Impact of SARS-CoV-2 spike stability and RBD exposure on antigenicity and immunogenicity.

The spike protein (S) of SARS-CoV-2 induces neutralizing antibodies and is the key component of current COVID-19 vaccines. The most efficacious COVID-19 vaccines are genetically-encoded spikes with a double proline substitution in the hinge region to stabilize S in the prefusion conformation (S-2P). A subunit vaccine can be a valuable addition to mRNA and viral vector-based vaccines but requires high stability of spike. In addition, further stabilization of the prefusion conformation of spike might improve immunogenicity. To test this, five spike proteins were designed and characterized, ranging from low to high stability. The immunogenicity of these proteins was assessed in mice, demonstrating that a spike (S-closed-2) with a high melting temperature, which still allowed ACE2 binding, induced the highest neutralization titers against homologous and heterologous strains (up to 16-fold higher than the least stabilized spike). In contrast, the most stable spike variant (S-locked), in which the receptor binding domains (RBDs) were locked in a closed conformation and thus not able to breathe, induced relatively low neutralizing antibody titers against heterologous strains. These data demonstrate that S protein stabilization with RBDs exposing highly conserved epitopes may be needed to increase the immunogenicity of spike proteins for future COVID-19 vaccines.

Intradermal delivery of the third dose of the mRNA-1273 SARS-CoV-2 vaccine: safety and immunogenicity of a fractional booster dose.

OBJECTIVES: The aim of this study was to assess the safety and immunogenicity of a dose-sparing fractional intradermal (ID) booster strategy with the mRNA-1273 COVID-19 vaccine. METHODS: COVID-19 naive adults aged 18-30 years were recruited from a previous study on primary vaccination regimens that compared 20 µg ID vaccinations with 100 µg intramuscular (IM) vaccinations with mRNA-1273 as the primary vaccination series. Participants previously immunized with ID regimens were randomly assigned (1:1) to receive a fractional ID booster dose (20 µg) or the standard-of-care intramuscular (IM) booster dose (50 µg) of the mRNA-1273 vaccine, 6 months after completing their primary series (ID-ID and ID-IM group, respectively). Participants that had received a full dose IM regimen as the primary series, received the IM standard-of-care booster dose (IM-IM group). In addition, COVID-19 naive individuals aged 18-40 years who had received an IM mRNA vaccine as the primary series were recruited from the general population to receive a fractional ID booster dose (IM-ID group). Immunogenicity was assessed using IgG anti-spike antibody responses and neutralizing capacity against SARS-CoV-2. Cellular immune responses were measured in a sub-group. Safety and tolerability were monitored. RESULTS: In January 2022, 129 participants were included in the study. Fractional ID boosting was safe and well tolerated, with fewer systemic adverse events compared with IM boosting. At day 28 post-booster, anti-spike S1 IgG geometric mean concentrations were 9106 (95% CI, 7150-11 597) binding antibody units (BAU)/mL in the IM-IM group and 4357 (3003-6322) BAU/mL; 6629 (4913-8946) BAU/mL; and 5264 (4032-6873) BAU/mL in the ID-IM, ID-ID, and IM-ID groups, respectively. DISCUSSION: Intradermal boosting provides robust immune responses and is a viable dose-sparing strategy for mRNA COVID-19 vaccines. The favourable side-effect profile supports its potential to reduce vaccine hesitancy. Fractional dosing strategies should be considered early in the clinical development of future mRNA vaccines to enhance vaccine availability and pandemic preparedness.

Deubiquitinating activity of SARS-CoV-2 papain-like protease does not influence virus replication or innate immune responses in vivo.

