Single-domain antigen-binding fragments of camelid antibodies, known as VHHs or nanobodies, are widely used affinity reagents. However, their production involving animal immunization is time- and ...resource-intensive. Starting from a sequence dataset of llama VHHs, we designed a novel scaffold, based on conserved framework sequences, suitable for bacterial nanobody expression and synthetic library construction. The consensus scaffold was validated by grafting the CDRs from two known nanobodies. While maintaining their binding properties, the two chimeric nanobodies showed higher levels of expression and solubility in
E. coli
when compared to the corresponding wild types. A proof-of-concept synthetic combinatorial library, suitable for ribosome display (RD) selection, was obtained by encoding three randomized complementarity determining regions within the consensus framework. The library, made of linear DNA fragments, has an estimated complexity of > 10
12
that is three orders of magnitude higher than common phage display libraries. The bacterial expression of several library clones showed a high production of soluble recombinant proteins. The high complexity of the library, confirmed by sequencing of a subset of clones, as well as a preliminary RD selection of a maltose binding protein binder, indicated this approach as a starting point in the construction of synthetic combinatorial libraries to be used as animal-free tools for the low-cost selection of target-specific nanobodies.
Bifidobacteria represent one of the dominant groups of microorganisms colonizing the human infant intestine. Commensal bacteria that interact with a eukaryotic host are believed to express adhesive ...molecules on their cell surface that bind to specific host cell receptors or soluble macromolecules. Whole-genome transcription profiling of Bifidobacterium bifidum PRL2010, a strain isolated from infant stool, revealed a small number of commonly expressed extracellular proteins, among which were genes that specify sortase-dependent pili. Expression of the coding sequences of these B. bifidum PRL2010 appendages in nonpiliated Lactococcus lactis enhanced adherence to human enterocytes through extracellular matrix protein and bacterial aggregation. Furthermore, such piliated L. lactis cells evoked a higher TNF-α response during murine colonization compared with their nonpiliated parent, suggesting that bifidobacterial sortase-dependent pili not only contribute to adherence but also display immunomodulatory activity.
Global burden of cervical cancer, the most common cause of mortality caused by human papillomavirus (HPV), is expected to increase during the next decade, mainly because current alternatives for HPV ...vaccination and cervical cancer screening programs are costly to be established in low-and-middle income countries. Recently, we described the development of the broadly protective, thermostable vaccine antigen Trx-8mer-OVX313 based on the insertion of eight different minor capsid protein L2 neutralization epitopes into a thioredoxin scaffold from the hyperthermophilic archaeon Pyrococcus furiosus and conversion of the resulting antigen into a nanoparticle format (median radius ~9 nm) upon fusion with the heptamerizing OVX313 module. Here we evaluated whether the engineered thioredoxin scaffold, in addition to humoral immune responses, can induce CD8.sup.+ T-cell responses upon incorporation of MHC-I-restricted epitopes. By systematically examining the contribution of individual antigen modules, we demonstrated that B-cell and T-cell epitopes can be combined into a single antigen construct without compromising either immunogenicity. While CD8.sup.+ T-cell epitopes had no influence on B-cell responses, the L2 polytope (8mer) and OVX313-mediated heptamerization of the final antigen significantly increased CD8.sup.+ T-cell responses. In a proof-of-concept experiment, we found that vaccinated mice remained tumor-free even after two consecutive tumor challenges, while unvaccinated mice developed tumors. A cost-effective, broadly protective vaccine with both prophylactic and therapeutic properties represents a promising option to overcome the challenges associated with prevention and treatment of HPV-caused diseases.
Innovative and highly performing smart voltammetric immunosensors for rapid and effective serological tests aimed at the determination of SARS-CoV-2 antibodies were developed and validated in human ...serum matrix. Two immunosensors were developed for the determination of immunoglobulins directed against either the nucleocapsid or the spike viral antigen proteins. The immunosensors were realized using disposable screen-printed electrodes modified with nanostructured materials for the immobilization of the antigens. Fast quantitative detection was achieved, with analysis duration being around 1 h. Signal readout was carried out through a smart, compact and battery-powered potentiostat, based on a Wi-Fi protocol and devised for the Internet of Things (IoT) paradigm. This device is used for the acquisition, storage and sharing of clinical data. Outstanding immunosensors’ sensitivity, specificity and accuracy (100%) were assessed, according to the diagnostic guidelines for epidemiological data. The overall performance of the sensing devices, combined with the portability of the IoT-based device, enables their suitability as a high-throughput diagnostic tool. Both of the immunosensors were validated using clinical human serum specimens from SARS-CoV-2 infected patients, provided by IRCCS Ospedale San Raffaele.
