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Vaccination of the global population against COVID-19 is a great scientific, logistical, and moral challenge. Despite the rapid development and authorization of several full-length Spike (S) protein vaccines, the global demand outweighs the current supply and there is a need for safe, potent, high-volume, affordable vaccines that can fill this gap, especially in low- and middle-income countries. Whether SARS-CoV-2 S-protein receptor-binding domain (RBD)-based vaccines could fill this gap has been debated, especially with regards to its suitability to protect against emerging viral variants of concern. Given a predominance for elicitation of neutralizing antibodies (nAbs) that target RBD following natural infection or vaccination, a key biomarker of protection, there is merit for selection of RBD as a sole vaccine immunogen. With its high-yielding production and manufacturing potential, RBD-based vaccines offer an abundance of temperature-stable doses at an affordable cost. In addition, as the RBD preferentially focuses the immune response to potent and recently recognized cross-protective determinants, this domain may be central to the development of future pan-sarbecovirus vaccines. In this study, we review the data supporting the non-inferiority of RBD as a vaccine immunogen compared to full-length S-protein vaccines with respect to humoral and cellular immune responses against both the prototype pandemic SARS-CoV-2 isolate and emerging variants of concern.

Global containment of COVID-19 still requires accessible and affordable vaccines for low- and middle-income countries (LMICs).1 Recently approved vaccines provide needed interventions, albeit at prices that may limit their global access.2 Subunit vaccines based on recombinant proteins are suited for large-volume microbial manufacturing to yield billions of doses annually, minimizing their manufacturing costs.3 These types of vaccines are well-established, proven interventions with multiple safe and efficacious commercial examples.4–6 Many vaccine candidates of this type for SARS-CoV-2 rely on sequences containing the receptor-binding domain (RBD), which mediates viral entry to cells via ACE2.7,8 Here we report an engineered sequence variant of RBD that exhibits high-yield manufacturability, high-affinity binding to ACE2, and enhanced immunogenicity after a single dose in mice compared to the Wuhan-Hu-1 variant used in current vaccines. Antibodies raised against the engineered protein exhibited heterotypic binding to the RBD from two recently reported SARS-CoV-2 variants of concern (501Y.V1/V2). Presentation of the engineered RBD on a designed virus-like particle (VLP) also reduced weight loss in hamsters upon viral challenge.

Extracellular vesicles (EVs) are secreted by myriad cells in culture and also by unicellular organisms, and their identification in mammalian fluids suggests that EV release also occurs at the organism level. However, although it is clearly important to better understand EVs' roles in organismal biology, EVs in solid tissues have received little attention. Here, we modified a protocol for EV isolation from primary neural cell culture to collect EVs from frozen whole murine and human neural tissues by serial centrifugation and purification on a sucrose gradient. Quantitative proteomics comparing brain-derived EVs from nontransgenic (NTg) and a transgenic amyotrophic lateral sclerosis (ALS) mouse model, superoxide dismutase 1 (SOD1)G93A, revealed that these EVs contain canonical exosomal markers and are enriched in synaptic and RNA-binding proteins. The compiled brain EV proteome contained numerous proteins implicated in ALS, and EVs from SOD1G93A mice were significantly depleted in myelin-oligodendrocyte glycoprotein compared with those from NTg animals. We observed that brain- and spinal cord–derived EVs, from NTg and SOD1G93A mice, are positive for the astrocyte marker GLAST and the synaptic marker SNAP25, whereas CD11b, a microglial marker, was largely absent. EVs from brains and spinal cords of the SOD1G93A ALS mouse model, as well as from human SOD1 familial ALS patient spinal cord, contained abundant misfolded and nonnative disulfide-cross-linked aggregated SOD1. Our results indicate that CNS-derived EVs from an ALS animal model contain pathogenic disease-causing proteins and suggest that brain astrocytes and neurons, but not microglia, are the main EV source.