The coronavirus papain-like protease (PLpro) is crucial for viral replicase polyprotein processing. Additionally, PLpro can subvert host defense mechanisms by its deubiquitinating (DUB) and deISGylating activities. To elucidate the role of these activities during SARS-CoV-2 infection, we introduced mutations that disrupt binding of PLpro to ubiquitin or ISG15. We identified several mutations that strongly reduced DUB activity of PLpro, without affecting viral polyprotein processing. In contrast, mutations that abrogated deISGylating activity also hampered viral polyprotein processing and when introduced into the virus these mutants were not viable. SARS-CoV-2 mutants exhibiting reduced DUB activity elicited a stronger interferon response in human lung cells. In a mouse model of severe disease, disruption of PLpro DUB activity did not affect lethality, virus replication, or innate immune responses in the lungs. This suggests that the DUB activity of SARS-CoV-2 PLpro is dispensable for virus replication and does not affect innate immune responses in vivo. Interestingly, the DUB mutant of SARS-CoV replicated to slightly lower titers in mice and elicited a diminished immune response early in infection, although lethality was unaffected. We previously showed that a MERS-CoV mutant deficient in DUB and deISGylating activity was strongly attenuated in mice. Here, we demonstrate that the role of PLpro DUB activity during infection can vary considerably between highly pathogenic coronaviruses. Therefore, careful considerations should be taken when developing pan-coronavirus antiviral strategies targeting PLpro.

The Main Protease of Middle East Respiratory Syndrome Coronavirus Induces Cleavage of Mitochondrial Antiviral Signaling Protein to Antagonize the Innate Immune Response.

Mitochondrial antiviral signaling protein (MAVS) is a crucial signaling adaptor in the sensing of positive-sense RNA viruses and the subsequent induction of the innate immune response. Coronaviruses have evolved multiple mechanisms to evade this response, amongst others, through their main protease (Mpro), which is responsible for the proteolytic cleavage of the largest part of the viral replicase polyproteins pp1a and pp1ab. Additionally, it can cleave cellular substrates, such as innate immune signaling factors, to dampen the immune response. Here, we show that MAVS is cleaved in cells infected with Middle East respiratory syndrome coronavirus (MERS-CoV), but not in cells infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). This cleavage was independent of cellular negative feedback mechanisms that regulate MAVS activation. Furthermore, MERS-CoV Mpro expression induced MAVS cleavage upon overexpression and suppressed the activation of the interferon-ß (IFN-ß) and nuclear factor-κB (NF-κB) response. We conclude that we have uncovered a novel mechanism by which MERS-CoV downregulates the innate immune response, which is not observed among other highly pathogenic coronaviruses.

Serological response and breakthrough infection after COVID-19 vaccination in patients with cirrhosis and post-liver transplant.

BACKGROUND: Vaccine hesitancy and lack of access remain major issues in disseminating COVID-19 vaccination to liver patients globally. Factors predicting poor response to vaccination and risk of breakthrough infection are important data to target booster vaccine programs. The primary aim of the current study was to measure humoral responses to 2 doses of COVID-19 vaccine. Secondary aims included the determination of factors predicting breakthrough infection. METHODS: COVID-19 vaccination and Biomarkers in cirrhosis And post-Liver Transplantation is a prospective, multicenter, observational case-control study. Participants were recruited at 4-10 weeks following first and second vaccine doses in cirrhosis [n = 325; 94% messenger RNA (mRNA) and 6% viral vaccine], autoimmune liver disease (AILD) (n = 120; 77% mRNA and 23% viral vaccine), post-liver transplant (LT) (n = 146; 96% mRNA and 3% viral vaccine), and healthy controls (n = 51; 72% mRNA, 24% viral and 4% heterologous combination). Serological end points were measured, and data regarding breakthrough SARS-CoV-2 infection were collected. RESULTS: After adjusting by age, sex, and time of sample collection, anti-Spike IgG levels were the lowest in post-LT patients compared to cirrhosis (p < 0.0001), AILD (p < 0.0001), and control (p = 0.002). Factors predicting reduced responses included older age, Child-Turcotte-Pugh B/C, and elevated IL-6 in cirrhosis; non-mRNA vaccine in AILD; and coronary artery disease, use of mycophenolate and dysregulated B-call activating factor, and lymphotoxin-α levels in LT. Incident infection occurred in 6.6%, 10.6%, 7.4%, and 15.6% of cirrhosis, AILD, post-LT, and control, respectively. The only independent factor predicting infection in cirrhosis was low albumin level. CONCLUSIONS: LT patients present the lowest response to the SARS-CoV-2 vaccine. In cirrhosis, the reduced response is associated with older age, stage of liver disease and systemic inflammation, and breakthrough infection with low albumin level.