There is an urgent need for new generation anti-SARS-Cov-2 vaccines in order to increase the efficacy of immunization and its broadness of protection against viral variants that are continuously ...arising and spreading. The effect of variants on protective immunity afforded by vaccination has been mostly analyzed with regard to B cell responses. This analysis revealed variable levels of cross-neutralization capacity for presently available SARS-Cov-2 vaccines. Despite the dampened immune responses documented for some SARS-Cov-2 mutations, available vaccines appear to maintain an overall satisfactory protective activity against most variants of concern (VoC). This may be attributed, at least in part, to cell-mediated immunity. Indeed, the widely multi-specific nature of CD8 T cell responses should allow to avoid VoC-mediated viral escape, because mutational inactivation of a given CD8 T cell epitope is expected to be compensated by the persistent responses directed against unchanged co-existing CD8 epitopes. This is particularly relevant because some immunodominant CD8 T cell epitopes are located within highly conserved SARS-Cov-2 regions that cannot mutate without impairing SARS-Cov-2 functionality. Importantly, some of these conserved epitopes are degenerate, meaning that they are able to associate with different HLA class I molecules and to be simultaneously presented to CD8 T cell populations of different HLA restriction. Based on these concepts, vaccination strategies aimed at potentiating the stimulatory effect on SARS-Cov-2-specific CD8 T cells should greatly enhance the efficacy of immunization against SARS-Cov-2 variants. Our review recollects, discusses and puts into a translational perspective all available experimental data supporting these “hot” concepts, with special emphasis on the structural constraints that limit SARS-CoV-2 S-protein evolution and on potentially invariant and degenerate CD8 epitopes that lend themselves as excellent candidates for the rational development of next-generation, CD8 T-cell response-reinforced, COVID-19 vaccines.
Abstract The N-terminal region of the human papillomavirus (HPV) L2 protein has been shown to contain immune epitopes able to induce the production of neutralizing and cross-neutralizing antibodies ...(Gambhira et al., 2007; Kawana et al., 1999). Using bacterial thioredoxin as a scaffold, we managed to enhance the immunogenicity of putative L2 neutralizing epitopes, but only a minor fraction of the resulting immune responses was found to be neutralizing (Rubio et al., 2009). To determine the recognition patterns for non-neutralizing, neutralizing and cross-neutralizing antibodies, we isolated and characterized a panel of 46 monoclonal antibodies directed against different HPV16 L2 epitopes. Four of such antibodies proved to be neutralizing, and two of them, both targeting the amino acid (aa) 20–38 region of L2, were found to cross-neutralize a broad range of papillomaviruses. The epitopes recognized by neutralizing and cross-neutralizing antibodies were mapped at high resolution and were found to be characterized by distinct recognition patterns. Even in the case of the L2 20–38 epitope, cross-neutralization of HPV31 pseudovirions proved to be extremely inefficient, and this was found to be primarily due to the lack of a proline residue at position 30. HPV16 specific amino acids in this region also appear to be responsible for the lack of cross-neutralizing activity, thus suggesting a potential immune escape mechanism. For the aa 71–80 region, instead, the data indicate that restriction of neutralization to HPV16 is due to sequence (or structural) differences laying outside of the epitope. Besides providing new insights on the molecular bases of L2-mediated immune reactivity, the present data may pave the way to novel vaccination approaches specifically evoking cross-neutralizing antibody responses.
Polycationic resurfaced proteins hold great promise as cell-penetrating bioreagents but their use as carriers for the intracellular delivery of peptide immuno-epitopes has not thus far been explored. ...Here, we report on the construction and functional characterization of a positively supercharged derivative of
Pyrococcus furiosus
thioredoxin (
Pf
Trx), a thermally hyperstable protein we have previously validated as a peptide epitope display and immunogenicity enhancing scaffold. Genetic conversion of 13 selected amino acids to lysine residues conferred to
Pf
Trx a net charge of +21 (starting from the -1 charge of the wild-type protein), along with the ability to bind nucleic acids. In its unfused form, +21
Pf
Trx was readily internalized by HeLa cells and displayed a predominantly cytosolic localization. A different intracellular distribution was observed for a +21
Pf
Trx-eGFP fusion protein, which although still capable of cell penetration was predominantly localized within endosomes. A mixed cytosolic/endosomal partitioning was observed for a +21
Pf
Trx derivative harboring three tandemly repeated copies of a previously validated HPV16-L2 (aa 20-38) B-cell epitope grafted to the display site of thioredoxin. Compared to its wild-type counterpart, the positively supercharged antigen induced a faster immune response and displayed an overall superior immunogenicity, including a substantial degree of self-adjuvancy. Altogether, the present data point to +21
Pf
Trx as a promising novel carrier for intracellular antigen delivery and the construction of potentiated recombinant subunit vaccines.