Vaccines against SARS-CoV-2 have been distributed at massive scale in developed countries, and have been effective at preventing COVID-19. Access to vaccines is limited, however, in low- and middle-income countries (LMICs) due to insufficient supply, high costs, and cold storage requirements. New vaccines that can be produced in existing manufacturing facilities in LMICs, can be manufactured at low cost, and use widely available, proven, safe adjuvants like alum, would improve global immunity against SARS-CoV-2. One such protein subunit vaccine is produced by the Serum Institute of India Pvt. Ltd. and is currently in clinical testing. Two protein components, the SARS-CoV-2 receptor binding domain (RBD) and hepatitis B surface antigen virus-like particles (VLPs), are each produced in yeast, which would enable a low-cost, high-volume manufacturing process. Here, we describe the design and preclinical testing of the RBD-VLP vaccine in cynomolgus macaques. We observed titers of neutralizing antibodies (>104) above the range of protection for other licensed vaccines in non-human primates. Interestingly, addition of a second adjuvant (CpG1018) appeared to improve the cellular response while reducing the humoral response. We challenged animals with SARS-CoV-2, and observed a ~3.4 and ~2.9 log10reduction in median viral loads in bronchoalveolar lavage and nasal mucosa, respectively, compared to sham controls. These results inform the design and formulation of current clinical COVID-19 vaccine candidates like the one described here, and future designs of RBD-based vaccines against variants of SARS-CoV-2 or other betacoronaviruses.

Significance Most of the global population resides in low- and middle-income countries, where current vaccines for COVID-19 remain largely unavailable. For the COVID-19 pandemic, the world will need access to >10 billion doses of vaccines, or more than double the annual volume of vaccines for all other diseases. Many vaccine candidates use the SARS-CoV-2 receptor-binding domain (RBD) antigen. Here, we present an engineered RBD with improved production titers in Pichia pastoris, a yeast commonly used for large-scale, low-cost manufacturing by vaccine manufacturers. The modified RBD also raises an enhanced immune response in mice relative to the Wuhan-Hu-1 sequence used in current candidates. These combined traits make it a promising candidate for next-generation vaccines addressing emerging variants of the virus., Global containment of COVID-19 still requires accessible and affordable vaccines for low- and middle-income countries (LMICs). Recently approved vaccines provide needed interventions, albeit at prices that may limit their global access. Subunit vaccines based on recombinant proteins are suited for large-volume microbial manufacturing to yield billions of doses annually, minimizing their manufacturing cost. These types of vaccines are well-established, proven interventions with multiple safe and efficacious commercial examples. Many vaccine candidates of this type for SARS-CoV-2 rely on sequences containing the receptor-binding domain (RBD), which mediates viral entry to cells via ACE2. Here we report an engineered sequence variant of RBD that exhibits high-yield manufacturability, high-affinity binding to ACE2, and enhanced immunogenicity after a single dose in mice compared to the Wuhan-Hu-1 variant used in current vaccines. Antibodies raised against the engineered protein exhibited heterotypic binding to the RBD from two recently reported SARS-CoV-2 variants of concern (501Y.V1/V2). Presentation of the engineered RBD on a designed virus-like particle (VLP) also reduced weight loss in hamsters upon viral challenge.

Advances in the understanding of Alzheimer’s disease (AD) suggest that pathogenesis is not directly related to plaque burden, but rather to soluble toxic amyloid-beta oligomers (AßO). Therapeutic antibodies targeting Aß monomers and/or plaque have shown limited efficacy and dose-limiting adverse events in clinical trials. These findings suggest that antibodies capable of selectively neutralizing toxic AßO may achieve improved efficacy and safety. To this end, we generated monoclonal antibodies against a conformational Aß epitope predicted by computational modeling to be presented on toxic AßO but not monomers or fibrils. The resulting lead antibody, PMN310, showed the desired AßO-selective binding profile. In vitro, PMN310 inhibited AßO propagation and toxicity. In vivo, PMN310 prevented AßO-induced loss of memory formation and reduced synaptic loss and inflammation. A humanized version (huPMN310) compared favorably to other Aß-directed antibodies showing a lack of adverse event-associated binding to Aß deposits in AD brains, and greater selective binding to AßO-enriched AD brain fractions that contain synaptotoxic Aß species. Systemic administration of huPMN310 in mice resulted in brain exposure and kinetics comparable to those of other therapeutic human monoclonal antibodies. Greater selectivity for AßO and the potential to safely administer high doses of huPMN310 are expected to result in enhanced safety and therapeutic potency.

