Journal of Virology, Ahead of Print.
Polyvalent bacteriophages show the feature of infecting bacteria across multiple species or even orders. Infectivity of a polyvalent phage is variable depending on the host bacteria, which can disclose differential inhibition of bacteria by the phage. In this study, a polyvalent phage CSP1 infecting both Cronobacter sakazakii ATCC 29544 and Escherichia coli MG1655 was isolated. CSP1 showed higher growth inhibition and adsorption rate in E. coli compared to C. sakazakii, and identification of host receptors revealed that CSP1 uses E. coli LamB (LamBE) as a receptor but that CSP1 requires both C. sakazakii LamB (LamBC) and lipopolysaccharide (LPS) core for C. sakazakii infection. The substitution of LamBC with LamBE in C. sakazakii enhanced CSP1 susceptibility and made C. sakazakii LPS core no more essential for CSP1 infection. Comparative analysis of LamBC and LamBE disclosed that the extra proline at amino acid residue 284 in LamBC made a structural distinction by forming a longer loop and that the deletion of 284P in LamBC aligns its structure and makes LamBC function like LamBE, enhancing CSP1 adsorption and growth inhibition of C. sakazakii. These results suggest that 284P of LamBC plays a critical role in determining the CSP1-host bacteria interaction. These findings could provide insight into the elucidation of molecular determinants in the interaction between polyvalent phages and host bacteria and help us to understand the phage infectivity for efficient phage application.
Importance: Polyvalent phages have the advantage of a broader host range, overcoming the limitation of the narrow host range of phages. However, the limited molecular biological understanding on the host bacteria-polyvalent phage interaction hinders its effective application. Here, we revealed that the ability of the polyvalent phage CSP1 to infect Cronobacter sakazakii ATCC 29544 is disturbed by a single proline residue in the LamB protein and that lipopolysaccharide is used as an auxiliary receptor for CSP1 to support the adsorption and the subsequent infection of C. sakazakii. These results can contribute to a better understanding of the interaction between polyvalent phages and host bacteria for efficient phage application.
The majority of naturally elicited antibodies against the HIV-1 envelope glycoproteins (Env) are non-neutralizing (nnAbs) because they are unable to recognize the Env trimer in its native "closed" conformation. Nevertheless, it has been shown that nnAbs have the potential to eliminate HIV-1-infected cells by antibody-dependent cellular cytotoxicity (ADCC) provided that Env is present on the cell surface in its "open" conformation. This is because most nnAbs recognize epitopes that become accessible only after Env interaction with CD4 and the exposure of epitopes that are normally occluded in the closed trimer. HIV-1 limits this vulnerability by downregulating CD4 from the surface of infected cells, thus preventing a premature encounter of Env with CD4. Small CD4-mimetics (CD4mc) sensitize HIV-1-infected cells to ADCC by opening the Env glycoprotein and exposing CD4-induced (CD4i) epitopes. There are two families of CD4i nnAbs, termed anti-cluster A and anti-CoRBS Abs, which are known to mediate ADCC in the presence of CD4mc. Here, we performed Fab competition experiments and found that anti-gp41 cluster I antibodies comprise a major fraction of the plasma ADCC activity in people living with HIV (PLWH). Moreover, addition of gp41 cluster I antibodies to cluster A and CoRBS antibodies greatly enhanced ADCC-mediated cell killing in the presence of a potent indoline CD4mc, CJF-III-288. This cocktail outperformed broadly neutralizing antibodies and even showed activity against HIV-1-infected monocyte-derived macrophages. Thus, combining CD4i antibodies with different specificities achieves maximal ADCC activity, which may be of utility in HIV cure strategies.IMPORTANCEThe elimination of HIV-1-infected cells remains an important medical goal. Although current antiretroviral therapy decreases viral loads below detection levels, it does not eliminate latently infected cells that form the viral reservoir. Here, we developed a cocktail of non-neutralizing antibodies targeting highly conserved Env regions and combined it with a potent indoline CD4mc. This combination exhibited potent ADCC activity against HIV-1-infected primary CD4 + T cells as well as monocyte-derived macrophages, suggesting its potential utility in decreasing the size of the viral reservoir.
