Memory B-cell and antibody responses to the SARS-CoV-2 spike protein contribute to long-term immune protection against severe COVID-19, which can also be prevented by antibody-based interventions. Here, wide SARS-CoV-2 immunoprofiling in Wuhan COVID-19 convalescents combining serological, cellular, and monoclonal antibody explorations revealed humoral immunity coordination. Detailed characterization of a hundred SARS-CoV-2 spike memory B-cell monoclonal antibodies uncovered diversity in their repertoire and antiviral functions. The latter were influenced by the targeted spike region with strong Fc-dependent effectors to the S2 subunit and potent neutralizers to the receptor-binding domain. Amongst those, Cv2.1169 and Cv2.3194 antibodies cross-neutralized SARS-CoV-2 variants of concern, including Omicron BA.1 and BA.2. Cv2.1169, isolated from a mucosa-derived IgA memory B cell demonstrated potency boost as IgA dimers and therapeutic efficacy as IgG antibodies in animal models. Structural data provided mechanistic clues to Cv2.1169 potency and breadth. Thus, potent broadly neutralizing IgA antibodies elicited in mucosal tissues can stem SARS-CoV-2 infection, and Cv2.1169 and Cv2.3194 are prime candidates for COVID-19 prevention and treatment.

Predicting how Leishmania will respond to control efforts requires an understanding of their transmission strategy. Using real-time quantitative PCR to quantify infectious metacyclic and non-metacyclic forms in mouse skin from single sandfly bites we show that most transmissions were highly enriched for infectious parasites. However, a quarter of sandflies were capable of transmitting high doses containing more non-infectious promastigotes from the vector’s midgut. Mouse infections replicating “high” to “low” quality, low-dose transmissions confirmed clear differences in the pathology of the infection and their onward transmissibility back to sandflies. Borrowing methods originally developed to account for exposure heterogeneity among hosts, we show how these high-dose, low-quality transmitters act as super-spreading vectors, capable of inflating Leishmania transmission potential by as much as six-fold. These results highlight the hidden potential of transmission of mixed Leishmania promastigote stages on disease prevalence and the role of dose heterogeneity as an underlying strategy for efficient transmission.

Leishmania parasites are transmitted to vertebrate hosts by female phlebotomine sand flies as they bloodfeed by lacerating the upper capillaries of the dermis with their barbed mouthparts. In the sand fly midgut secreted proteophosphoglycans from Leishmania form a biological plug known as the promastigote secretory gel (PSG), which blocks the gut and facilitates the regurgitation of infective parasites. The interaction between the wound created by the sand fly bite and PSG is not known. Here we nanoinjected a sand fly egested dose of PSG into BALB/c mouse skin that lead to the differential expression of 7,907 transcripts. These transcripts were transiently up-regulated during the first 6 hours post-wound and enriched for pathways involved in inflammation, cell proliferation, fibrosis, epithelial cell differentiation and wound remodelling. We found that PSG significantly accelerated wound healing in vitro and in mice; which was associated with an early up-regulation of transcripts involved in inflammation (IL-1β, IL-6, IL-10, TNFα) and inflammatory cell recruitment (CCL2, CCL3, CCL4, CXCL2), followed 6 days later by enhanced expression of transcripts associated with epithelial cell proliferation, fibroplasia and fibrosis (FGFR2, EGF, EGFR, IGF1). Dermal expression of IGF1 was enhanced following an infected sand fly bite and was acutely responsive to the deposition of PSG but not the inoculation of parasites or sand fly saliva. Antibody blockade of IGF1 ablated the gel’s ability to promote wound closure in mouse ears and significantly reduced the virulence of Leishmania mexicana infection delivered by an individual sand fly bite. Dermal macrophages recruited to air-pouches on the backs of mice revealed that IGF1 was pivotal to the PSG’s ability to promote macrophage alternative activation and Leishmania infection. Our data demonstrate that through the regurgitation of PSG Leishmania exploit the wound healing response of the host to the vector bite by promoting the action of IGF1 to drive the alternative activation of macrophages. Female phlebotomine sand flies efficiently transmit Leishmania parasites, yet the underlying mechanisms remain poorly understood. During transmission, promastigote secretory gel (PSG) regurgitated from the blocked sand fly gut promote Leishmania infection and exacerbates disease. Here we investigate mouse ear-skin response to PSG and find that a significant proportion of up-regulated transcripts are involved in wound healing. Following a wound to skin preconditioned with PSG, key transcripts associated with inflammation and cell recruitment were enhanced by 4 hours and those associated with epithelial cell differentiation, proliferation and fibrosis were enhanced in the late phase of wound healing; including insulin growth factor-1 (IGF1) and its receptor. The presence of PSG significantly accelerated wound closure in vitro and in vivo, and this was directed by the presence of IGF1. IGF1 was essential to both the wound healing and disease-exacerbating properties of PSG, arising from its ability to alternatively activate macrophages. IGF1-blockade efficiently reduced the virulence of Leishmania infection from sand fly bite transmission indicating that it is essential to the function of PSG during natural infection. Our findings open the dual possibility of exploiting PSG, or defined components thereof, as a novel wound healing therapy and provide new targets for anti-leishmanial therapeutic design.

