Integrating form and function for design: Antibodies are broadly used in therapies and as research tools because they can be generated against a wide range of targets. Efficacy can often be increased by clustering antibodies in multivalent assemblies. Divine et al. designed antibody nanocages from two components: One is an antibody-binding homo-oligomic protein and the other is the antibody itself. Computationally designed proteins drive the assembly of antibody nanocages in a range of architectures, allowing control of the symmetry and the antibody valency. The multivalent display enhances antibody-dependent signaling, and nanocages displaying antibodies against the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein effectively neutralize pseudovirus. Science , this issue p. eabd9994

The vector-borne disease leishmaniasis is transmitted to humans by infected female sand flies, which transmits Leishmania parasites together with saliva during blood feeding. In Iran, cutaneous leishmaniasis (CL) is caused by Leishmania (L.) major and L. tropica, and their main vectors are Phlebotomus (Ph.) papatasi and Ph. sergenti, respectively. Previous studies have demonstrated that mice immunized with the salivary gland homogenate (SGH) of Ph. papatasi or subjected to bites from uninfected sand flies are protected against L. major infection. In this work we tested the immune response in BALB/c mice to 14 different plasmids coding for the most abundant salivary proteins of Ph. sergenti. The plasmid coding for the salivary protein PsSP9 induced a DTH response in the presence of a significant increase of IFN-γ expression in draining lymph nodes (dLN) as compared to control plasmid and no detectable PsSP9 antibody response. Animals immunized with whole Ph. sergenti SGH developed only a saliva-specific antibody response and no DTH response. Mice immunized with whole Ph. sergenti saliva and challenged intradermally with L. tropica plus Ph. sergenti SGH in their ears, exhibited no protective effect. In contrast, PsSP9-immunized mice showed protection against L. tropica infection resulting in a reduction in nodule size, disease burden and parasite burden compared to controls. Two months post infection, protection was associated with a significant increase in the ratio of IFN-γ to IL-5 expression in the dLN compared to controls. This study demonstrates that while immunity to the whole Ph. sergenti saliva does not induce a protective response against cutaneous leishmaniasis in BALB/c mice, PsSP9, a member of the PpSP15 family of Ph. sergenti salivary proteins, provides protection against L. tropica infection. These results suggest that this family of proteins in Ph. sergenti, Ph. duboscqi and Ph. papatasi may have similar immunogenic and protective properties against different Leishmania species. Indeed, this anti-saliva immunity may act as an adjuvant to accelerate the cell-mediated immune response to co-administered Leishmania antigens, or even cause the activation of infected macrophages to remove parasites more efficiently. These findings highlight the idea of applying arthropod saliva components in vaccination approaches for diseases caused by vector-borne pathogens. Leishmaniasis is a vector-borne disease transmitted to humans by an infected sand fly bite, through which Leishmania parasites and saliva are co-delivered into the host skin. Despite the numerous studies performed in this area, no vaccine is yet available to control this neglected disease in humans. During the past two decades, saliva of sand flies has been tested for possible application as a vaccine against leishmaniasis. Exposure to specific salivary proteins or sand fly bites can induce a protective cell-mediated immunity. Immunization with Ph. papatasi saliva or recombinant PpSP15 has been previously reported to provide protection against L. major infection. In this study, the efficiency of immunization with Ph. sergenti saliva or plasmid coding for Ph. sergenti salivary proteins in protecting the BALB/c mice against L. tropica was explored. Here we show that although immunization with whole saliva induces a humoral response, this immune response is unable to protect mice against infection; in contrast, immunization with a plasmid coding for Ph. sergenti PsSP9 salivary protein induces a Th1 immune response characterized by a strong DTH response, no detectable antibody response, and a high expression ratio of IFN-γ to IL-5 in lymph nodes. The Th1 induced immunity in PsSP9-immunized mice correlates with the protection observed against infection with L. tropica, which was associated with a significant reduction in ear thickness and decrease in parasite load in the ear of protected animals in comparison to control group. Our data suggest that instead of using the whole sand fly salivary proteins, a more effective approach is the use of a single Ph. sergenti salivary protein.

High yield recombinant protein production is highly desirable for biotechnological purposes. In the design of recombinant expression conditions, a number of essential central elements such as expression strain, type of medium, bioprocess optimization, and mathematical modeling should be considered. Well-designed industrial scale production of one recombinant protein with optimized influential parameters and yield can address the cost and production reproducibility issues. In the present study, statistical experimental design methodology was used to investigate the effect of fermentation conditions (dissolved oxygen, IPTG, and temperature) on rPDT production by Escherichia coli. rPDT is a recombinant fusion protein consisting of three different protein domains including the N-terminal 179 amino acid fragment of the S1 subunit of pertussis toxin, the full-length genetically detoxified diphtheria toxin (CRM197), and the 50 kDa tetanus toxin fragment C. A 15 Box–Behnken design augmented with center points revealed that IPTG and DO at the center point and low temperature will result in high yield. The optimal condition for rPDT production were found to be 100 µM IPTG, DO 30% and temperature 20 °C.

Antibodies are widely used in biology and medicine, and there has been considerable interest in multivalent antibody formats to increase binding avidity and enhance signaling pathway agonism. However, there are currently no general approaches for forming precisely oriented antibody assemblies with controlled valency. We describe the computational design of two-component nanocages that overcome this limitation by uniting form and function. One structural component is any antibody or Fc fusion and the second is a designed Fc-binding homo-oligomer that drives nanocage assembly. Structures of 8 antibody nanocages determined by electron microscopy spanning dihedral, tetrahedral, octahedral, and icosahedral architectures with 2, 6, 12, and 30 antibodies per nanocage match the corresponding computational models. Antibody nanocages targeting cell-surface receptors enhance signaling compared to free antibodies or Fc-fusions in DR5-mediated apoptosis, Tie2-mediated angiogenesis, CD40 activation, and T cell proliferation; nanocage assembly also increases SARS-CoV-2 pseudovirus neutralization by α-SARS-CoV-2 monoclonal antibodies and Fc-ACE2 fusion proteins. We anticipate that the ability to assemble arbitrary antibodies without need for covalent modification into highly ordered assemblies with different geometries and valencies will have broad impact in biology and medicine.