Broad Epitope Coverage of Therapeutic Multi-Antibody Combinations Targeting SARS-CoV-2 Boosts In Vivo Protection and Neutralization Potency to Corner an Immune-Evading Virus.

Therapeutic antibodies (Abs) which act on a broader range of epitopes may provide more durable protection against the genetic drift of a target, typical of viruses or tumors. When these Abs exist concurrently on the targeted antigen, several mechanisms of action (MoAs) can be engaged, boosting therapeutic potency. This study selected combinations of four and five Abs with non- or partially overlapping epitopes to the SARS-CoV-2 spike glycoprotein, on or outside the crucial receptor binding domain (RBD), to offer resilience to emerging variants and trigger multiple MoAs. The combinations were derived from a pool of unique-sequence scFv Ab fragments retrieved from two SARS-CoV-2-naïve human phage display libraries. Following recombinant expression to full-length human IgG1 candidates, a biolayer interferometric analysis mapped epitopes to bins and confirmed that up to four Abs from across the bins can exist simultaneously on the spike glycoprotein trimer. Not all the bins of Abs interfered with the spike protein binding to angiotensin converting enzyme 2 (ACE2) in competitive binding assays, nor neutralized the pseudovirus or authentic virus in vitro, but when combined in vivo, their inclusion resulted in a much stronger viral clearance in the lungs of intranasally challenged hamsters, compared to that of those treated with mono ACE2 blockers. In addition, the Ab mixtures activated in vitro reporter cells expressing Fc-gamma receptors (FcγRs) involved in antibody-dependent cellular cytotoxicity (ADCC) and phagocytosis (ADCP). The best four-Ab combination neutralized seventeen variants of concern from Wuhan-Hu1 to Omicron BA.4/BA.5 in vitro.

Temperature-Sensitive Salmonella enterica Serovar Enteritidis PT13a Expressing Essential Proteins of Psychrophilic Bacteria.

Synthetic genes based on deduced amino acid sequences of the NAD-dependent DNA ligase (ligA) and CTP synthetase (pyrG) of psychrophilic bacteria were substituted for their native homologues in the genome of Salmonella enterica serovar Enteritidis phage type 13a (PT13a). The resulting strains were rendered temperature sensitive (TS) and did not revert to temperature resistance at a detectable level. At permissive temperatures, TS strains grew like the parental strain in broth medium and in macrophage-like cells, but their growth was slowed or stopped when they were shifted to a restrictive temperature. When injected into BALB/c mice at the base of the tail, representing a cool site of the body, the strains with restrictive temperatures of 37, 38.5, and 39°C persisted for less than 1 day, 4 to 7 days, and 20 to 28 days, respectively. The wild-type strain persisted at the site of inoculation for at least 28 days. The wild-type strain, but not the TS strains, was also found in spleen-plus-liver homogenates within 1 day of inoculation of the tail and was detectable in these organs for at least 28 days. Intramuscular vaccination of White Leghorn chickens with the PT13a strain carrying the psychrophilic pyrG gene provided some protection against colonization of the reproductive tract and induced an anti-S. enterica antibody response.

The biochemical properties of the Francisella pathogenicity island (FPI)-encoded proteins IglA, IglB, IglC, PdpB and DotU suggest roles in type VI secretion.

