A systems immunology study comparing innate and adaptive immune responses in adults to COVID-19 mRNA and adenovirus vectored vaccines.

Identifying the molecular mechanisms that promote optimal immune responses to coronavirus disease 2019 (COVID-19) vaccination is critical for future rational vaccine design. Here, we longitudinally profile innate and adaptive immune responses in 102 adults after the first, second, and third doses of mRNA or adenovirus-vectored COVID-19 vaccines. Using a multi-omics approach, we identify key differences in the immune responses induced by ChAdOx1-S and BNT162b2 that correlate with antigen-specific antibody and T cell responses or vaccine reactogenicity. Unexpectedly, we observe that vaccination with ChAdOx1-S, but not BNT162b2, induces an adenoviral vector-specific memory response after the first dose, which correlates with the expression of proteins with roles in thrombosis with potential implications for thrombosis with thrombocytopenia syndrome (TTS), a rare but serious adverse event linked to adenovirus-vectored vaccines. The COVID-19 Vaccine Immune Responses Study thus represents a major resource that can be used to understand the immunogenicity and reactogenicity of these COVID-19 vaccines.

Protocol to assess the impact of early-life antibiotic exposure on murine longevity.

We present this protocol using a mouse model to assess the impact of early-life antibiotic exposure on mammalian lifespan and the composition of the gut microbiota over time. We describe longitudinal fecal sampling and health monitoring following early-life antibiotic exposure. We detail DNA extraction and 16S rRNA gene sequencing to longitudinally profile the composition of the fecal microbiota. Finally, we discuss how to address potential confounders such as the stochastic recolonization of the gut microbiota following antibiotic exposure. For complete details on the use and execution of this protocol, please refer to Lynn et al. (2021).

Long-term perturbation of the peripheral immune system months after SARS-CoV-2 infection.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a highly infectious respiratory virus which is responsible for the coronavirus disease 2019 (COVID-19) pandemic. It is increasingly clear that recovered individuals, even those who had mild COVID-19, can suffer from persistent symptoms for many months after infection, a condition referred to as "long COVID", post-acute sequelae of COVID-19 (PASC), post-acute COVID-19 syndrome, or post COVID-19 condition. However, despite the plethora of research on COVID-19, relatively little is known about the molecular underpinnings of these long-term effects. METHODS: We have undertaken an integrated analysis of immune responses in blood at a transcriptional, cellular, and serological level at 12, 16, and 24 weeks post-infection (wpi) in 69 patients recovering from mild, moderate, severe, or critical COVID-19 in comparison to healthy uninfected controls. Twenty-one of these patients were referred to a long COVID clinic and > 50% reported ongoing symptoms more than 6 months post-infection. RESULTS: Anti-Spike and anti-RBD IgG responses were largely stable up to 24 wpi and correlated with disease severity. Deep immunophenotyping revealed significant differences in multiple innate (NK cells, LD neutrophils, CXCR3+ monocytes) and adaptive immune populations (T helper, T follicular helper, and regulatory T cells) in convalescent individuals compared to healthy controls, which were most strongly evident at 12 and 16 wpi. RNA sequencing revealed significant perturbations to gene expression in COVID-19 convalescents until at least 6 months post-infection. We also uncovered significant differences in the transcriptome at 24 wpi of convalescents who were referred to a long COVID clinic compared to those who were not. CONCLUSIONS: Variation in the rate of recovery from infection at a cellular and transcriptional level may explain the persistence of symptoms associated with long COVID in some individuals.

Protocol to colonize gnotobiotic mice in early life and assess the impact on early life immune programming.

Understanding how changes in gut microbiota in early life impact immune programming can be difficult to study due to variations in the assembly of the microbiota. In this protocol, we describe how to colonize gnotobiotic/germ-free mice in early life with different microbiota community types (e.g., PAMI and PAMII). We detail several assays to determine whether differential colonization alters immune programming in early life. We also describe how to propagate mouse fecal microbiota transplant material if the donor fecal sample is limited. For complete details on the use and execution of this protocol, please refer to Lynn et al. (2021).1.

Modulation of immune responses to vaccination by the microbiota: implications and potential mechanisms.