Multiantigen pan-sarbecovirus DNA vaccines generate protective T cell immune responses.

SARS-CoV-2 is the third zoonotic coronavirus to cause a major outbreak in humans in recent years, and many more SARS-like coronaviruses with pandemic potential are circulating in several animal species. Vaccines inducing T cell immunity against broadly conserved viral antigens may protect against hospitalization and death caused by outbreaks of such viruses. We report the design and preclinical testing of 2 T cell-based pan-sarbecovirus vaccines, based on conserved regions within viral proteins of sarbecovirus isolates of human and other carrier animals, like bats and pangolins. One vaccine (CoVAX_ORF1ab) encoded antigens derived from nonstructural proteins, and the other (CoVAX_MNS) encoded antigens from structural proteins. Both multiantigen DNA vaccines contained a large set of antigens shared across sarbecoviruses and were rich in predicted and experimentally validated human T cell epitopes. In mice, the multiantigen vaccines generated both CD8+ and CD4+ T cell responses to shared epitopes. Upon encounter of full-length spike antigen, CoVAX_MNS-induced CD4+ T cells were responsible for accelerated CD8+ T cell and IgG Ab responses specific to the incoming spike, irrespective of its sarbecovirus origin. Finally, both vaccines elicited partial protection against a lethal SARS-CoV-2 challenge in human angiotensin-converting enzyme 2-transgenic mice. These results support clinical testing of these universal sarbecovirus vaccines for pandemic preparedness.

Engineering potent live attenuated coronavirus vaccines by targeted inactivation of the immune evasive viral deubiquitinase.

Coronaviruses express a papain-like protease (PLpro) that is required for replicase polyprotein maturation and also serves as a deubiquitinating enzyme (DUB). In this study, using a Middle East respiratory syndrome virus (MERS-CoV) PLpro modified virus in which the DUB is selectively inactivated, we show that the PLpro DUB is an important MERS-CoV interferon antagonist and virulence factor. Although the DUB-negative rMERS-CoVMA replicates robustly in the lungs of human dipeptidyl peptidase 4 knock-in (hDPP4 KI) mice, it does not cause clinical symptoms. Interestingly, a single intranasal vaccination with DUB-negative rMERS-CoVMA induces strong and sustained neutralizing antibody responses and sterilizing immunity after a lethal wt virus challenge. The survival of naïve animals also significantly increases when sera from animals vaccinated with the DUB-negative rMERS-CoVMA are passively transferred, prior to receiving a lethal virus dose. These data demonstrate that DUB-negative coronaviruses could be the basis of effective modified live attenuated vaccines.

Immune Determinants of Viral Clearance in Hospitalised COVID-19 Patients: Reduced Circulating Naïve CD4+ T Cell Counts Correspond with Delayed Viral Clearance.

Virus-specific cellular and humoral responses are major determinants for protection from critical illness after SARS-CoV-2 infection. However, the magnitude of the contribution of each of the components to viral clearance remains unclear. Here, we studied the timing of viral clearance in relation to 122 immune parameters in 102 hospitalised patients with moderate and severe COVID-19 in a longitudinal design. Delayed viral clearance was associated with more severe disease and was associated with higher levels of SARS-CoV-2-specific (neutralising) antibodies over time, increased numbers of neutrophils, monocytes, basophils, and a range of pro-inflammatory cyto-/chemokines illustrating ongoing, partially Th2 dominating, immune activation. In contrast, early viral clearance and less critical illness correlated with the peak of neutralising antibodies, higher levels of CD4 T cells, and in particular naïve CD4+ T cells, suggesting their role in early control of SARS-CoV-2 possibly by proving appropriate B cell help. Higher counts of naïve CD4+ T cells also correlated with lower levels of MIF, IL-9, and TNF-beta, suggesting an indirect role in averting prolonged virus-induced tissue damage. Collectively, our data show that naïve CD4+ T cell play a critical role in rapid viral T cell control, obviating aberrant antibody and cytokine profiles and disease deterioration. These data may help in guiding risk stratification for severe COVID-19.

