An urgent global quest for effective therapies to prevent and treat coronavirus disease 2019 (COVID-19) is ongoing. We previously described REGN-COV2, a cocktail of two potent neutralizing antibodies ...(REGN10987 and REGN10933) that targets nonoverlapping epitopes on the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein. In this report, we evaluate the in vivo efficacy of this antibody cocktail in both rhesus macaques, which may model mild disease, and golden hamsters, which may model more severe disease. We demonstrate that REGN-COV-2 can greatly reduce virus load in the lower and upper airways and decrease virus-induced pathological sequelae when administered prophylactically or therapeutically in rhesus macaques. Similarly, administration in hamsters limits weight loss and decreases lung titers and evidence of pneumonia in the lungs. Our results provide evidence of the therapeutic potential of this antibody cocktail.
Monoclonal antibodies against SARS-CoV-2 are a clinically validated therapeutic option against COVID-19. Because rapidly emerging virus mutants are becoming the next major concern in the fight ...against the global pandemic, it is imperative that these therapeutic treatments provide coverage against circulating variants and do not contribute to development of treatment-induced emergent resistance. To this end, we investigated the sequence diversity of the spike protein and monitored emergence of virus variants in SARS-COV-2 isolates found in COVID-19 patients treated with the two-antibody combination REGEN-COV, as well as in preclinical in vitro studies using single, dual, or triple antibody combinations, and in hamster in vivo studies using REGEN-COV or single monoclonal antibody treatments. Our study demonstrates that the combination of non-competing antibodies in REGEN-COV provides protection against all current SARS-CoV-2 variants of concern/interest and also protects against emergence of new variants and their potential seeding into the population in a clinical setting.
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•REGEN-COV retains neutralization potency against current variants of concern/interest•In vitro escape studies can predict emergence of viral variants in animals and humans•3 noncompeting mAb in combination reduce variant risk compared to a combination of 2•Treatment with REGEN-COV in humans does not lead to emergence of viral variants
Treatment with monoclonal antibody combinations limits generation of SARS-CoV-2 escape variants in humans and model animals and protects against current variants of concern.
Molecular epidemiological assessments, drug treatment optimization, and development of immunological interventions all depend on understanding pathogen adaptation and genetic variation, which differ ...for specific pathogens. Mycobacterium tuberculosis is an exceptionally successful human pathogen, yet beyond knowledge that this bacterium has low overall genomic variation but acquires drug resistance mutations, little is known of the factors that drive its population genomic characteristics. Here, we compared the genetic diversity of the bacteria that established infection to the bacterial populations obtained from infected tissues during murine M. tuberculosis pulmonary infection and human disseminated M. bovis BCG infection. We found that new mutations accumulate during in vitro culture, but that in vivo, purifying selection against new mutations dominates, indicating that M. tuberculosis follows a dominant lineage model of evolution. Comparing bacterial populations passaged in T cell-deficient and immunocompetent mice, we found that the presence of T cells is associated with an increase in the diversity of the M. tuberculosis genome. Together, our findings put M. tuberculosis genetic evolution in a new perspective and clarify the impact of T cells on sequence diversity of M. tuberculosis.
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Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The presentation of virus-derived peptides by HLA class I molecules on the surface of an infected cell and the recognition of these HLA-peptide complexes by, and subsequent activation of, CD8
...cytotoxic T cells provides an important mechanism for immune protection against viruses. Recent advances in proteogenomics have allowed researchers to discover a growing number of unique HLA-restricted viral peptides, resulting in a rapidly expanding repertoire of targets for immunotherapeutics (i.e. bispecific antibodies, engineered T-cell receptors (TCRs), chimeric antigen receptor T-cells (CAR-Ts)) to infected tissues. However, genomic variability between viral strains, such as Hepatitis-B virus (HBV), in combination with differences in patient HLA alleles, make it difficult to develop therapeutics against these targets. To address this challenge, we developed a novel proteogenomics approach for generating patient-specific databases that enable the identification of viral peptides based on the viral transcriptomes sequenced from individual patient liver samples. We also utilized DNA sequencing of patient samples to identify HLA genotypes and assist in target selection. Liver samples from 48 HBV infected patients, primarily from Asia, were examined to reconstruct patient-specific HBV genomes, identify regions within the human chromosomes targeted by HBV integrations and obtain a comprehensive view of HBV peptide epitopes using our HLA class-I (HLA-I) immunopeptidomics discovery platform. Two previously reported HLA associated HBV-derived peptides, HLA-A02 binder FLLTRILTI (S
) from the large surface antigen and HLA-A11 binder STLPETTVVRR (C
) from the capsid protein were validated by our discovery platform, but both were detected at very low frequencies. In addition, we identified and validated, using heavy peptide analogues, novel strain-specific HBV-HLA associated peptides, such as GSLPQEHIVQK (P
) and variants. Overall, our novel approach can guide the development of bispecific antibody, TCR-T, or CAR-T based therapeutics for the treatment of HBV-related HCC and inform vaccine development.