The need for highly effective vaccines that induce robust and long-lasting immunity has never been more apparent. However, for reasons that are still poorly understood, immune responses to vaccination are highly variable between different individuals and different populations. Furthermore, vaccine immunogenicity is frequently suboptimal in the very populations who are at most risk from infectious disease, including infants, the elderly, and those living in low-income and middle-income countries. Although many factors have the potential to influence vaccine immunogenicity and therefore vaccine effectiveness, increasing evidence from clinical studies and animal models now suggests that the composition and function of the gut microbiota are crucial factors modulating immune responses to vaccination. In this Review, we synthesize this evidence, discuss the immunological mechanisms that potentially mediate these effects and consider the potential of microbiota-targeted interventions to optimize vaccine effectiveness.

Immunisation with the BCG and DTPw vaccines induces different programs of trained immunity in mice.

In addition to providing pathogen-specific immunity, vaccines can also confer nonspecific effects (NSEs) on mortality and morbidity unrelated to the targeted disease. Immunisation with live vaccines, such as the BCG vaccine, has generally been associated with significantly reduced all-cause infant mortality. In contrast, some inactivated vaccines, such as the diphtheria, tetanus, whole-cell pertussis (DTPw) vaccine, have been controversially associated with increased all-cause mortality especially in female infants in high-mortality settings. The NSEs associated with BCG have been attributed, in part, to the induction of trained immunity, an epigenetic and metabolic reprograming of innate immune cells, increasing their responsiveness to subsequent microbial encounters. Whether non-live vaccines such as DTPw induce trained immunity is currently poorly understood. Here, we report that immunisation of mice with DTPw induced a unique program of trained immunity in comparison to BCG immunised mice. Altered monocyte and DC cytokine responses were evident in DTPw immunised mice even months after vaccination. Furthermore, splenic cDCs from DTPw immunised mice had altered chromatin accessibility at loci involved in immunity and metabolism, suggesting that these changes were epigenetically mediated. Interestingly, changing the order in which the BCG and DTPw vaccines were co-administered to mice altered subsequent trained immune responses. Given these differences in trained immunity, we also assessed whether administration of these vaccines altered susceptibility to sepsis in two different mouse models. Immunisation with either BCG or a DTPw-containing vaccine prior to the induction of sepsis did not significantly alter survival. Further studies are now needed to more fully investigate the potential consequences of DTPw induced trained immunity in different contexts and to assess whether other non-live vaccines also induce similar changes.

The composition of the gut microbiota following early-life antibiotic exposure affects host health and longevity in later life.

Studies investigating whether there is a causative link between the gut microbiota and lifespan have largely been restricted to invertebrates or to mice with a reduced lifespan because of a genetic deficiency. We investigate the effect of early-life antibiotic exposure on otherwise healthy, normal chow-fed, wild-type mice, monitoring these mice for more than 700 days in comparison with untreated control mice. We demonstrate the emergence of two different low-diversity community types, post-antibiotic microbiota (PAM) I and PAM II, following antibiotic exposure. PAM II but not PAM I mice have impaired immunity, increased insulin resistance, and evidence of increased inflammaging in later life as well as a reduced lifespan. Our data suggest that differences in the composition of the gut microbiota following antibiotic exposure differentially affect host health and longevity in later life.

The immunotoxicity, but not anti-tumor efficacy, of anti-CD40 and anti-CD137 immunotherapies is dependent on the gut microbiota.

Immune agonist antibodies (IAAs) are promising immunotherapies that target co-stimulatory receptors to induce potent anti-tumor immune responses, particularly when combined with checkpoint inhibitors. Unfortunately, their clinical translation is hampered by serious dose-limiting, immune-mediated toxicities, including high-grade and sometimes fatal liver damage, cytokine release syndrome (CRS), and colitis. We show that the immunotoxicity, induced by the IAAs anti-CD40 and anti-CD137, is dependent on the gut microbiota. Germ-free or antibiotic-treated mice have significantly reduced colitis, CRS, and liver damage following IAA treatment compared with conventional mice or germ-free mice recolonized via fecal microbiota transplant. MyD88 signaling is required for IAA-induced CRS and for anti-CD137-induced, but not anti-CD40-induced, liver damage. Importantly, antibiotic treatment does not impair IAA anti-tumor efficacy, alone or in combination with anti-PD1. Our results suggest that microbiota-targeted therapies could overcome the toxicity induced by IAAs without impairing their anti-tumor activity.