A third vaccination with a single T cell epitope confers protection in a murine model of SARS-CoV-2 infection.

Understanding the mechanisms and impact of booster vaccinations are essential in the design and delivery of vaccination programs. Here we show that a three dose regimen of a synthetic peptide vaccine elicits an accruing CD8+ T cell response against one SARS-CoV-2 Spike epitope. We see protection against lethal SARS-CoV-2 infection in the K18-hACE2 transgenic mouse model in the absence of neutralizing antibodies, but two dose approaches are insufficient to confer protection. The third vaccine dose of the single T cell epitope peptide results in superior generation of effector-memory T cells and tissue-resident memory T cells, and these tertiary vaccine-specific CD8+ T cells are characterized by enhanced polyfunctional cytokine production. Moreover, fate mapping shows that a substantial fraction of the tertiary CD8+ effector-memory T cells develop from re-migrated tissue-resident memory T cells. Thus, repeated booster vaccinations quantitatively and qualitatively improve the CD8+ T cell response leading to protection against otherwise lethal SARS-CoV-2 infection.

Humoral response to SARS-CoV-2 infection among liver transplant recipients.

OBJECTIVE: Immunosuppressive agents are known to interfere with T and/or B lymphocytes, which are required to mount an adequate serologic response. Therefore, we aim to investigate the antibody response to SARS-CoV-2 in liver transplant (LT) recipients after COVID-19. DESIGN: Prospective multicentre case-control study, analysing antibodies against the nucleocapsid protein, spike (S) protein of SARS-CoV-2 and their neutralising activity in LT recipients with confirmed SARS-CoV-2 infection (COVID-19-LT) compared with immunocompetent patients (COVID-19-immunocompetent) and LT recipients without COVID-19 symptoms (non-COVID-19-LT). RESULTS: Overall, 35 LT recipients were included in the COVID-19-LT cohort. 35 and 70 subjects fulfilling the matching criteria were assigned to the COVID-19-immunocompetent and non-COVID-19-LT cohorts, respectively. We showed that LT recipients, despite immunosuppression and less symptoms, mounted a detectable antinucleocapsid antibody titre in 80% of the cases, although significantly lower compared with the COVID-19-immunocompetent cohort (3.73 vs 7.36 index level, p<0.001). When analysing anti-S antibody response, no difference in positivity rate was found between the COVID-19-LT and COVID-19-immunocompetent cohorts (97.1% vs 100%, p=0.314). Functional antibody testing showed neutralising activity in 82.9% of LT recipients (vs 100% in COVID-19-immunocompetent cohort, p=0.024). CONCLUSIONS: Our findings suggest that the humoral response of LT recipients is only slightly lower than expected, compared with COVID-19 immunocompetent controls. Testing for anti-S antibodies alone can lead to an overestimation of the neutralising ability in LT recipients. Altogether, routine antibody testing against separate SARS-CoV-2 antigens and functional testing show that the far majority of LT patients are capable of mounting an adequate antibody response with neutralising ability.

Prolonged activation of nasal immune cell populations and development of tissue-resident SARS-CoV-2-specific CD8+ T cell responses following COVID-19.

Systemic immune cell dynamics during coronavirus disease 2019 (COVID-19) are extensively documented, but these are less well studied in the (upper) respiratory tract, where severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) replicates1-6. Here, we characterized nasal and systemic immune cells in individuals with COVID-19 who were hospitalized or convalescent and compared the immune cells to those seen in healthy donors. We observed increased nasal granulocytes, monocytes, CD11c+ natural killer (NK) cells and CD4+ T effector cells during acute COVID-19. The mucosal proinflammatory populations positively associated with peripheral blood human leukocyte antigen (HLA)-DRlow monocytes, CD38+PD1+CD4+ T effector (Teff) cells and plasmablasts. However, there was no general lymphopenia in nasal mucosa, unlike in peripheral blood. Moreover, nasal neutrophils negatively associated with oxygen saturation levels in blood. Following convalescence, nasal immune cells mostly normalized, except for CD127+ granulocytes and CD38+CD8+ tissue-resident memory T cells (TRM). SARS-CoV-2-specific CD8+ T cells persisted at least 2 months after viral clearance in the nasal mucosa, indicating that COVID-19 has both transient and long-term effects on upper respiratory tract immune responses.