Extracellular vesicles (EVs) are secreted by myriad cells in culture and also by unicellular organisms, and their identification in mammalian fluids suggests that EV release also occurs at the organism level. However, although it is clearly important to better understand EVs' roles in organismal biology, EVs in solid tissues have received little attention. Here, we modified a protocol for EV isolation from primary neural cell culture to collect EVs from frozen whole murine and human neural tissues by serial centrifugation and purification on a sucrose gradient. Quantitative proteomics comparing brain-derived EVs from nontransgenic (NTg) and a transgenic amyotrophic lateral sclerosis (ALS) mouse model, superoxide dismutase 1 (SOD1)(G93A), revealed that these EVs contain canonical exosomal markers and are enriched in synaptic and RNA-binding proteins. The compiled brain EV proteome contained numerous proteins implicated in ALS, and EVs from SOD1(G93A) mice were significantly depleted in myelin-oligodendrocyte glycoprotein compared with those from NTg animals. We observed that brain- and spinal cord–derived EVs, from NTg and SOD1(G93A) mice, are positive for the astrocyte marker GLAST and the synaptic marker SNAP25, whereas CD11b, a microglial marker, was largely absent. EVs from brains and spinal cords of the SOD1(G93A) ALS mouse model, as well as from human SOD1 familial ALS patient spinal cord, contained abundant misfolded and nonnative disulfide-cross-linked aggregated SOD1. Our results indicate that CNS-derived EVs from an ALS animal model contain pathogenic disease-causing proteins and suggest that brain astrocytes and neurons, but not microglia, are the main EV source.

Amyotrophic lateral sclerosis (ALS), a fatal adult-onset degenerative neuromuscular disorder with a poorly defined etiology, progresses in an orderly spatiotemporal manner from one or more foci within the nervous system, reminiscent of prion disease pathology. We have previously shown that misfolded mutant Cu/Zn superoxide dismutase (SOD1), mutation of which is associated with a subset of ALS cases, can induce endogenous wild-type SOD1 misfolding in the intracellular environment in a templating fashion similar to that of misfolded prion protein. Our recent observations further extend the prion paradigm of pathological SOD1 to help explain the intercellular transmission of disease along the neuroaxis. It has been shown that both mutant and misfolded wild-type SOD1 can traverse cell-to-cell either as protein aggregates that are released from dying cells and taken up by neighboring cells via macropinocytosis, or released to the extracellular environment on the surface of exosomes secreted from living cells. Furthermore, once propagation of misfolded wild-type SOD1 has been initiated in human cell culture, it continues over multiple passages of transfer and cell growth. Propagation and transmission of misfolded wild-type SOD1 is therefore a potential mechanism in the systematic progression of ALS pathology.

Leishmania disease expression has been linked to IL-10. In this study, we investigated the regulation of IL-10 production by macrophages infected with Leishmania donovani. Infection of either murine or human macrophages brought about selective phosphorylation of Akt-2 in a PI3K-dependent manner. These events were linked to phosphorylation and inactivation of glycogen synthase kinase-3β (GSK-3β) at serine 9, as the latter was abrogated by inhibition of either PI3K or Akt. One of the transcription factors that is negatively regulated by GSK-3β is CREB, which itself positively regulates IL-10 expression. Infection of macrophages with leishmania induced phosphorylation of CREB at serine 133, and this was associated with enhanced CREB DNA binding activity and induction of IL-10. Similar to phosphorylation of GSK-3β, both phosphorylation of CREB at serine 133 and CREB DNA binding activity were abrogated in cells treated with inhibitors of either PI3K or Akt prior to infection. Furthermore, disruption of this pathway either by inhibition of Akt or by overexpression of GSK-3β markedly attenuated IL-10 production in response to leishmania. Thus, GSK-3β negatively regulates myeloid cell IL-10 production in response to leishmania. Switching off GSK-3β promotes disease pathogenesis.