Extensive rewiring of the EGFR network in colorectal cancer cells expressing transforming levels of KRASG13D.

Protein-protein-interaction networks (PPINs) organize fundamental biological processes, but how oncogenic mutations impact these interactions and their functions at a network-level scale is poorly understood. Here, we analyze how a common oncogenic KRAS mutation (KRASG13D) affects PPIN structure and function of the Epidermal Growth Factor Receptor (EGFR) network in colorectal cancer (CRC) cells. Mapping >6000 PPIs shows that this network is extensively rewired in cells expressing transforming levels of KRASG13D (mtKRAS). The factors driving PPIN rewiring are multifactorial including changes in protein expression and phosphorylation. Mathematical modelling also suggests that the binding dynamics of low and high affinity KRAS interactors contribute to rewiring. PPIN rewiring substantially alters the composition of protein complexes, signal flow, transcriptional regulation, and cellular phenotype. These changes are validated by targeted and global experimental analysis. Importantly, genetic alterations in the most extensively rewired PPIN nodes occur frequently in CRC and are prognostic of poor patient outcomes.

Transcriptional and metabolic rewiring of colorectal cancer cells expressing the oncogenic KRASG13D mutation.

BACKGROUND: Activating mutations in KRAS frequently occur in colorectal cancer (CRC) patients, leading to resistance to EGFR-targeted therapies. METHODS: To better understand the cellular reprogramming which occurs in mutant KRAS cells, we have undertaken a systems-level analysis of four CRC cell lines which express either wild type (wt) KRAS or the oncogenic KRASG13D allele (mtKRAS). RESULTS: RNAseq revealed that genes involved in ribosome biogenesis, mRNA translation and metabolism were significantly upregulated in mtKRAS cells. Consistent with the transcriptional data, protein synthesis and cell proliferation were significantly higher in the mtKRAS cells. Targeted metabolomics analysis also confirmed the metabolic reprogramming in mtKRAS cells. Interestingly, mtKRAS cells were highly transcriptionally responsive to EGFR activation by TGFα stimulation, which was associated with an unexpected downregulation of genes involved in a range of anabolic processes. While TGFα treatment strongly activated protein synthesis in wtKRAS cells, protein synthesis was not activated above basal levels in the TGFα-treated mtKRAS cells. This was likely due to the defective activation of the mTORC1 and other pathways by TGFα in mtKRAS cells, which was associated with impaired activation of PKB signalling and a transient induction of AMPK signalling. CONCLUSIONS: We have found that mtKRAS cells are substantially rewired at the transcriptional, translational and metabolic levels and that this rewiring may reveal new vulnerabilities in oncogenic KRAS CRC cells that could be exploited in future.

Investigation of molecular alterations of AKT-3 in triple-negative breast cancer.

AIMS: Triple-negative breast cancer (TNBC) is responsible for a disproportionate number of breast cancer (BC) deaths, owing to its intrinsic aggressiveness and a lack of treatment options, especially targeted therapies. Thus, there is an urgent need for the development of better targeted treatments for TNBC. Molecular alteration of AKT-3 was previously reported in oestrogen receptor (ER)-positive BC. AKT-3 has also been suggested to play a role in hormone-unresponsive BC. The aim of this study was to investigate molecular alterations of AKT-3 in TNBC, to perform associated survival analysis, and to compare these findings with the incidence of AKT-3 molecular alterations in ER-positive BC. RESULTS: Our study revealed AKT-3 amplification and deletions in 11% (9/82) and 13% (11/82) of TNBCs, respectively. In contrast, 1% (2/209) of ER-positive BCs were found to have AKT-3 amplifications and deletions. A higher prevalence of AKT-3 copy number gains was observed in TNBC [26% (21/82)] than in ER-positive BC [9% (19/209)]. AKT-3 amplification together with Akt-3 protein expression was negatively associated with recurrence-free survival in TNBC. Furthermore, a negative association between high AKT-3 copy number and recurrence-free survival was observed. CONCLUSION: AKT-3 amplification could represent a potentially relevant oncogenic event in a subset of TNBCs that may, in turn, select cells sensitive to Akt-3 inhibitors.