A Yellow Fever 17D Virus Replicon-Based Vaccine Platform for Emerging Coronaviruses.

The tremendous global impact of the current SARS-CoV-2 pandemic, as well as other current and recent outbreaks of (re)emerging viruses, emphasize the need for fast-track development of effective vaccines. Yellow fever virus 17D (YF17D) is a live-attenuated virus vaccine with an impressive efficacy record in humans, and therefore, it is a very attractive platform for the development of novel chimeric vaccines against various pathogens. In the present study, we generated a YF17D-based replicon vaccine platform by replacing the prM and E surface proteins of YF17D with antigenic subdomains from the spike (S) proteins of three different betacoronaviruses: MERS-CoV, SARS-CoV and MHV. The prM and E proteins were provided in trans for the packaging of these RNA replicons into single-round infectious particles capable of expressing coronavirus antigens in infected cells. YF17D replicon particles expressing the S1 regions of the MERS-CoV and SARS-CoV spike proteins were immunogenic in mice and elicited (neutralizing) antibody responses against both the YF17D vector and the coronavirus inserts. Thus, YF17D replicon-based vaccines, and their potential DNA- or mRNA-based derivatives, may constitute a promising and particularly safe vaccine platform for current and future emerging coronaviruses.

Two-Component Nanoparticle Vaccine Displaying Glycosylated Spike S1 Domain Induces Neutralizing Antibody Response against SARS-CoV-2 Variants.

Vaccines pave the way out of the SARS-CoV-2 pandemic. Besides mRNA and adenoviral vector vaccines, effective protein-based vaccines are needed for immunization against current and emerging variants. We have developed a virus-like particle (VLP)-based vaccine using the baculovirus-insect cell expression system, a robust production platform known for its scalability, low cost, and safety. Baculoviruses were constructed encoding SARS-CoV-2 spike proteins: full-length S, stabilized secreted S, or the S1 domain. Since subunit S only partially protected mice from SARS-CoV-2 challenge, we produced S1 for conjugation to bacteriophage AP205 VLP nanoparticles using tag/catcher technology. The S1 yield in an insect-cell bioreactor was ∼11 mg/liter, and authentic protein folding, efficient glycosylation, partial trimerization, and ACE2 receptor binding was confirmed. Prime-boost immunization of mice with 0.5 µg S1-VLPs showed potent neutralizing antibody responses against Wuhan and UK/B.1.1.7 SARS-CoV-2 variants. This two-component nanoparticle vaccine can now be further developed to help alleviate the burden of COVID-19. IMPORTANCE Vaccination is essential to reduce disease severity and limit the transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Protein-based vaccines are useful to vaccinate the world population and to boost immunity against emerging variants. Their safety profiles, production costs, and vaccine storage temperatures are advantageous compared to mRNA and adenovirus vector vaccines. Here, we use the versatile and scalable baculovirus expression vector system to generate a two-component nanoparticle vaccine to induce potent neutralizing antibody responses against SARS-CoV-2 variants. These nanoparticle vaccines can be quickly adapted as boosters by simply updating the antigen component.

Immunogenicity and efficacy of one and two doses of Ad26.COV2.S COVID vaccine in adult and aged NHP.

Safe and effective coronavirus disease-19 (COVID-19) vaccines are urgently needed to control the ongoing pandemic. While single-dose vaccine regimens would provide multiple advantages, two doses may improve the magnitude and durability of immunity and protective efficacy. We assessed one- and two-dose regimens of the Ad26.COV2.S vaccine candidate in adult and aged nonhuman primates (NHPs). A two-dose Ad26.COV2.S regimen induced higher peak binding and neutralizing antibody responses compared with a single dose. In one-dose regimens, neutralizing antibody responses were stable for at least 14 wk, providing an early indication of durability. Ad26.COV2.S induced humoral immunity and T helper cell (Th cell) 1-skewed cellular responses in aged NHPs that were comparable to those in adult animals. Aged Ad26.COV2.S-vaccinated animals challenged 3 mo after dose 1 with a SARS-CoV-2 spike G614 variant showed near complete lower and substantial upper respiratory tract protection for both regimens. Neutralization of variants of concern by NHP sera was reduced for B.1.351 lineages while maintained for the B.1.1.7 lineage independent of Ad26.COV2.S vaccine regimen.

