A recombinant herpes virus expressing influenza hemagglutinin confers protection and induces antibody-dependent cellular cytotoxicity

Despite widespread yearly vaccination, influenza leads to significant morbidity and mortality across the globe. To make a more broadly protective influenza vaccine, it may be necessary to elicit antibodies that can activate effector functions in immune cells, such as antibody-dependent cellular cytotoxicity (ADCC). There is growing evidence supporting the necessity for ADCC in protection against influenza and herpes simplex virus (HSV), among other infectious diseases. An HSV-2 strain lacking the essential glycoprotein D (gD), was used to create ΔgD-2, which is a highly protective vaccine against lethal HSV-1 and HSV-2 infection in mice. It also elicits high levels of IgG2c antibodies that bind FcγRIV, a receptor that activates ADCC. To make an ADCC-eliciting influenza vaccine, we cloned the hemagglutinin (HA) gene from an H1N1 influenza A strain into the ΔgD-2 HSV vector. Vaccination with ΔgD-2::HA_PR8 was protective against homologous influenza challenge and elicited an antibody response against HA that inhibits hemagglutination (HAI⁺), is predominantly IgG2c, strongly activates FcγRIV, and protects against influenza challenge following passive immunization of naïve mice. Prior exposure of mice to HSV-1, HSV-2, or a replication-defective HSV-2 vaccine (dl5-29) does not reduce protection against influenza by ΔgD-2::HA_PR8. This vaccine also continues to elicit protection against both HSV-1 and HSV-2, including high levels of IgG2c antibodies against HSV-2. Mice lacking the interferon-α/β receptor and mice lacking the interferon-γ receptor were also protected against influenza challenge by ΔgD-2::HA_PR8. Our results suggest that ΔgD-2 can be used as a vaccine vector against other pathogens, while also eliciting protective anti-HSV immunity.

Influenza remains a global health threat. Seasonal strains of influenza A and B cause an estimated 5 million cases of severe infections and 500,000 deaths per year (1). Influenza pandemics have caused even greater morbidity and mortality. During the H1N1 pandemic of 1918 to 1919, 500 million people, approximately one-third of the world’s population at that time, were estimated to have been infected with this strain, leading to 50 million deaths (2). The H1N1 pandemic of 2009 is estimated to have caused up to 575,000 deaths (2). Currently, three types of influenza vaccines are offered annually in the United States: a recombinant virus expressing influenza proteins, chemically inactivated virus, and live attenuated virus (3). Regardless of the vaccine type, multiple strains are included to increase the chances of developing sufficient protection against major circulating influenza strains. However, these vaccines primarily elicit a neutralizing antibody response that is sensitive to changes in the influenza virus due to antigenic drift and shift (4). Antigenic drift results from an accumulation of random mutations in influenza antigens, like hemagglutinin (HA), altering sites recognized by the immune system (4). Influenza A strains can also undergo antigenic shift, whereby two different influenza strains infect the same cell to form a reassortant virus with new antigenic properties (4). Due to limited immunity in the population, these new strains are highly virulent, causing widespread epidemics and disease (4). With antigenic drift and shift, vaccine-mediated protection against circulating strains has been insufficient (5). Influenza vaccines that elicit more robust and long-term protection are therefore needed. Notably, if an influenza vaccine with ≥75% efficacy were to be broadly used in the United States, an estimated 19,500 deaths a year could be prevented and direct healthcare costs reduced by $3.5 billion (6).For many years, efforts to improve influenza vaccines have focused on eliciting an immune response for full, broad protection against both circulating and future strains of the virus. These studies have shown that, in general, neutralizing antibodies are sufficient for homologous protection (7). However, achieving heterologous protection may require more broadly neutralizing antibodies or nonneutralizing antibodies able to activate effector immune cells (5). Previous studies have found that passively transferred