The Francisella pathogenicity island (FPI) encodes proteins thought to compose a type VI secretion system (T6SS) that is required for the intracellular growth of Francisella novicida. In this work we used deletion mutagenesis and genetic complementation to determine that the intracellular growth of F. novicida was dependent on 14 of the 18 genes in the FPI. The products of the iglABCD operon were localized by the biochemical fractionation of F. novicida, and Francisella tularensis LVS. Sucrose gradient separation of water-insoluble material showed that the FPI-encoded proteins IglA, IglB and IglC were found in multiple fractions, especially in a fraction that did not correspond to a known membrane fraction. We interpreted these data to suggest that IglA, IglB and IglC are part of a macromolecular structure. Analysis of published structural data suggested that IglC is an analogue of Hcp, which is thought to form long nano-tubes. Thus the fractionation properties of IglA, IglB and IglC are consistent with the current model of the T6SS apparatus, which supposes that IglA and IglB homologues form an outer tube structure that surrounds an inner tube composed of Hcp (IglC) subunits. Fractionation of F. novicida expressing FLAG-tagged DotU (IcmH homologue) and PdpB (IcmF homologue) showed that these proteins localize to the inner membrane. Deletion of dotU led to the cleavage of PdpB, suggesting an interaction of these two proteins that is consistent with results obtained with other T6SSs. Our results may provide a mechanistic basis for many of the studies that have examined the virulence properties of Francisella mutants in FPI genes, namely that the observed phenotypes of the mutants are the result of the disruption of the FPI-encoded T6SS structure.

Human body temperature and new approaches to constructing temperature-sensitive bacterial vaccines.

Many of the live human and animal vaccines that are currently in use are attenuated by virtue of their temperature-sensitive (TS) replication. These vaccines are able to function because they can take advantage of sites in mammalian bodies that are cooler than the core temperature, where TS vaccines fail to replicate. In this article, we discuss the distribution of temperature in the human body, and relate how the temperature differential can be exploited for designing and using TS vaccines. We also examine how one of the coolest organs of the body, the skin, contains antigen-processing cells that can be targeted to provoke the desired immune response from a TS vaccine. We describe traditional approaches to making TS vaccines, and highlight new information and technologies that are being used to create a new generation of engineered TS vaccines. We pay particular attention to the recently described technology of substituting essential genes from Arctic bacteria for their homologues in mammalian pathogens as a way of creating TS vaccines.

Temperature-sensitive bacterial pathogens generated by the substitution of essential genes from cold-loving bacteria: potential use as live vaccines.

Temperature-sensitive (TS) viruses have been used for decades as vaccines capable of limited replication in their hosts. Although attenuated bacteria, such as the Bacille Calmette-Guérin anti-tuberculosis vaccine, have been used for almost a century, it is only recently that there has been progress in using TS bacterial strains as live vaccines. Decades of work on essential bacterial genes and the recent explosion in the number of available bacterial genomic sequences set the groundwork for the identification of essential genes from diverse bacteria. This knowledge has allowed for the substitution of essential genes from cold-loving bacteria into the chromosomes of pathogenic bacteria. Many of these gene substitutions generated TS pathogenic bacterial strains, and some were demonstrated to provide protective immunity in mice. This work opens the possibility of engineering many pathogenic bacteria to create TS strains that can be used as vaccines.

Essential genes from Arctic bacteria used to construct stable, temperature-sensitive bacterial vaccines.

All bacteria share a set of evolutionarily conserved essential genes that encode products that are required for viability. The great diversity of environments that bacteria inhabit, including environments at extreme temperatures, place adaptive pressure on essential genes. We sought to use this evolutionary diversity of essential genes to engineer bacterial pathogens to be stably temperature-sensitive, and thus useful as live vaccines. We isolated essential genes from bacteria found in the Arctic and substituted them for their counterparts into pathogens of mammals. We found that substitution of nine different essential genes from psychrophilic (cold-loving) bacteria into mammalian pathogenic bacteria resulted in strains that died below their normal-temperature growth limits. Substitution of three different psychrophilic gene orthologs of ligA, which encode NAD-dependent DNA ligase, resulted in bacterial strains that died at 33, 35, and 37 degrees C. One ligA gene was shown to render Francisella tularensis, Salmonella enterica, and Mycobacterium smegmatis temperature-sensitive, demonstrating that this gene functions in both Gram-negative and Gram-positive lineage bacteria. Three temperature-sensitive F. tularensis strains were shown to induce protective immunity after vaccination at a cool body site. About half of the genes that could be tested were unable to mutate to temperature-resistant forms at detectable levels. These results show that psychrophilic essential genes can be used to create a unique class of bacterial temperature-sensitive vaccines for important human pathogens, such as S. enterica and Mycobacterium tuberculosis.