Ad26.COV2.S protects Syrian hamsters against G614 spike variant SARS-CoV-2 and does not enhance respiratory disease.

Previously we have shown that a single dose of recombinant adenovirus serotype 26 (Ad26) vaccine expressing a prefusion stabilized SARS-CoV-2 spike antigen (Ad26.COV2.S) is immunogenic and provides protection in Syrian hamster and non-human primate SARS-CoV-2 infection models. Here, we investigated the immunogenicity, protective efficacy, and potential for vaccine-associated enhanced respiratory disease (VAERD) mediated by Ad26.COV2.S in a moderate disease Syrian hamster challenge model, using the currently most prevalent G614 spike SARS-CoV-2 variant. Vaccine doses of 1 × 109 and 1 × 1010 VP elicited substantial neutralizing antibodies titers and completely protected over 80% of SARS-CoV-2 inoculated Syrian hamsters from lung infection and pneumonia but not upper respiratory tract infection. A second vaccine dose further increased neutralizing antibody titers that was associated with decreased infectious viral load in the upper respiratory tract after SARS-CoV-2 challenge. Suboptimal non-protective immune responses elicited by low-dose A26.COV2.S vaccination did not exacerbate respiratory disease in SARS-CoV-2-inoculated Syrian hamsters with breakthrough infection. In addition, dosing down the vaccine allowed to establish that binding and neutralizing antibody titers correlate with lower respiratory tract protection probability. Overall, these preclinical data confirm efficacy of a one-dose vaccine regimen with Ad26.COV2.S in this G614 spike SARS-CoV-2 virus variant Syrian hamster model, show the added benefit of a second vaccine dose, and demonstrate that there are no signs of VAERD under conditions of suboptimal immunity.

Capsid-like particles decorated with the SARS-CoV-2 receptor-binding domain elicit strong virus neutralization activity.

The rapid development of a SARS-CoV-2 vaccine is a global priority. Here, we develop two capsid-like particle (CLP)-based vaccines displaying the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein. RBD antigens are displayed on AP205 CLPs through a split-protein Tag/Catcher, ensuring unidirectional and high-density display of RBD. Both soluble recombinant RBD and RBD displayed on CLPs bind the ACE2 receptor with nanomolar affinity. Mice are vaccinated with soluble RBD or CLP-displayed RBD, formulated in Squalene-Water-Emulsion. The RBD-CLP vaccines induce higher levels of serum anti-spike antibodies than the soluble RBD vaccines. Remarkably, one injection with our lead RBD-CLP vaccine in mice elicits virus neutralization antibody titers comparable to those found in patients that had recovered from COVID-19. Following booster vaccinations, the virus neutralization titers exceed those measured after natural infection, at serum dilutions above 1:10,000. Thus, the RBD-CLP vaccine is a highly promising candidate for preventing COVID-19.

Ad26 vector-based COVID-19 vaccine encoding a prefusion-stabilized SARS-CoV-2 Spike immunogen induces potent humoral and cellular immune responses.

Development of effective preventative interventions against SARS-CoV-2, the etiologic agent of COVID-19 is urgently needed. The viral surface spike (S) protein of SARS-CoV-2 is a key target for prophylactic measures as it is critical for the viral replication cycle and the primary target of neutralizing antibodies. We evaluated design elements previously shown for other coronavirus S protein-based vaccines to be successful, e.g., prefusion-stabilizing substitutions and heterologous signal peptides, for selection of a S-based SARS-CoV-2 vaccine candidate. In vitro characterization demonstrated that the introduction of stabilizing substitutions (i.e., furin cleavage site mutations and two consecutive prolines in the hinge region of S2) increased the ratio of neutralizing versus non-neutralizing antibody binding, suggestive for a prefusion conformation of the S protein. Furthermore, the wild-type signal peptide was best suited for the correct cleavage needed for a natively folded protein. These observations translated into superior immunogenicity in mice where the Ad26 vector encoding for a membrane-bound stabilized S protein with a wild-type signal peptide elicited potent neutralizing humoral immunity and cellular immunity that was polarized towards Th1 IFN-γ. This optimized Ad26 vector-based vaccine for SARS-CoV-2, termed Ad26.COV2.S, is currently being evaluated in a phase I clinical trial (ClinicalTrials.gov Identifier: NCT04436276).