An IoT-WiFi smart and portable electrochemical immunosensor for the quantification of SARS-CoV-2 spike protein was developed with integrated machine learning features. The immunoenzymatic sensor is ...based on the immobilization of monoclonal antibodies directed at the SARS-CoV-2 S1 subunit on Screen-Printed Electrodes functionalized with gold nanoparticles. The analytical protocol involves a single-step sample incubation. Immunosensor performance was validated in a viral transfer medium which is commonly used for the desorption of nasopharyngeal swabs. Remarkable specificity of the response was demonstrated by testing H1N1 Hemagglutinin from swine-origin influenza A virus and Spike Protein S1 from Middle East respiratory syndrome coronavirus. Machine learning was successfully used for data processing and analysis. Different support vector machine classifiers were evaluated, proving that algorithms affect the classifier accuracy. The test accuracy of the best classification model in terms of true positive/true negative sample classification was 97.3%. In addition, the ML algorithm can be easily integrated into cloud-based portable Wi-Fi devices. Finally, the immunosensor was successfully tested using a third generation replicating incompetent lentiviral vector pseudotyped with SARS-CoV-2 spike glycoprotein, thus proving the applicability of the immunosensor to whole virus detection.
Abstract
Skin colonization by human papillomavirus (HPV) is typically related to inconspicuous cutaneous infections without major disease or complications in immunocompetent individuals. However, in ...immunosuppressed patients, especially organ transplanted recipients, cutaneous HPV infections may cause massive, highly spreading and recurrent skin lesions upon synergism with UV-exposure. Current HPV prophylactic vaccines are not effective against cutaneous HPV types (cHPV). By applying a modular polytope-based approach, in this work, we explored different vaccine candidates based on selected, tandemly arranged cHPV-L2 epitopes fused to thioredoxin (Trx) as a scaffold protein. Upon conversion to heptameric nanoparticles with the use of a genetically fused oligomerization domain, our candidate Trx-L2 vaccines induce broadly neutralizing immune responses against 19 cHPV in guinea pigs. Similar findings were obtained in mice, where protection against virus challenge was also achieved via passive transfer of immune sera. Remarkably, immunization with the candidate cHPV vaccines also induced immune responses against several mucosal low- and high-risk HPV types, including HPV16 and 18. Based on cumulative immunogenicity data but also on ease and yield of production, we identified a lead vaccine candidate bearing 12 different cHPV-L2 epitopes that holds great promise as a scalable and GMP production-compatible lead molecule for the prevention of post-transplantation skin lesions caused by cHPV infection.
The human papillomavirus minor capsid protein L2 is being extensively explored in pre-clinical studies as an attractive vaccine antigen capable of inducing broad-spectrum prophylactic antibody ...responses. Recently, we have developed two HPV vaccine antigens - PANHPVAX and CUT-PANHPVAX- both based on heptameric nanoparticle antigens displaying polytopes of the L2 major cross-neutralizing epitopes of eight mucosal and twelve cutaneous HPV types, respectively. Prompted by the variable neutralizing antibody responses against some of the HPV types targeted by the antigens observed in previous studies, here we investigated the influence on immunogenicity of six distinct glycine-proline spacers inserted upstream to a specific L2 epitope. We show that spacer variants differentially influence antigen immunogenicity in a mouse model, with the antigen constructs M8merV6 and C12merV6 displaying a superior ability in the induction of neutralizing antibodies as determined by pseudovirus-based neutralization assays (PBNAs). L2-peptide enzyme-linked immunosorbent assay (ELISA) assessments determined the total anti-L2 antibody level for each antigen variant, showing for the majority of sera a correlation with their repective neutralizing antibody level. Surface Plasmon Resonance revealed that L2 epitope-specific, neutralizing monoclonal antibodies (mAbs) display distinct avidities to different antigen spacer variants. Furthermore, mAb affinity toward individual spacer variants was well correlated with their neutralizing antibody induction capacity, indicating that the mAb affinity assay predicts L2-based antigen immunogenicity. These observations provide insights on the development and optimization of L2-based HPV vaccines.
PDF