nonneutralizing monoclonal antibodies can be potently protective in a mouse influenza challenge model (8–10). Several novel strategies have attempted to generate a nonneutralizing response against influenza. For example, vaccines have been created to specifically target the conserved stem region of HA (11–13).Nonneutralizing antibodies stimulate effector cell mechanisms, including antibody-mediated phagocytosis and antibody-dependent cellular cytotoxicity (ADCC), both of which require activation of the Fcγ receptors (FcγRs) (14). Specific isotypes of IgG antibodies are associated with FcγR modulation and subsequent ADCC activation, including the IgG1 and IgG3 subtypes in humans, as well as IgG2a and IgG2c subtypes in mice (15–19). IgG2a and IgG2c isotypes are functionally equivalent and mouse strain-dependent, with IgG2c present in C57BL/6J mice (20). Recent studies have demonstrated that natural infection by influenza and vaccination elicit nonneutralizing antibodies with effector functions that contribute to protection (5, 9, 21–27). In mouse and nonhuman primate challenge models, ADCC-mediating antibodies have demonstrated protection against both homologous and heterologous influenza challenge (9, 28).Recently, we developed a single-cycle herpes simplex virus (HSV) vaccine that completely protects against vaginal, skin, and ocular challenges by HSV-1 and HSV-2 (29, 30). Protection elicited by this vaccine, designated ΔgD-2 for its lack of the essential glycoprotein D (gD) gene, is transferable via passive infusion of immune sera to naïve wild-type mice but not to mice lacking the Fcγ common chain (30). The immune response elicited by ΔgD-2 primarily elicits nonneutralizing antibodies with high levels of FcγRIV-activating function.We asked whether ΔgD-2 could be used as a vaccine platform to induce broadly protective FcγRIV-activating antibodies against a heterologous antigen, such as influenza HA. In this study, we demonstrate that our recombinant vaccine, ΔgD-2::HA_PR8, elicits protection against influenza with a high proportion of FcγRIV-activating antibodies. Additionally, anticipating the use of ΔgD-2 as a vaccine vector against other pathogens, we tested whether our construct would still be protective in mice lacking interferon (IFN) function. Many humans have inborn errors in their IFN signaling pathways, leading to more lethal outcomes in infection (31). Patients with such deficiencies are disproportionately represented among HSV encephalitis cases and are often diagnosed only after presenting with serious symptoms (32–38). This at-risk population underscores the importance of eliciting protection against HSV in the absence of a functional IFN-α/β response. Additionally, many pathogens, such as dengue virus, require mouse models lacking IFN function, and for ease of testing, an efficacious vaccine should remain functional in these mice (39–41). In this study, we demonstrate that ΔgD-2 is a versatile, immunogenic vaccine vector that provides a strong FcγRIV-activating immune response against heterologous pathogens, while maintaining its protective benefit against HSV, in both wild-type and IFN-deficient mice.     

Redox potential of the terminal quinone electron acceptor QB in photosystem II reveals the mechanism of electron transfer regulation

Photosystem II (PSII) extracts electrons from water at a Mn₄CaO₅ cluster using light energy and then transfers them to two plastoquinones, the primary quinone electron acceptor Q_A and the secondary quinone electron acceptor Q_B. This forward electron transfer is an essential process in light energy conversion. Meanwhile, backward electron transfer is also significant in photoprotection of PSII proteins. Modulation of the redox potential (E_m) gap of Q_A and Q_B mainly regulates the forward and backward electron transfers in PSII. However, the full scheme of electron transfer regulation remains unresolved due to the unknown E_m value of Q_B. Here, for the first time (to our knowledge), the E_m value of Q_B reduction was measured directly using spectroelectrochemistry in combination with light-induced Fourier transform infrared difference spectroscopy. The E_m(Q_B⁻/Q_B) was determined to be approximately +90 mV and was virtually unaffected by depletion of the Mn₄CaO₅ cluster. This insensitivity of E_m(Q_B⁻/Q_B), in combination with the known large