SipB-SipC complex is essential for translocon formation.

The delivery of effector proteins by Salmonella across the host cell membrane requires a subset of effectors secreted by the type III secretion system (TTSS) known as translocators. SipC and SipB are translocator proteins that are inserted into host membranes and presumably form a channel that translocates type III effectors into the host cell. The molecular events of how these translocators insert into the host cell membrane remain unknown. We have previously shown that the SipC C-terminal amino acid region (321-409) is required for the translocation of effectors into host cells. In this study, we demonstrate that the ability to form SipC-SipB complex is essential for their insertion into the host membrane. The SipB-interacting domain of SipC is near its C-terminal amino acid region (340-409). In the absence of SipB, SipC was not detected in the membrane fraction. Furthermore, SipC mutants that no longer interact with SipB are defective in inserting into the host cell membrane. We propose a mechanism whereby SipC binds SipB through its C-terminal region to facilitate membrane-insertion and subsequent translocon formation in the host cell membrane.

Insight into bacterial virulence mechanisms against host immune response via the Yersinia pestis-human protein-protein interaction network.

A Yersinia pestis-human protein interaction network is reported here to improve our understanding of its pathogenesis. Up to 204 interactions between 66 Y. pestis bait proteins and 109 human proteins were identified by yeast two-hybrid assay and then combined with 23 previously published interactions to construct a protein-protein interaction network. Topological analysis of the interaction network revealed that human proteins targeted by Y. pestis were significantly enriched in the proteins that are central in the human protein-protein interaction network. Analysis of this network showed that signaling pathways important for host immune responses were preferentially targeted by Y. pestis, including the pathways involved in focal adhesion, regulation of cytoskeleton, leukocyte transendoepithelial migration, and Toll-like receptor (TLR) and mitogen-activated protein kinase (MAPK) signaling. Cellular pathways targeted by Y. pestis are highly relevant to its pathogenesis. Interactions with host proteins involved in focal adhesion and cytoskeketon regulation pathways could account for resistance of Y. pestis to phagocytosis. Interference with TLR and MAPK signaling pathways by Y. pestis reflects common characteristics of pathogen-host interaction that bacterial pathogens have evolved to evade host innate immune response by interacting with proteins in those signaling pathways. Interestingly, a large portion of human proteins interacting with Y. pestis (16/109) also interacted with viral proteins (Epstein-Barr virus [EBV] and hepatitis C virus [HCV]), suggesting that viral and bacterial pathogens attack common cellular functions to facilitate infections. In addition, we identified vasodilator-stimulated phosphoprotein (VASP) as a novel interaction partner of YpkA and showed that YpkA could inhibit in vitro actin assembly mediated by VASP.

The C terminus of SipC binds and bundles F-actin to promote Salmonella invasion.

Salmonella enterica serovar Typhimurium invade non-phagocytic cells by injecting bacterial effector proteins to exploit the host actin cytoskeleton network. SipC is such a Salmonella effector known to nucleate actin, bundle F-actin, and translocate type III effectors. The molecular mechanism of how SipC bundles F-actin and SipC domains responsible for these activities are not well characterized. We successfully separated these activities through a series of genetic deletion/insertions in SipC. We found that the C terminus (amino acids 200-409) of SipC bundled actin filaments using in vitro biochemical assays. We further demonstrated that amino acid residues 221-260 and 381-409 of full-length SipC were indispensable for its actin binding and bundling activities. Furthermore, Salmonella mutant strains lacking the actin bundling activity were less invasive into HeLa cells. These studies indicate that the C terminus of SipC bundles F-actin to promote Salmonella invasion.