After loading with live Leishmania (L) amazonensis amastigotes, mouse myeloid dendritic leucocytes/DLs are known to undergo reprogramming of their immune functions. In the study reported here, we investigated whether the presence of live L. amazonensis amastigotes in mouse bone marrow-derived DLs is able to trigger re-programming of DL lipid, and particularly neutral lipid metabolism. Affymetrix-based transcriptional profiles were determined in C57BL/6 and DBA/2 mouse bone marrow-derived DLs that had been sorted from cultures exposed or not to live L. amazonensis amastigotes. This showed that live amastigote-hosting DLs exhibited a coordinated increase in: (i) long-chain fatty acids (LCFA) and cholesterol uptake/transport, (ii) LCFA and cholesterol (re)-esterification to triacyl-sn-glycerol (TAG) and cholesteryl esters (CE), respectively. As these neutral lipids are known to make up the lipid body (LB) core, oleic acid was added to DL cultures and LB accumulation was compared in live amastigote-hosting versus amastigote-free DLs by epi-fluorescence and transmission electron microscopy. This showed that LBs were both significantly larger and more numerous in live amastigote-hosting mouse dendritic leucocytes. Moreover, many of the larger LB showed intimate contact with the membrane of the parasitophorous vacuoles hosting the live L. amazonensis amastigotes. As leucocyte LBs are known to be more than simple neutral lipid repositories, we set about addressing two related questions. Could LBs provide lipids to live amastigotes hosted within the DL parasitophorous vacuole and also deliver? Could LBs impact either directly or indirectly on the persistence of L. amazonensis amastigotes in rodent skin? Once they have gained entry to mammals, live Leishmania (L) amazonensis amastigotes are known to subvert both macrophages and dendritic leucocytes (DLs) as host cells. These L. amazonensis amastigotes then may or may not proliferate in these two phagocytic leucocyte lineages, but in both cases the otherwise versatile differentiation program of these lineages is known to be rapidly remodeled. Here, we describe the rapid reprogramming of C57BL/6 and DBA/2 mouse bone marrow-derived DLs, with a special focus on cytosolic lipid bodies (LBs) that are known to store neutral lipids such as triacyl-sn-glycerol (TAG) and cholesteryl esters (CE). After extracting RNA from carefully sorted amastigote-free DLs and L. amazonensis amastigote-hosting DLs, an Affymetrix-based analysis clearly showed a singular and coordinated increase in DL transcripts involved in (i) long-chain fatty acid uptake, transport and esterification to TAG and (ii) cholesterol uptake and esterification to cholesteryl esters. Oleic acid was added to check that neutral lipid metabolism was both rapidly increased and reprogrammed in amastigote-hosting DLs. It should be noted that the LBs in live amastigote-hosting DLs were more numerous, and that the largest of these LBs were in contact with live amastigote- hosting parasitophorous vacuoles. We further discuss these findings in the context of live L. amazonensis amastigote-rodent host interactions.

Parasitic infections such as leishmaniasis induce a cascade of host physiological responses, including metabolic and immunological changes. Infection with Leishmania major parasites causes cutaneous leishmaniasis in humans, a neglected tropical disease that is difficult to manage. To understand the determinants of pathology, we studied L. major infection in two mouse models: the self-healing C57BL/6 strain and the nonhealing BALB/c strain. Metabolic profiling of urine, plasma, and feces via proton NMR spectroscopy was performed to discover parasite-specific imprints on global host metabolism. Plasma cytokine status and fecal microbiome were also characterized as additional metrics of the host response to infection. Results demonstrated differences in glucose and lipid metabolism, distinctive immunological phenotypes, and shifts in microbial composition between the two models. We present a novel approach to integrate such metrics using correlation network analyses, whereby self-healing mice demonstrated an orchestrated interaction between the biological measures shortly after infection. In contrast, the response observed in nonhealing mice was delayed and fragmented. Our study suggests that trans-system communication across host metabolism, the innate immune system, and gut microbiome is key for a successful host response to L. major and provides a new concept, potentially translatable to other diseases.

Leishmania (L.) spp are intracellular eukaryotic parasites responsible for cutaneous or visceral leishmaniasis, replicating predominantly in macrophages (MF). In C57BL/6 mice virulence with L. amazonensis has been associated with inhibition of Th1 immune responses and an uncontrolled lesion development, whereas DBA/2 mice control any lesion. Parasitic clearance by the MFs requires the activation of proper immune responses. One of the immune related genes expressed in immune cells including MF, codes for osteopontin (OPN). OPN is a secreted glycoprotein, acting as an immune regulator. Its implication in promoting Th1 immunity in response to infectious microorganisms and its known protective effect against viral and bacterial infections via activation of the immune response, render OPN a molecule of interest in the study of the host response to L. amazonensis. We examined the host response to L. amazonensis of opn mutant and wild type C57BL/6 mice. Bone marrow derived MFs were infected with the parasites in vitro, and opn mutant and wild type mice were inoculated in vivo by intradermal injection in the ears. The DBA/2 strain known to control L. amazonensis infection was also used for comparison. Our data indicate that the parasites increased opn gene expression and OPN protein while parasitic proliferation was contained in the presence of OPN. In the presence of parasites the expression of inflammation-related transcripts was inhibited. Interleukin-1-beta (IL-1β), and transcripts of the NLR–family (NLRC4, NLRP3) were down regulated after L. amazonensis infection. In the absence of OPN, the inhibition by the parasites of IL-1β transcripts was less efficient and a pyroptosis-like cell phenotype was detected in vitro, suggesting a central role of OPN in the host-response to L. amazonensis. Similarly, in vivo, in the absence of OPN, while the clinical inflammatory phenotype is more severe, an increase of these transcripts was observed. L. amazonensis infection induces opn gene expression and protein, which in turn participates in shaping the host response to the parasites, seemingly by decreasing the activation of inflammation. OPN, further evaluated as a target for Leishmaniasis control represents an additional interest in improving vaccination strategies against the parasites.