upshift of E_m(Q_A⁻/Q_A), explains the mechanism of PSII photoprotection with an impaired Mn₄CaO₅ cluster, in which a large decrease in the E_m gap between Q_A and Q_B promotes rapid charge recombination via Q_A⁻.In oxygenic photosynthesis in plants and cyanobacteria, photosystem II (PSII) has an important function in light-driven water oxidation, a process that leads to the generation of electrons and protons for CO₂ reduction and ATP synthesis, respectively (1–3). Photosynthetic water oxidation also produces molecular oxygen as a byproduct, which is the source of atmospheric oxygen and sustains virtually all life on Earth. PSII reactions are initiated by light-induced charge separation between a chlorophyll (Chl) dimer (P680) and a pheophytin (Pheo) electron acceptor, leading to the formation of a P680⁺Pheo⁻ radical pair (4, 5). An electron hole on P680⁺ is transferred to a Mn₄CaO₅ cluster, the catalytic center of water oxidation, via the redox-active tyrosine, Y_Z (D1-Tyr161). At the Mn₄CaO₅ cluster, water oxidation proceeds through a cycle of five intermediates denoted S_n states (n = 0–4) (6, 7). On the electron acceptor side, the electron is transferred from Pheo⁻ to the primary quinone electron acceptor Q_A and then to the secondary quinone electron acceptor Q_B (8, 9). Q_A and Q_B have many similarities: they consist of plastoquinone (PQ), are located symmetrically around a nonheme iron center, and interact with D2 and D1 proteins, respectively, in a similar manner (Fig. 1) (10, 11). However, they play significantly different roles in PSII (8, 9). Q_A is only singly reduced to transfer an electron to Q_B, whereas Q_B accepts one or two electrons. When Q_B is doubly reduced, the resultant Q_B²⁻ takes up two protons to form plastoquinol (PQH₂), which is then released into thylakoid membranes. Differences between Q_A and Q_B could be caused by differences in the molecular interactions of PQ with surrounding proteins in Q_A and Q_B pockets, although the detailed mechanism remains to be clarified (12, 13).Open in a separate windowFig. 1.Redox cofactors in PSII and the electron transfer pathway (blue arrows). For the PSII structure, the X-ray crystallographic structure at 1.9-Å resolution (Protein Data Bank ID code 3ARC) (9) was used. The electron acceptor side is expanded, showing the arrangements of Q_A, Q_B, and the nonheme iron with their molecular interactions. Nearby carboxylic groups are also shown.Electron transfer reactions in PSII are highly regulated by the spatial localization of redox components and their redox potentials (E_m values). Both forward and backward electron transfers are important; backward electron transfers control charge recombination in PSII, and this serves as photoprotection for PSII proteins (5, 14–17). PSII involves specific mechanisms to regulate forward and backward electron transfer reactions in response to environmental changes. For instance, in strong light, some species of cyanobacteria increase the E_m of Pheo to facilitate charge recombination. Specifically, they exchange D1 subunits originating from different psbA genes to change the hydrogen bond interactions of Pheo (16–20). On the other hand, it was found that impairment of the Mn₄CaO₅ cluster led to a significant increase in the E_m of Q_A by ∼150 mV (21–27). This potential increase was thought to inhibit forward electron transfer to Q_B to promote direct relaxation of Q_A⁻ without forming triplet-state Chl, a precursor of harmful singlet oxygen (2, 5, 14, 15, 17, 23). In addition, charge recombination of Q_A⁻ with P680⁺ prevents oxidative damage by high-potential P680⁺ (5). However, the full mechanism of photoprotection by the regulation of the quinone electron acceptor E_m values remains to be resolved, because the E_m of Q_B has not been determined conclusively, and the effect of Mn₄CaO₅ cluster inactivation on it has not been examined (5).Although the E_m of the single reduction of Q_B has been estimated to be ∼80 mV higher than that of Q_A from kinetic and thermodynamic data (28–32), so far no reports have measured the E_m of Q_B directly. In contrast, the E_m of Q_A was measured extensively using chemical or electrochemical titrations and determined to be approximately −100 mV for oxygen-evolving PSII (21–27). The main reason for this difference is due to the fact that the Q_A reaction can be