Brucella are facultative intracellular bacteria that chronically infect humans and animals causing brucellosis. Brucella are able to invade and replicate in a broad range of cell lines in vitro, ...however the cells supporting bacterial growth in vivo are largely unknown. In order to identify these, we used a Brucella melitensis strain stably expressing mCherry fluorescent protein to determine the phenotype of infected cells in spleen and liver, two major sites of B. melitensis growth in mice. In both tissues, the majority of primary infected cells expressed the F4/80 myeloid marker. The peak of infection correlated with granuloma development. These structures were mainly composed of CD11b⁺ F4/80⁺ MHC-II⁺ cells expressing iNOS/NOS2 enzyme. A fraction of these cells also expressed CD11c marker and appeared similar to inflammatory dendritic cells (DCs). Analysis of genetically deficient mice revealed that differentiation of iNOS⁺ inflammatory DC, granuloma formation and control of bacterial growth were deeply affected by the absence of MyD88, IL-12p35 and IFN-γ molecules. During chronic phase of infection in susceptible mice, we identified a particular subset of DC expressing both CD11c and CD205, serving as a reservoir for the bacteria. Taken together, our results describe the cellular nature of immune effectors involved during Brucella infection and reveal a previously unappreciated role for DC subsets, both as effectors and reservoir cells, in the pathogenesis of brucellosis.
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Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
In many bacteria, the phosphotransferase system (PTS) is a key player in the regulation of the assimilation of alternative carbon sources notably through catabolic repression. The intracellular ...pathogens Brucella spp. possess four PTS proteins (EINtr, NPr, EIIANtr and an EIIA of the mannose family) but no PTS permease suggesting that this PTS might serve only regulatory functions.
In vitro biochemical analyses and in vivo detection of two forms of EIIANtr (phosphorylated or not) established that the four PTS proteins of Brucella melitensis form a functional phosphorelay. Moreover, in vitro the protein kinase HprK/P phosphorylates NPr on a conserved serine residue, providing an additional level of regulation to the B. melitensis PTS. This kinase activity was inhibited by inorganic phosphate and stimulated by fructose-1,6 bisphosphate. The genes encoding HprK/P, an EIIAMan-like protein and NPr are clustered in a locus conserved among α-proteobacteria and also contain the genes for the crucial two-component system BvrR-BvrS. RT-PCR revealed a transcriptional link between these genes suggesting an interaction between PTS and BvrR-BvrS. Mutations leading to the inactivation of EINtr or NPr significantly lowered the synthesis of VirB proteins, which form a type IV secretion system. These two mutants also exhibit a small colony phenotype on solid media. Finally, interaction partners of PTS proteins were identified using a yeast two hybrid screen against the whole B. melitensis ORFeome. Both NPr and HprK/P were shown to interact with an inorganic pyrophosphatase and the EIIAMan-like protein with the E1 component (SucA) of 2-oxoglutarate dehydrogenase.
The B. melitensis can transfer the phosphoryl group from PEP to the EIIAs and a link between the PTS and the virulence of this organism could be established. Based on the protein interaction data a preliminary model is proposed in which this regulatory PTS coordinates also C and N metabolism.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The HLA (human leukocyte antigen) molecules that present pathogen-derived epitopes to T cells are highly diverse. Correspondingly, many pathogens such as HIV evolve epitope variants in order to evade ...immune recognition. In contrast, another persistent human pathogen, Mycobacterium tuberculosis, has highly conserved epitope sequences. This raises the question whether there is also a difference in the ability of these pathogens' epitopes to bind diverse HLA alleles, referred to as an epitope's binding promiscuity. To address this question, we compared the in silico HLA binding promiscuity of T cell epitopes from pathogens with distinct infection strategies and outcomes of human exposure.