Characterization of the pathogenicity island protein PdpA and its role in the virulence of Francisella novicida.

Francisella tularensis is a highly virulent, intracellular pathogen that causes the disease tularaemia. A research surrogate for F. tularensis is Francisella novicida, which causes a tularaemia-like disease in mice, grows similarly in macrophages, and yet is unable to cause disease in humans. Both Francisella species contain a cluster of genes referred to as the Francisella pathogenicity island (FPI). Pathogenicity determinant protein A (PdpA), encoded by the pdpA gene, is located within the FPI and has been associated with the virulence of Francisella species. In this work we examined the properties of PdpA protein expression and localization as well as the phenotype of a F. novicida pdpA deletion mutant. Monoclonal antibody detection of PdpA showed that it is a soluble protein that is upregulated in iron-limiting conditions and undetectable in an mglA or mglB mutant background. Deletion of pdpA resulted in a strain that was highly attenuated for virulence in chicken embryos and mice.

A Francisella novicida pdpA mutant exhibits limited intracellular replication and remains associated with the lysosomal marker LAMP-1.

Several genes contained in the Francisella pathogenicity island (FPI) encode proteins needed for intracellular growth and virulence of Francisella tularensis. The pdpA gene is the first cistron in the larger of the two operons found in the FPI. In this work we studied the intracellular growth phenotype of a Francisella novicida mutant in the pdpA gene. The DeltapdpA strain was capable of a small amount of intracellular replication but, unlike wild-type F. novicida, remained associated with the lysosomal marker LAMP-1, suggesting that PdpA is necessary for progression from the early phagosome phase of infection. Strains with in cis complementation of the DeltapdpA lesion showed a restoration of intracellular growth to wild-type levels. Infection of macrophages with the DeltapdpA mutant generated a host-cell mRNA profile distinct from that generated by infection with wild-type F. novicida. The transcriptional response of the host macrophage indicates that PdpA functions directly or indirectly to suppress macrophage ability to signal via growth factors, cytokines and adhesion ligands.

The Francisella pathogenicity island protein PdpD is required for full virulence and associates with homologues of the type VI secretion system.

Francisella tularensis is a highly infectious, facultative intracellular bacterial pathogen that is the causative agent of tularemia. Nearly a century ago, researchers observed that tularemia was often fatal in North America but almost never fatal in Europe and Asia. The chromosomes of F. tularensis strains carry two identical copies of the Francisella pathogenicity island (FPI), and the FPIs of North America-specific biotypes contain two genes, anmK and pdpD, that are not found in biotypes that are distributed over the entire Northern Hemisphere. In this work, we studied the contribution of anmK and pdpD to virulence by using F. novicida, which is very closely related to F. tularensis but which carries only one copy of the FPI. We showed that anmK and pdpD are necessary for full virulence but not for intracellular growth. This is in sharp contrast to most other FPI genes that have been studied to date, which are required for intracellular growth. We also showed that PdpD is localized to the outer membrane. Further, overexpression of PdpD affects the cellular distribution of FPI-encoded proteins IglA, IglB, and IglC. Finally, deletions of FPI genes encoding proteins that are homologues of known components of type VI secretion systems abolished the altered distribution of IglC and the outer membrane localization of PdpD.

Genetic elements for selection, deletion mutagenesis and complementation in Francisella spp.

Francisella novicida is a gram-negative pathogen that can induce disease in mice that mimics human tularemia, and is nearly identical to Francisella tularensis at the genomic level. In this work a number of antibiotic marker cassettes that incorporate a strong F. novicida promoter is constructed, which greatly enhances selection in F. novicida and F. tularensis. Two low-copy plasmid vectors based on a broad-host-range plasmid, and an integrating vector have also been made, and these can be used for genetic complementation. Two general approaches to deletion mutagenesis in F. novicida is also described.