Upon binding to the host cell receptor, CD4, the pretriggered (State-1) conformation of the human immunodeficiency virus (HIV-1) envelope glycoprotein (Env) trimer undergoes transitions to downstream conformations important for virus entry. State 1 is targeted by most broadly neutralizing antibodies (bNAbs), whereas downstream conformations elicit immunodominant, poorly neutralizing antibody (pNAb) responses. Extraction of Env from the membranes of viruses or Env-expressing cells disrupts the metastable State-1 Env conformation, even when detergent-free approaches like styrene-maleic acid lipid nanoparticles (SMALPs) are used. Here, we combine three strategies to solubilize and purify mature membrane Envs that are antigenically native (i.e., recognized by bNAbs and not pNAbs): (1) solubilization of Env with a novel amphipathic copolymer, Amphipol A18; (2) use of stabilized pretriggered Env mutants; and (3) addition of the State-1-stabilizing entry inhibitor, BMS-806. Amphipol A18 was superior to the other amphipathic copolymers tested (SMA and AASTY 11-50) for preserving a native Env conformation. A native antigenic profile of A18 Env-lipid-nanodiscs was maintained for at least 7 days at 4°C and 2 days at 37°C in the presence of BMS-806 and was also maintained for at least 1 h at 37°C in a variety of adjuvants. The damaging effects of a single cycle of freeze-thawing on the antigenic profile of the A18 Env-lipid-nanodiscs could be prevented by the addition of 10% sucrose or 10% glycerol. These results underscore the importance of the membrane environment to the maintenance of a pretriggered (State-1) Env conformation and provide strategies for the preparation of lipid-nanodiscs containing native membrane Envs.IMPORTANCEThe human immunodeficiency virus (HIV-1) envelope glycoproteins (Envs) mediate virus entry into the host cell and are targeted by neutralizing antibodies elicited by natural infection or vaccines. Detailed studies of membrane proteins like Env rely on purification procedures that maintain their natural conformation. In this study, we show that an amphipathic copolymer A18 can directly extract HIV-1 Env from a membrane without the use of detergents. A18 promotes the formation of nanodiscs that contain Env and membrane lipids. Env in A18-lipid nanodiscs largely preserves features recognized by broadly neutralizing antibodies (bNAbs) and conceals features potentially recognized by poorly neutralizing antibodies (pNAbs). Our results underscore the importance of the membrane environment to the native conformation of HIV-1 Env. Purification methods that bypass the need for detergents could be useful for future studies of HIV-1 Env structure, interaction with receptors and antibodies, and immunogenicity.
Nipah virus (NiV) is a highly pathogenic paramyxovirus capable of causing severe respiratory and neurologic disease in humans. Currently, there are no licensed vaccines or therapeutics against NiV, underscoring the urgent need for the development of countermeasures. The NiV surface-displayed glycoproteins, NiV-G and NiV-F, mediate host cell attachment and fusion, respectively, and are heavily targeted by host antibodies. Here, we describe a vaccination-derived neutralizing monoclonal antibody, mAb92, that targets NiV-F. Structural characterization of the Fab region bound to NiV-F (NiV-F-Fab92) by cryo-electron microscopy analysis reveals an epitope in the DIII domain at the membrane distal apex of NiV-F, an established site of vulnerability on the NiV surface. Further, prophylactic treatment of hamsters with mAb92 offered complete protection from NiV disease, demonstrating beneficial activity of mAb92 in vivo. This work provides support for targeting NiV-F in the development of vaccines and therapeutics against NiV.IMPORTANCENipah virus (NiV) is a highly lethal henipavirus (HNV) that causes severe respiratory and neurologic disease in humans. Currently, there are no licensed vaccines or therapeutics against NiV, highlighting a need to develop countermeasures. The NiV surface displays the receptor binding protein (NiV-G, or RBP) and the fusion protein (NiV-F), which allow the virus to attach and enter cells. These proteins can be targeted by vaccines and antibodies to prevent disease. This work describes a neutralizing antibody (mAb92) that targets NiV-F. Structural characterization by cryo-electron microscopy analysis reveals where the antibody binds to NiV-F to neutralize the virus. This study also shows that prophylactic treatment of hamsters with mAb92 completely protected against developing NiV disease. This work shows how targeting NiV-F can be useful to preventing NiV disease, supporting future studies in the development of vaccines and therapeutics.
2C is a highly conserved picornaviral non-structural protein with ATPase activity and plays a multifunctional role in the viral life cycle as a promising target for anti-picornavirus drug development. While the structure-function of enteroviral 2Cs have been well studied, cardioviral 2Cs remain largely uncharacterized. Here, an endogenous ATP molecule was identified in the crystal structure of 2C from encephalomyocarditis virus (EMCV, Cardiovirus A). The ATP is bound into the ATPase active site with a unique compact conformation. Notably, the γ-phosphate of ATP directly interacts with Arg311 (conserved in cardioviral 2Cs), and its mutation significantly inhibits the ATPase activity. Unexpectedly, this mutation remarkably promotes 2C self-oligomerization and viral replication efficiency. Molecular dynamic simulations showed that the Arg311 side chain is highly dynamic, indicating it may function as a switch between the activation state and the inhibition state of ATPase activity. A hexameric ring model of EMCV 2C full length indicated that the C-terminal helix may get close to the N-terminal amphipathic helices to form a continuous positive region for RNA binding. The RNA-binding studies of EMCV 2C revealed that the RNA length is closely associated with the RNA-binding affinities and indicated that the substrate may wrap around the outer surface of the hexamer. Our studies provide a biochemical framework to guide the characterization of EMCV 2C and the essential role of arginine in cardioviral 2C functions.