Differential quantitative proteomic profiling of Leishmania infantum and Leishmania mexicana density gradient separated membranous fractions.

Leishmaniasis, caused by infection with Leishmania, is a major public health concern affecting more than 20million people globally. Leishmania has a digenetic lifecycle consisting of an extracellular flagellated promastigote, adapted to live in the mid-gut of the sand fly host and an aflagellated intracellular amastigote that resides within the macrophage of the mammalian host. Leishmania mexicana and Leishmania infantum are causative agents of cutaneous and visceral leishmaniasis, respectively. Membrane proteins play a pivotal role in host-pathogen interactions and in regulatory pathways. As the genome of Leishmania is essentially constitutively expressed, regulation of protein expression during differentiation occurs post-transcriptionally and/or post-translationally. Quantitative mass spectrometry using iTRAQ labeling identified differences in the proteomes of density gradient separated membranous fractions of promastigote and amastigote life-stages. We identified 189 L. infantum and 107 L. mexicana non-redundant proteins of which 20-40% showed differential expression levels between promastigote and amastigote lifecycle stages. Differentially expressed proteins mapped to several pathways including cell motility, metabolism, and infectivity as well as virulence factors such as eEF-1α, amastin and leishmanolysin (GP63). Western blot analysis validated iTRAQ quantitation for leishmanolysin. Focusing on differentially expressed proteins essential for pathogenesis, may ultimately lead to the identification of novel potential therapeutic targets. BIOLOGICAL SIGNIFICANCE: Leishmania, protozoan parasites of the Trypanosomatidae family, are the causative agents of leishmaniasis that represents a major public health concern affecting more than 20million people globally Membrane associated proteins play a pivotal role in host-pathogen interactions and in regulatory pathways. Quantitative proteomic analysis of the membranous fractions from L. mexicana and L. infantum (causative agents of cutaneous and visceral leishmaniasis, respectively) identified a number of proteins that may have important stage-specific functions in either the sand fly or mammalian host. The function of these proteins includes roles in virulence, as well as differences in metabolic process between life stages. Many of the proteins identified may act as virulence factors playing significant roles in parasite invasion, host-parasite interaction or parasite survival and thus may have therapeutic potential as drug target candidates.

Effect of BMAP-28 antimicrobial peptides on Leishmania major promastigote and amastigote growth: role of leishmanolysin in parasite survival.

BACKGROUND: Protozoan parasites, such as Leishmania, still pose an enormous public health problem in many countries throughout the world. Current measures are outdated and have some associated drug resistance, prompting the search into novel therapies. Several innovative approaches are under investigation, including the utilization of host defence peptides (HDPs) as emerging anti-parasitic therapies. HDPs are characterised by their small size, amphipathic nature and cationicity, which induce permeabilization of cell membranes, whilst modulating the immune response of the host. Recently, members of the cathelicidin family of HDPs have demonstrated significant antimicrobial activities against various parasites including Leishmania. The cathelicidin bovine myeloid antimicrobial peptide 28 (BMAP-28) has broad antimicrobial activities and confers protection in animal models of bacterial infection or sepsis. We tested the effectiveness of the use of BMAP-28 and two of its isomers the D-amino acid form (D-BMAP-28) and the retro-inverso form (RI-BMAP-28), as anti-leishmanial agents against the promastigote and amastigote intracellular Leishmania major lifecycle stages. METHODOLOGY/PRINCIPAL FINDINGS: An MTS viability assay was utilized to show the potent antiparasitic activity of BMAP-28 and its protease resistant isomers against L. major promastigotes in vitro. Cell membrane permeability assays, caspase 3/7, Tunel assays and morphologic studies suggested that this was a late stage apoptotic cell death with early osmotic cell lysis caused by the antimicrobial peptides. Furthermore, BMAP-28 and its isomers demonstrated anti-leishmanial activities against intracellular amastigotes within a macrophage infection model. CONCLUSIONS/SIGNIFICANCE: Interestingly, D-BMAP-28 appears to be the most potent antiparasitic of the three isomers against wild type L. major promastigotes and amastigotes. These exciting results suggest that BMAP-28 and its protease resistant isomers have significant therapeutic potential as novel anti-leishmanials.