We used computer algorithms to predict the binding affinity of experimentally-verified microbial epitope peptides to diverse HLA-DR, HLA-A and HLA-B alleles. We then analyzed binding promiscuity of epitopes derived from HIV and M. tuberculosis. We also analyzed promiscuity of epitopes from Streptococcus pyogenes, which is known to exhibit epitope diversity, and epitopes of Bacillus anthracis and Clostridium tetani toxins, as these bacteria do not depend on human hosts for their survival or replication, and their toxin antigens are highly immunogenic human vaccines.
We found that B. anthracis and C. tetani epitopes were the most promiscuous of the group that we analyzed. However, there was no consistent difference or trend in promiscuity in epitopes contained in HIV, M. tuberculosis, and S. pyogenes.
Our results show that human pathogens with distinct immune evasion strategies and epitope diversities exhibit equivalent levels of T cell epitope promiscuity. These results indicate that differences in epitope promiscuity do not account for the observed differences in epitope variation and conservation.
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Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Antibodies targeting the spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) present a promising approach to combat the coronavirus disease 2019 (COVID-19) pandemic; ...however, concerns remain that mutations can yield antibody resistance. We investigated the development of resistance against four antibodies to the spike protein that potently neutralize SARS-CoV-2, individually as well as when combined into cocktails. These antibodies remain effective against spike variants that have arisen in the human population. However, novel spike mutants rapidly appeared after in vitro passaging in the presence of individual antibodies, resulting in loss of neutralization; such escape also occurred with combinations of antibodies binding diverse but overlapping regions of the spike protein. Escape mutants were not generated after treatment with a noncompeting antibody cocktail.
•Whole-genome sequencing has enabled real-time genomic surveillance of S. aureus.•Of the 40,000 available S. aureus genomes in NCBI only 0.4% are fully assembled.•S. aureus genomic heterogeneity ...encompasses mutation and mobile genetic elements.•Long-read sequencing is required to capture mobile genetic element diversity.•Methods for accurately annotating of S. aureus genomes are desperately needed.
The genome of Staphylococcus aureus has rapidly become one the most frequently sequenced among bacteria, with more than 40000 genome sequences uploaded to public databases. Computational resources required for analysis and quality assessment have lagged behind accumulation of sequence data. Improved analytic pipelines, in combination with the development of customized S. aureus reference databases, can be used to inform S. aureus biology and potentially predict clinical outcome. Here, we review the currently available data about S. aureus genome in public databases, and discuss their potential utility for understanding S. aureus evolution. Also discussed are ways to overcome challenges to the application of whole-genome sequencing data for prevention and management of S. aureus disease.
Abstract Mycobacterium bovis bacillus Calmette-Guerin (BCG), the only vaccine currently used against tuberculosis, is an attenuated derivative of M. bovis that has been propagated in vitro for more ...than 40 years. We have previously reported that the experimentally-verified human T cell epitopes of the M. tuberculosis complex (MTBC) are the most conserved elements of the genome; whether immune recognition is the force driving the conservation of epitopes in the MTBC is unknown. Therefore, we sequenced the genomes of 12 BCG strains to determine whether T cell epitopes were under selection pressure during BCG in vitro evolution. We constructed a genome-wide phylogeny and refined the previously-determined BCG phylogeny. Notably, we identified a new cluster between BCG Japan and BCG Russia, and repositioned the relationships of several strains within the lineage. We also compared the sequence diversity of 1530 experimentally verified human T cell epitopes in the BCG vaccines with those in the MTBC. We found 23% of the known T cell epitopes are absent, and that the majority (82%) of the absent epitopes in BCG are contained in 6 proteins encoded in 2 regions of difference (RD) unique to BCG strains. We also found that T cell epitope sequences in BCG are more conserved than non-epitope sequences in the same gene. Finally, we find evidence that epitope sequence variation in BCG potentially affects human T cell recognition. These findings provide new insight into sequence variation in a slow-growing bacterium closely related to the MTBC that has been subjected to prolonged passage outside of a mammalian host, and indicate little difference in the extent of variation in vivo and in vitro.
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