Importance: Encephalomyocarditis virus (Cardiovirus A) is the causative agent of the homonymous disease, which may induce myocarditis, encephalitis, and reproductive disorders in various mammals. 2C protein is functionally indispensable and a promising target for drug development involving broad-spectrum picornaviral inhibitors. Here, an endogenous ATP molecule with a unique conformation was discovered by a combination of protein crystallography and high-performance liquid chromatography in the encephalomyocarditis virus (EMCV) 2C structure. Biochemical and structural characterization analysis of EMCV 2C revealed the critical role of conserved Arg311 in ATPase activity and self-oligomerization of EMCV 2C. The viral replication kinetics and infectivity study suggested that the residue negatively regulated the infectivity titer and virus encapsulation efficiency of EMCV and is, therefore, crucial for 2C protein to promote viral replication. Our systemic structure-function analysis provides unique insights into the function and regulation mechanism of cardioviral 2C protein.
VRC01-class broadly neutralizing antibodies (bnAbs) have been isolated from people with HIV-1, but they have not yet been elicited by vaccination. They are extensively somatically mutated and sometimes accumulate CDRL1 deletions. Such indels may allow VRC01-class antibodies to accommodate the glycans expressed on a conserved N276 N-linked glycosylation site in loop D of the gp120 subunit. These glycans constitute a major obstacle in the development of VRC01-class antibodies, as unmutated antibody forms are unable to accommodate them. Although immunizations of knock-in mice expressing human VRC01-class B-cell receptors (BCRs) with specifically designed Env-derived immunogens lead to the accumulation of somatic mutations in VRC01-class BCRs, CDRL1 deletions are rarely observed, and the elicited antibodies display narrow neutralizing activities. The lack of broad neutralizing potential could be due to the absence of deletions, the lack of appropriate somatic mutations, or both. To address this point, we modified our previously determined prime-boost immunization with a germline-targeting immunogen nanoparticle (426c.Mod.Core), followed by a heterologous core nanoparticle (HxB2.WT.Core), by adding a final boost with a cocktail of various stabilized soluble Env trimers. We isolated VRC01-like antibodies with extensive somatic mutations and, in one case, a seven-amino acid CDRL1 deletion. We generated chimeric antibodies that combine the vaccine-elicited somatic mutations with CDRL1 deletions present in human mature VRC01 bnAbs. We observed that CDRL1 indels did not improve the neutralizing antibody activities. Our study indicates that CDRL1 length by itself is not sufficient for the broadly neutralizing phenotype of this class of antibodies.
Importance: HIV-1 broadly neutralizing antibodies will be a key component of an effective HIV-1 vaccine, as they prevent viral acquisition. Over the past decade, numerous broadly neutralizing antibodies (bnAbs) have been isolated from people with HIV. Despite an in-depth knowledge of their structures, epitopes, ontogenies, and, in a few rare cases, their maturation pathways during infection, bnAbs have, so far, not been elicited by vaccination. This necessitates the identification of key obstacles that prevent their elicitation by immunization and overcoming them. Here we examined whether CDRL1 shortening is a prerequisite for the broadly neutralizing potential of VRC01-class bnAbs, which bind within the CD4 receptor binding site of Env. Our findings indicate that CDRL1 shortening by itself is important but not sufficient for the acquisition of neutralization breadth, and suggest that particular combinations of amino acid mutations, not elicited so far by vaccination, are most likely required for the development of such a feature.
Dengue virus (DENV) gains genetic mutations during continuous transmission and evolution, making the virus more adaptive and virulent. The clade of DENV-1 genotype I has expanded and become the predominant genotype in Asia and the Pacific areas, but the underlying mechanisms are unclear. A combined analysis of nonsynonymous mutations in domain III of the envelope protein and their biological effects on virus pathogenesis and transmission was evaluated. Phylogenetic analyses found three nonsynonymous mutations (V324I, V351L, and V380I) in domain III of the envelope protein, which emerged in 1970s-1990s and stably inherited and expanded in contemporary strains after 2000. We generated reverse-mutated viruses (I324V, L351V, and I380V) based on an infectious clone of an epidemic DENV-1 strain (NIID02-20), and the results suggested that the infectivity of the contemporary epidemic virus (wild type, WT) has increased compared to the reverse mutant viruses in mammalian hosts but not mosquito vectors. The WT virus showed a higher binding affinity to host cells and increased virion stability. In addition, weaker immunogenicity and higher resistance to neutralizing antibodies of the WT virus indicated a trend of immune escape. The data suggested that nonsynonymous mutations of the E protein (V324I, V351L, and V380I) promote infectivity and immune evasion of DENV-1 genotype I, which may facilitate its onward transmission on a global scale.
