and VH1-24 gene usage. A subset of the antibodies was able to potently inhibit authentic SARS-CoV-2 infection at a concentration as low as 0.007 micrograms per milliliter. Competition and electron microscopy studies illustrate that the SARS-CoV-2 spike protein contains multiple distinct antigenic sites, including several receptor-binding domain (RBD) epitopes as well as non-RBD epitopes. In addition to providing guidance for vaccine design, the antibodies described here are promising candidates for COVID-19 treatment and prevention.

Development of broadly reactive influenza vaccines by targeting the conserved regions of the hemagglutinin stem and head domains.

INTRODUCTION: Influenza virus infections cause serious illness in millions of people each year. Although influenza virus vaccines are available, they are not optimally effective due to mismatches between the influenza virus strains used for the vaccine and the circulating strains. To improve protection by vaccines, a broadly protective or universal vaccine may be required. Strategies to develop universal vaccines aim to elicit broadly reactive antibodies, which target regions on the viral hemagglutinin (HA) protein which are conserved between strains. Broadly reactive antibodies have helped to identify such targets and can guide the design of such a vaccine. AREAS COVERED: The first part of this review provides an in-depth overview of broadly reactive anti-HA antibodies, discussing their origin, breadth and their mechanisms of protection. The second part discusses the technical design and mode of action of potential universal vaccine candidates that aim to elicit these broadly reactive antibodies and provide protection against a majority of influenza strains. EXPERT OPINION: While great strides have been made in the development of universal influenza vaccine candidates, real-life use still requires improvement of stability, enhancement of their breadth of protection and ease of production, while efficacies need to be determined in human trials.

Quantitative in vitro-to-in vivo extrapolation (QIVIVE) of estrogenic and anti-androgenic potencies of BPA and BADGE analogues.

The goal of the present study was to obtain an in vivo relevant prioritization method for the endocrine potencies of different polycarbonate monomers, by combining in vitro bioassay data with physiologically based kinetic (PBK) modelling. PBK models were developed for a selection of monomers, including bisphenol A (BPA), two bisphenol F (BPF) isomers and four different bisphenol A diglycidyl ethers (BADGEs), using in vitro input data. With these models, the plasma concentrations of the compounds were simulated, providing means to estimate the dose levels at which the in vitro endocrine effect concentrations are reached. The results revealed that, whereas the in vitro relative potencies of different BADGEs (predominantly anti-androgenic effects) can be up to fourfold higher than BPA, the estimated in vivo potencies based on the oral equivalent doses are one to two orders of magnitude lower than BPA because of fast detoxification of the BADGEs. In contrast, the relative potencies of 2,2-BPF and 4,4-BPF increase when accounting for the in vivo availability. 4,4-BPF is estimated to be fivefold more potent than BPA in humans in vivo in inducing estrogenic effects and both 2,2-BPF and 4,4-BPF are estimated to be, respectively, 7 and 11-fold more potent in inducing anti-androgenic effects. These relative potencies were considered to be first-tier estimates, particularly given that the potential influence of intestinal metabolism on the in vivo availability was not accounted for. Overall, it can be concluded that both 2,2-BPF and 4,4-BPF are priority compounds.

Glucocorticoid receptor and nuclear factor kappa-b affect three-dimensional chromatin organization.

BACKGROUND: The impact of signal-dependent transcription factors, such as glucocorticoid receptor and nuclear factor kappa-b, on the three-dimensional organization of chromatin remains a topic of discussion. The possible scenarios range from remodeling of higher order chromatin architecture by activated transcription factors to recruitment of activated transcription factors to pre-established long-range interactions. RESULTS: Using circular chromosome conformation capture coupled with next generation sequencing and high-resolution chromatin interaction analysis by paired-end tag sequencing of P300, we observed agonist-induced changes in long-range chromatin interactions, and uncovered interconnected enhancer-enhancer hubs spanning up to one megabase. The vast majority of activated glucocorticoid receptor and nuclear factor kappa-b appeared to join pre-existing P300 enhancer hubs without affecting the chromatin conformation. In contrast, binding of the activated transcription factors to loci with their consensus response elements led to the increased formation of an active epigenetic state of enhancers and a significant increase in long-range interactions within pre-existing enhancer networks. De novo enhancers or ligand-responsive enhancer hubs preferentially interacted with ligand-induced genes. CONCLUSIONS: We demonstrate that, at a subset of genomic loci, ligand-mediated induction leads to active enhancer formation and an increase in long-range interactions, facilitating efficient regulation of target genes. Therefore, our data suggest an active role of signal-dependent transcription factors in chromatin and long-range interaction remodeling.

Widespread genomic incompatibilities in Caenorhabditis elegans.

In the Bateson-Dobzhansky-Muller (BDM) model of speciation, incompatibilities emerge from the deleterious interactions between alleles that are neutral or advantageous in the original genetic backgrounds, i.e., negative epistatic effects. Within species such interactions are responsible for outbreeding depression and F2 (hybrid) breakdown. We sought to identify BDM incompatibilities in the nematode Caenorhabditis elegans by looking for genomic regions that disrupt egg laying; a complex, highly regulated, and coordinated phenotype. Investigation of introgression lines and recombinant inbred lines derived from the isolates CB4856 and N2 uncovered multiple incompatibility quantitative trait loci (QTL). These QTL produce a synthetic egg-laying defective phenotype not seen in CB4856 and N2 nor in other wild isolates. For two of the QTL regions, results are inconsistent with a model of pairwise interaction between two loci, suggesting that the incompatibilities are a consequence of complex interactions between multiple loci. Analysis of additional life history traits indicates that the QTL regions identified in these screens are associated with effects on other traits such as lifespan and reproduction, suggesting that the incompatibilities are likely to be deleterious. Taken together, these results indicate that numerous BDM incompatibilities that could contribute to reproductive isolation can be detected and mapped within C. elegans.

Structural and biochemical insights to the role of the CCR4-NOT complex and DDX6 ATPase in microRNA repression.

MicroRNAs (miRNAs) control gene expression by regulating mRNA translation and stability. The CCR4-NOT complex is a key effector of miRNA function acting downstream of GW182/TNRC6 proteins. We show that miRNA-mediated repression requires the central region of CNOT1, the scaffold protein of CCR4-NOT. A CNOT1 domain interacts with CNOT9, which in turn interacts with the silencing domain of TNRC6 in a tryptophan motif-dependent manner. These interactions are direct, as shown by the structure of a CNOT9-CNOT1 complex with bound tryptophan. Another domain of CNOT1 with an MIF4G fold recruits the DEAD-box ATPase DDX6, a known translational inhibitor. Structural and biochemical approaches revealed that CNOT1 modulates the conformation of DDX6 and stimulates ATPase activity. Structure-based mutations showed that the CNOT1 MIF4G-DDX6 interaction is important for miRNA-mediated repression. These findings provide insights into the repressive steps downstream of the GW182/TNRC6 proteins and the role of the CCR4-NOT complex in posttranscriptional regulation in general.