monitored readily by fluorescence measurement in that an increase in fluorescence indicates Q_A⁻ formation (8, 33–35). However, the fluorescence method cannot be used easily to monitor Q_B reduction. Although UV-Vis absorption and electron spin resonance have also been used to monitor Q_A⁻ in redox titration (summarized in ref. 22), so far these methods have not been used to monitor the titration of Q_B, likely because Q_A⁻ and Q_B⁻ give similar signals (36–39). Another spectroscopic method that can be used to monitor Q_A and Q_B reactions is Fourier transform infrared (FTIR) difference spectroscopy, which detects reaction-induced changes in the molecular vibrations of a cofactor and its environment in proteins (40–45). It was previously shown that comparison of FTIR difference spectra upon Q_A⁻ and Q_B⁻ formation showed some characteristic differences in spectral features (46). In particular, bands at 1,721 and 1,745 cm⁻¹, which were assigned to ester C=O vibrations of nearby Pheo molecules affected by the reduction of Q_A and Q_B, respectively, were suggested to be good markers for discriminating between Q_A and Q_B reactions (46).In this study, we directly measured the E_m of Q_B in PSII using spectroelectrochemistry and light-induced FTIR difference spectroscopy. The effect of Mn₄CaO₅ cluster depletion on the E_m value was also examined. Spectroelectrochemistry has been used to accurately measure the E_m values of cofactors in various redox proteins (47, 48) including redox cofactors in PSII (24, 25, 49, 50). FTIR spectroelectrochemistry, which has the additional merit of being able to provide structural information, has also been used to investigate redox reactions of biomolecules and proteins (48, 50–54). This method was recently applied to the nonheme iron center of PSII to examine the effect of Mn depletion on the E_m value and obtain structural information around the nonheme iron (50). The results in our study showed that the E_m of the first reduction of Q_B [E_m(Q_B⁻/Q_B)] was much higher than previously estimated, and the E_m of the second reduction [E_m(PQH₂/Q_B⁻)] was higher than the first reduction. Furthermore, we showed that Mn depletion hardly affected the E_m values of Q_B, in contrast to the large change in the E_m of Q_A (21–27). With these results, the mechanism of photoprotection of PSII when the Mn₄CaO₅ cluster is inactivated is now clearly explained.     

Induction of broadly neutralizing antibodies using a secreted form of the hepatitis C virus E1E2 heterodimer as a vaccine candidate

Hepatitis C virus (HCV) is a global disease burden, and a preventive vaccine is needed to control or eradicate the virus. Despite the advent of effective antiviral therapy, this treatment is not accessible to many patients and does not prevent reinfection, making chronic hepatitis C an ongoing global health problem. Thus, development of a prophylactic vaccine will represent a significant step toward global eradication of HCV. HCV exhibits high genetic variability, which leads frequently to immune escape. However, a considerable challenge faced in HCV vaccine development is designing an antigen that elicits broadly neutralizing antibodies. Here, we characterized the immunogenicity of a vaccine based on a soluble, secreted form of the E1E2 envelope heterodimer (sE1E2.LZ). Sera from mice immunized with sE1E2.LZ exhibited an anti-E1E2–specific response comparable to mice immunized with membrane-bound E1E2 (mbE1E2) or a soluble E2 ectodomain (sE2). In competition-inhibition ELISA using antigenic domain-specific neutralizing and nonneutralizing antibodies, sera from sE1E2.LZ-immunized mice showed nearly identical or stronger competition toward neutralizing antibodies when compared with mbE1E2. In contrast, sera from mice immunized with sE2, and to a lesser extent mbE1E2, competed more effectively with nonneutralizing antibodies. An assessment of neutralization activity using both HCV pseudoparticles and cell culture–derived infectious HCV showed that immunization with sE1E2.LZ elicited the broadest neutralization activity of the three antigens, and sE1E2.LZ induced neutralization activity against all genotypes. These results indicate that our native-like soluble glycoprotein design, sE1E2.LZ, induces broadly neutralizing antibodies and serves as a promising vaccine candidate for further development.