The release of exosomes and other microvesicles by diverse prokaryotic and eukaryotic cells and organisms was first appreciated early in the 20th century. The functional properties of these organelles, however, have only recently been the focus of rigorous investigation. In this review, we discuss the release of microvesicles of varying complexity by diverse microbial pathogens. This includes vesicle secretion by Gram-negative bacteria, eukaryotic parasites of the kinetoplast lineage and opportunistic fungal pathogens of both the ascomycetes and basidiomycetes lineages. We also discuss vesicle release from mammalian cells brought about as a result of infection with bacteria, viruses and prions. In addition, we review the evidence showing that in their specific microenvironments, release of these organelles from diverse pathogens contributes to pathogenesis. Germane to this and based upon recent findings with Leishmania, we propose a model whereby exosome release by an intracellular pathogen serves as a general mechanism for effector molecule delivery from eukaryotic pathogen to host cell cytosol. These new findings linking exosomes and other microvesicles to infection biology have important implications for understanding the immune response to infection and for the design of research strategies aimed at the development of novel therapeutics and vaccines.

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.

Specialized secretion systems are used by numerous bacterial pathogens to export virulence factors into host target cells. Leishmania and other eukaryotic intracellular pathogens also deliver effector proteins into host cells; however, the mechanisms involved have remained elusive. In this report, we identify exosome-based secretion as a general mechanism for protein secretion by Leishmania, and show that exosomes are involved in the delivery of proteins into host target cells. Comparative quantitative proteomics unambiguously identified 329 proteins in Leishmania exosomes, accounting for >52% of global protein secretion from these organisms. Our findings demonstrate that infection-like stressors (37°C ± pH 5.5) upregulated exosome release more than twofold and also modified exosome protein composition. Leishmania exosomes and exosomal proteins were detected in the cytosolic compartment of infected macrophages and incubation of macrophages with exosomes selectively induced secretion of IL-8, but not TNF-α. We thus provide evidence for an apparently broad-based mechanism of protein export by Leishmania. Moreover, we describe a mechanism for the direct delivery of Leishmania molecules into macrophages. These findings suggest that, like mammalian exosomes, Leishmania exosomes function in long-range communication and immune modulation.

Amyotrophic lateral sclerosis (ALS) is a fatal adult-onset neuromuscular degenerative disorder with a poorly defined etiology. ALS patients experience motor weakness, which starts focally and spreads throughout the nervous system, culminating in paralysis and death within a few years of diagnosis. While the vast majority of clinical ALS is sporadic with no known cause, mutations in human copper-zinc superoxide dismutase 1 (SOD1) cause about 20 % of inherited cases of ALS. ALS with SOD1 mutations is caused by a toxic gain of function associated with the propensity of mutant SOD1 to misfold, presenting a non-native structure. The mechanisms responsible for the progressive spreading of ALS pathology have been the focus of intense study. We have shown that misfolded SOD1 protein can seed misfolding and aggregation of endogenous wild-type SOD1 similar to amyloid-β and prion protein seeding. Our recent observations demonstrate a transfer of the misfolded SOD1 species from cell to cell, modeling the intercellular transmission of disease through the neuroaxis. We have shown that both mutant and misfolded wild-type SOD1 can traverse cell-to-cell, either as protein aggregates that are released from dying cells and taken up by neighboring cells via macropinocytosis, or in association with vesicles which are released into the extracellular environment. Furthermore, once misfolding of wild-type SOD1 has been initiated in a human cell culture, it can induce misfolding in naive cell cultures over multiple passages of media transfer long after the initial misfolding template is degraded. Herein we review the data on mechanisms of intercellular transmission of misfolded SOD1.