Importance: We provide evidence that minor sequence variation among dengue virus (DENV) strains can result in increased adaptability and virulence, impacting both the biology of the virus and the antiviral immune response. The genetic mutations of DENV-1 gained during continuous transmission and evolution will offer new clues for the design of novel vaccines against flaviviruses.
CD4-mimetics (CD4mcs) are small molecule compounds that mimic the interaction of the CD4 receptor with HIV-1 envelope glycoproteins (Env). Env from primary viruses normally samples a "closed" conformation that occludes epitopes recognized by CD4-induced (CD4i) non-neutralizing antibodies (nnAbs). CD4mcs induce conformational changes on Env resulting in the exposure of these otherwise inaccessible epitopes. Here, we evaluated the capacity of plasma from a cohort of 50 people living with HIV to recognize HIV-1-infected cells and eliminate them by antibody-dependent cellular cytotoxicity (ADCC) in the presence of a potent indoline CD4mc. We observed a marked heterogeneity among plasma samples. By measuring the levels of different families of CD4i Abs, we found that the levels of anti-cluster A, anti-coreceptor binding site, and anti-gp41 cluster I antibodies are responsible for plasma-mediated ADCC in the presence of CD4mc.
Importance: There are several reasons that make it difficult to target the HIV reservoir. One of them is the capacity of infected cells to prevent the recognition of HIV-1 envelope glycoproteins (Env) by commonly elicited antibodies in people living with HIV. Small CD4-mimetic compounds expose otherwise occluded Env epitopes, thus enabling their recognition by non-neutralizing antibodies (nnAbs). A better understanding of the contribution of these antibodies to eliminate infected cells in the presence of CD4mc could lead to the development of therapeutic cure strategies.
The continued emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants necessitates updating coronavirus disease 2019 (COVID-19) vaccines to match circulating strains. The immunogenicity and efficacy of these vaccines must be tested in pre-clinical animal models. In Syrian hamsters, we measured the humoral and cellular immune response after immunization with the nanoparticle recombinant Spike (S) protein-based COVID-19 vaccine (Novavax, Inc.). We also compared the efficacy of the updated monovalent XBB.1.5 variant vaccine with previous COVID-19 vaccines for the induction of XBB.1.5 and EG.5.1 neutralizing antibodies and protection against a challenge with the EG.5.1 variant of SARS-CoV-2. Immunization induced high levels of S-specific IgG and IgA antibody-secreting cells and antigen-specific CD4+ T cells. The XBB.1.5 and XBB.1.16 vaccines, but not the Prototype vaccine, induced high levels of neutralizing antibodies against the XBB.1.5, EG.5.1, and JN.1 variants of SARS-CoV-2. Upon challenge with the Omicron EG.5.1 variant, the XBB.1.5 and XBB.1.16 vaccines reduced the virus load in the lungs, nasal turbinates, trachea, and nasal washes. The bivalent vaccine (Prototype rS + BA.5 rS) continued to offer protection in the trachea and lungs, but protection was reduced in the upper airways. By contrast, the monovalent Prototype vaccine no longer offered good protection, and breakthrough infections were observed in all animals and tissues. Thus, based on these study results, the protein-based XBB.1.5 vaccine is immunogenic and increased the breadth of protection against the Omicron EG.5.1 variant in the Syrian hamster model.
Importance: As SARS-CoV-2 continues to evolve, there is a need to assess the immunogenicity and efficacy of updated vaccines against newly emerging variants in pre-clinical models such as mice and hamsters. Here, we compared the immunogenicity and efficacy between the updated XBB.1.5, the original Prototype Wuhan-1, and the bivalent Prototype + BA.5 vaccine against a challenge with the EG.5.1 Omicron variant of SARS-CoV-2 in hamsters. The XBB.1.5 and bivalent vaccine, but not the Prototype, induced serum-neutralizing antibodies against EG.5.1, albeit the titers were higher in the XBB.1.5 immunized hamsters. The presence of neutralizing antibodies was associated with complete protection against EG.5.1 infection in the lower airways and reduced virus titers in the upper airways. Compared with the bivalent vaccine, immunization with XBB.1.5 improved viral control in the nasal turbinates. Together, our data show that the updated vaccine is immunogenic and that it offers better protection against recent variants of SARS-CoV-2.
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