Leishmania exosomes modulate innate and adaptive immune responses through effects on monocytes and dendritic cells.

We investigated the properties of leishmania exosomes with respect to influencing innate and adaptive immune responses. Exosomes from Leishmania donovani modulated human monocyte cytokine responses to IFN-γ in a bimodal fashion by promoting IL-10 production and inhibiting that of TNF-α. Moreover, these vesicles were inhibitory with respect to cytokine responses (IL-12p70, TNF-α, and IL-10) by human monocyte-derived dendritic cells. Exosomes from wild-type (WT) L. donovani failed to prime monocyte-derived dendritic cells to drive the differentiation of naive CD4 T cells into IFN-γ-producing Th1 cells. In contrast, vesicles from heat shock protein (HSP)100(-/-) L. donovani showed a gain-of-function and proinflammatory phenotype and promoted the differentiation of naive CD4 lymphocytes into Th1 cells. Proteomic analysis showed that exosomes from WT and HSP100(-/-) leishmania had distinct protein cargo, suggesting that packaging of proteins into exosomes is dependent in part on HSP100. Treatment of C57BL/6 mice with WT L. donovani exosomes prior to challenge with WT organisms exacerbated infection and promoted IL-10 production in the spleen. In contrast, HSP100(-/-) exosomes promoted spleen cell production of IFN-γ and did not adversely affect hepatic parasite burdens. Furthermore, the proparasitic properties of WT exosomes were not species specific because BALB/c mice exposed to Leishmania major exosomes showed increased Th2 polarization and exacerbation of disease in response to infection with L. major. These findings demonstrate that leishmania exosomes are predominantly immunosuppressive. Moreover, to our knowledge, this is the first evidence to suggest that changes in the protein cargo of exosomes may influence the impact of these vesicles on myeloid cell function.

The Leishmania mexicana A600 genes are functionally required for amastigote replication.

Leishmania parasites, the causative agent of leishmaniasis, have a digenetic lifecycle consisting of the morphologically distinct insect vector stage (promastigote) and the mammalian infective amastigote stage. Differentiation of promastigotes to the amastigote stage involves significant morphological and biochemical changes, however, very few genes have been characterised as being differentially expressed in the two stages. The Leishmania A600 genes are one of the few gene families that exhibit stage-specific expression and, as such, they are of interest as potential virulent factors. In this study, we characterize the A600 family in several Leishmania species and investigate their role in amastigote differentiation and proliferation. Four open reading frames, A600-1, A600-2, A600-3, and A600-4, were identified at the multi-gene L. mexicana A600 locus via cloning and restriction mapping. Homology searching identified A600 homologues in other Leishmania species, L. major, L. braziliensis and L. infantum but not in the closely related Trypanosoma family. A targeted gene deletion approach was utilized to determine the cellular function of the L. mexicanaA600 genes. A600(-/-) promastigotes differentiated to axenic amastigotes in response to temperature shift and acidification of culture media, but showed significant growth arrest. Similarly, during in vitro macrophage infection studies, A600(-/-) promastigotes established an early infection, but were deficient in their ability to proliferate as intracellular amastigotes. The ability of A600(-/-) amastigotes to proliferate in mouse peritoneal macrophages was restored by re-introduction of the A600-1 gene, but not the A600-4 gene. The results from these experiments show that the A600-1 gene is essential for continued proliferation of amastigotes, and potentially for development of chronic leishmaniasis. Furthermore, these results suggest a potential role for the L. mexicana A600-deficient mutant as a vaccine candidate.

Leishmania: conserved evolution--diverse diseases.

The landmark completion of the Leishmania major genome sequence and the recent publication of the L. infantum and L. braziliensis genomes revealed the surprising result that, although separated by 15-50 million years of evolution, the Leishmania genomes are highly conserved and have less than 1% species-specific genes. Yet, these three species of Leishmania cause distinctive and diverse diseases in humans. Here, we discuss these findings together with recent microarray and proteomics studies and highlight their importance in understanding Leishmania disease phenotypes.