Amyotrophic lateral sclerosis (ALS) is predominantly sporadic, but associated with heritable genetic mutations in 5-10% of cases, including those in Cu/Zn superoxide dismutase (SOD1). We previously showed that misfolding of SOD1 can be transmitted to endogenous human wild-type SOD1 (HuWtSOD1) in an intracellular compartment. Using NSC-34 motor neuron-like cells, we now demonstrate that misfolded mutant and HuWtSOD1 can traverse between cells via two nonexclusive mechanisms: protein aggregates released from dying cells and taken up by macropinocytosis, and exosomes secreted from living cells. Furthermore, once HuWtSOD1 propagation has been established, misfolding of HuWtSOD1 can be efficiently and repeatedly propagated between HEK293 cell cultures via conditioned media over multiple passages, and to cultured mouse primary spinal cord cells transgenically expressing HuWtSOD1, but not to cells derived from nontransgenic littermates. Conditioned media transmission of HuWtSOD1 misfolding in HEK293 cells is blocked by HuWtSOD1 siRNA knockdown, consistent with human SOD1 being a substrate for conversion, and attenuated by ultracentrifugation or incubation with SOD1 misfolding-specific antibodies, indicating a relatively massive transmission particle which possesses antibody-accessible SOD1. Finally, misfolded and protease-sensitive HuWtSOD1 comprises up to 4% of total SOD1 in spinal cords of patients with sporadic ALS (SALS). Propagation of HuWtSOD1 misfolding, and its subsequent cell-to-cell transmission, is thus a candidate process for the molecular pathogenesis of SALS, which may provide novel treatment and biomarker targets for this devastating disease.

Evasion or subversion of host immune responses is a well-established paradigm in infection with visceralizing leishmania. In this review, we summarize current findings supporting a model in which leishmania target host regulatory molecules and pathways, such as the PTP SHP-1 and the PI3K/Akt signaling cascade, to prevent effective macrophage activation. Furthermore, we describe how virulence factors, secreted by leishmania, interfere with macrophage intracellular signaling. Finally, we discuss mechanisms of secretion and provide evidence that leishmania use a remarkably adept, exosome-based secretion mechanism to export and deliver effector molecules to host cells. In addition to representing a novel mechanism for trafficking of virulence factors across membranes, recent findings indicate that leishmania exosomes may have potential as vaccine candidates.

Herein, we review evidence supporting a role for leishmania exosomes during early infection. We suggest a model in which leishmania secreted microvesicles released into the extracellular milieu deliver effector cargo to host target cells. This cargo mediates immunosuppression and functionally primes host cells for leishmania invasion. Leishmania ssp. release microvesicles and the amount of vesicle release and the specific protein cargo of the vesicles is sensitive to changes in environmental conditions that mimic infection. Leishmania exosomes influence the phenotype of treated immune cells. For example, ; wild-type (WT) exosomes attenuate interferon-γ-induced pro-inflammatory cytokine production (TNF-α) by leishmania-infected monocytes while conversely enhancing production of the anti-inflammatory cytokine IL-10. The leishmania proteins GP63 and elongation factor-1α (EF-1α) are found in secreted vesicles and are likely important effectors responsible for these changes in phenotype. GP63 and EF-1α access host cell cytosol and activate multiple host protein tyrosine phosphatases (PTPs). Activation of these PTPs negatively regulates interferon-γ signaling and this prevents effective expression of the macrophage microbicidal arsenal, including TNF-α and nitric oxide. In addition to changing macrophage phenotype, WT vesicles dampen the immune response of monocyte-derived dendritic cells and CD4+ T lymphocytes. This capacity is lost when the protein cargo of the vesicles is modified, specifically when the amount of GP63 and EF-1α in the vesicles is reduced. It appears that exosome delivery of effector proteins results in activation of host PTPs and the negative regulatory effects of the latter creates a pro-parasitic environment. The data suggest that leishmania exosomes secreted upon initial infection are capable of delivering effector cargo to naïve target cells wherein the cargo primes host cells for infection by interfering with host cell signaling pathways.