Hepatitis C virus (HCV) is a global disease burden, with an estimated 71 million people infected worldwide (1, 2). Roughly 75% of HCV infections become chronic (3–5), and in severe cases can result in cirrhosis or hepatocellular carcinoma (6). Viral infection can be cured at high rates by direct acting antivirals (DAAs), but several issues have blunted their effectiveness in eradicating HCV. In particular, multiple public health and financial barriers (7, 8) restrict access to DAAs in areas with high incidence of infection and DAAs do not prevent reinfection. Moreover, HCV infection is largely asymptomatic and often does not generate sterilizing immunity, thereby contributing to reinfection or continued disease progression (7, 9, 10). Collectively, these issues have resulted in a continued rise in HCV infections.Acute HCV infections can be cleared by host immunity in ∼25% of cases. Among individuals who clear their first infection, the rate of clearance rises to 80% for subsequent infections, indicating an effective immune memory response (11–14). This type of natural protective immunity to HCV requires the induction of broadly neutralizing antibodies to E1E2 ectodomains and T cell responses to the structural and nonstructural proteins (15–17). The above clinical observations suggest that, if a vaccine candidate could induce broadly neutralizing antibody and cell-mediated immune responses equivalent to that seen in spontaneous clearance, such a vaccine would be highly effective at preventing HCV infection. An HCV vaccine therefore remains an essential proactive measure to protect against viral spread, yet vaccine developments against the virus have been unsuccessful to date (17, 18).A number of challenges exist that have thus far limited progress toward developing a prophylactic vaccine against HCV. One major challenge in developing a successful vaccine for HCV has been the remarkable genetic diversity of the virus which has six major genotypes (genotypes 1 to 6), in addition to two less-common genotypes (19) (genotypes 7 and 8), and intragenotypic diversity resulting in 90 total subtypes (20). Moreover, shielding of important neutralizing epitopes with glycans (21, 22), and the presence of immunodominant nonneutralizing epitopes (23–26) deflect the immune response from conserved regions that mediate virus neutralization. Multiple studies in chimpanzees and humans have used E1E2 formulations to induce a humoral immune response, but their success in generating high titers of broadly neutralizing antibody (bnAb) responses has been limited. In particular, immunological assessment in chimpanzees of an E1E2 vaccine produced superior immune responses as compared with E2 administered alone and resulted in sterilizing immunity against homologous virus challenge (27, 28), but with less cross-neutralization capacity against heterologous isolates (29). In addition, an E1E2 formulation tested in humans is well-tolerated (30). However, due to the limited neutralization breadth observed in the human clinical trial (31, 32), using native E1E2 as a vaccine is not likely to provide sufficient protection from HCV infection. Rather, optimization of E1E2 to improve its immunogenicity and capacity to elicit bnAbs through rational design appears to be the preferred path for developing an effective B cell-based vaccine (33).An additional bottleneck contributing to the difficulty in generating protective B cell immune responses required for an effective HCV vaccine is preparation of a homogeneous E1E2 antigen. HCV envelope glycoproteins E1 and E2 form a heterodimer on the surface of the virion (34–36). Furthermore, E1E2 assembly has been proposed to form a trimer of heterodimers (37) mediated by hydrophobic C-terminal transmembrane domains (TMDs) (36, 38, 39) and interactions between E1 and E2 ectodomains (40–42). These glycoproteins are necessary for viral entry and infection, as E2 attaches to the CD81 and scavenger receptor type B class I (SR-B1) coreceptors as part of a multistep entry process on the surface of hepatocytes (43–46). Neutralizing antibody (nAb) responses to HCV infection target epitopes in E1, E2, or the E1E2 heterodimer (25, 47–52). A significant impediment to the uniform production of an immunogenic E1E2 heterodimer that could be utilized for vaccine development is the association of the antigen with the membrane via the TMDs (36, 53). Progress has been made in the production and purification of the membrane-bound E1E2 complex via immunoaffinity purification (54, 55) or the use of tags that allow protein A (56) or anti-Flag (57) chromatography. While these methods produce high-quality samples, they all involve harsh elution conditions. How such conditions might influence sample quality at a scale required for vaccine trials is unclear. Furthermore, intracellular expression and membrane extraction limits the ability to produce large quantities of sufficient homogeneity required for both basic research and vaccine production.In contrast, viral glycoproteins of influenza hemagglutinin (58), respiratory syncytial virus (RSV) (59), SARS-CoV-2 (60), and others (61, 62) have been stabilized in soluble form using a C-terminal attached foldon trimerization domain to facilitate assembly. In addition, HIV gp120-gp41 proteins have been designed as soluble SOSIP trimers in part by introducing a furin cleavage site to facilitate native-like assembly when cleaved by the enzyme (63, 64). Recent efforts have made strides toward liberating the E1E2 complex from the membrane in its native form (65, 66). In particular, our previous work (66) showed that a soluble E1E2 (sE1E2) using the Fos/Jun leucine zipper (LZ) coiled-coil as a scaffold (sE1E2.LZ) is antigenically intact, as the protein is recognized by E1E2-specific mAbs AR4A and AR5A (67). Moreover, sE1E2.LZ elicited nAbs in mice immunized with the antigen, making this scaffold a promising potential platform for engineering of additional HCV vaccine candidates.Here, we describe the immunogenicity of our native-like secreted E1E2 construct sE1E2.LZ and compare it with the membrane-bound E1E2 complex (mbE1E2) and a secreted form of the E2 ectodomain (sE2). Immunization of mice with sE1E2.LZ produced sera possessing anti-E1E2 antibodies at levels comparable to mice immunized with mbE1E2 or sE2. Moreover, the antibody response in sE1E2.LZ-immunized mice is skewed more toward nAbs relative to non-nAbs than the other two antigens. Remarkably, sera from sE1E2.LZ-immunized mice exhibited broader neutralization activity than either mbE1E2 or sE2 when assessed using both pseudotyped HCV particles (HCVpp) and cell culture-derived HCV (HCVcc), suggesting that this sE1E2 platform represents a favorable starting point for developing scaffolded E1E2 vaccine candidates.     

Immunogenicity and efficacy of the COVID-19 candidate vector vaccine MVA-SARS-2-S in preclinical vaccination

Severe acute respiratory syndrome (SARS) coronavirus 2 (SARS-CoV-2) has emerged as the infectious agent causing the pandemic coronavirus disease 2019 (COVID-19) with dramatic consequences for global human health and economics. Previously, we reached clinical evaluation with our vector vaccine based on modified vaccinia virus Ankara (MVA) against the Middle East respiratory syndrome coronavirus (MERS-CoV), which causes an infection in humans similar to SARS and COVID-19. Here, we describe the construction and preclinical characterization of a recombinant MVA expressing full-length SARS-CoV-2 spike (S) protein (MVA-SARS-2-S). Genetic stability and growth characteristics of MVA-SARS-2-S, plus its robust expression of S protein as antigen, make it a suitable candidate vaccine for industrial-scale production. Vaccinated mice produced S-specific CD8⁺ T cells and serum antibodies binding to S protein that neutralized SARS-CoV-2. Prime-boost vaccination with MVA-SARS-2-S protected mice sensitized with a human ACE2-expressing adenovirus from SARS-CoV-2 infection. MVA-SARS-2-S is currently being investigated in a phase I clinical trial as aspirant for developing a safe and efficacious vaccine against COVID-19.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causal agent of coronavirus disease 2019 (COVID-19), first emerged in late 2019 in China (1). SARS-CoV-2 exhibits extremely efficient human-to-human transmission, the new pathogen rapidly spread worldwide, and within months it caused a global pandemic, changing daily life for billions of people. The COVID-19 case fatality rate of ∼2–5% makes the development of countermeasures a global priority. In fact, the development of COVID-19 vaccine candidates is advancing at an international level with unprecedented speed. About 1 y after the first known cases of COVID-19, we can account for >80 SARS-CoV-2–specific vaccines in clinical evaluations and >10 candidate vaccines already in phase III trials (2–4). However, we still lack information on the key immune mechanisms needed for protection against COVID-19. A better understanding of the types of immune response elicited upon natural SARS-CoV-2 infections has become an essential component to assess the promise of various vaccination strategies (5).The SARS-CoV-2 spike (S) protein serves as the most important target antigen for vaccine development based on preclinical research on candidate vaccines against SARS-CoV or Middle East respiratory syndrome coronavirus (MERS-CoV). The trimeric S protein is a prominent structure at the virion surface and essential for SARS-CoV-2 cell entry. As a class I viral fusion protein, it mediates virus interaction with the cellular receptor angiotensin-converting enzyme 2 (ACE2), and fusion with the host cell membrane, both key steps in infection. Thus, infection can be prevented by S-specific antibodies neutralizing the virus (6–9).Among the front-runner vaccines are new technologies such as messenger RNA (mRNA)-based vaccines and nonreplicating adenovirus vector vaccines (10–13). First reports from these SARS-CoV-2-S–specific vaccines in phase 1/2 clinical studies demonstrated acceptable safety and promising immunogenicity profiles, and by now data from large phase 3 clinical trials show promising levels of protective efficacy (4, 12–14). In December 2020, the first mRNA-based COVID-19 vaccines received emergency use authorization or conditional licensing by the US Food and Drug Administration and European Medicines Agency (11, 15, 16). By March 2021, two adenovirus vector-based COVID-10 vaccines had been approved by regulatory authorities (17, 18). This is good news because efficacious vaccines will provide a strategy to change SARS-CoV-2 transmission dynamics. In addition, multiple vaccine types will be advantageous to meet specific demands across different target populations. This includes the possibility of using heterologous immunization strategies depending on an individual’s health status, boosting capacities, and the need for balanced humoral and Th1-directed cellular immune responses.MVA, a highly attenuated strain of vaccinia virus originating from growth selection on chicken embryo tissue cultures, shows a characteristic replication defect in mammalian cells but allows unimpaired production of heterologous proteins (19). At present, MVA serves as an advanced vaccine technology platform for developing new vector vaccines against infectious disease including emerging viruses and cancer (20). In response to the ongoing pandemic, the MVA vector vaccine platform allows rapid generation of experimental SARS-CoV-2–specific vaccines (21). Previous work from our laboratory addressed the development of an MVA candidate vaccine against MERS with immunizations in animal models demonstrating the safety, immunogenicity, and protective efficacy of MVA-induced MERS-CoV S-antigen–specific immunity (22–25). Clinical safety and immunogenicity of the MVA-MERS-S candidate vaccine was established in a first-in-human phase I clinical study under funding from the German Center for Infection Research (DZIF) (26).Here, we show that a recombinant MVA produces the full-length S protein of SARS-CoV-2 as ∼190- to 200-kDa N-glycosylated protein. Our studies confirmed cleavage of the mature full-length S protein into an amino-terminal domain (S1) and a ∼80- to 100-kDa carboxyl-terminal domain (S2) that is anchored to the membrane. When tested as a vaccine in BALB/c mice, recombinant MVA expressing the S protein induced SARS-CoV-2–specific T cells and antibodies, and robustly protected vaccinated animals against lung infection upon SARS-CoV-2 challenge.     

Mechanism of the formation of proton transfer pathways in photosynthetic reaction centers

In photosynthetic reaction centers from purple bacteria (PbRCs) from Rhodobacter sphaeroides, the secondary quinone Q_B accepts two electrons and two protons via electron-coupled proton transfer (PT). Here, we identify PT pathways that proceed toward the Q_B binding site, using a quantum mechanical/molecular mechanical approach. As the first electron is transferred to Q_B, the formation of the Grotthuss-like pre-PT H-bond network is observed along Asp-L213, Ser-L223, and the distal Q_B carbonyl O site. As the second electron is transferred, the formation of a low-barrier H-bond is observed between His-L190 at Fe and the proximal Q_B carbonyl O site, which facilitates the second PT. As Q_BH₂ leaves PbRC, a chain of water molecules connects protonated Glu-L212 and deprotonated His-L190 forms, which serves as a pathway for the His-L190 reprotonation. The findings of the second pathway, which does not involve Glu-L212, and the third pathway, which proceeds from Glu-L212 to His-L190, provide a mechanism for PT commonly used among PbRCs.

Purple bacterial photosynthetic reaction centers (PbRCs) have special pair bacteriochlorophylls (P_L/P_M), accessory bacteriochlorophylls (B_L/B_M), bacteriopheophytins (H_L/H_M), and ubiquinones (Q_A/Q_B) in the heterodimeric L/M protein subunit pair. P_L and P_M form the electronically coupled special pair [P_LP_M]. The electronic excitation of [P_LP_M] leads to the formation of the charge-separated state, [P_LP_M]^•+B_L^•−, and subsequent electron transfer occurs to Q_B via H_L and Q_A (1–3). Q_B accepts two electrons via Q_A and two protons via the proton transfer (PT) pathways, forming Q_BH₂, and leaves the PbRC.The first and second electron transfers from Q_A to Q_B occur with rates k_AB⁽¹⁾ (10⁴ s⁻¹) and k_AB⁽²⁾ (10³ s⁻¹), respectively (4). The first PT leads to the protonation of the distal and carbonyl O site of Q_B (with respect to the nonheme Fe). The proton donor of the distal Q_B O site is Ser-L223, for which the H-bond donor is Asp-L213. The Ser-L223 side chain, which donates an H-bond with Asp-L213 due to the highly polarized carboxyl O site, reorients toward the distal Q_B O site in response to Q_B^•− formation (5, 6). Notably, in photosystem II (PSII), D1-Ser264 and D1-His252, which correspond to Ser-L223 and Asp-L213 in the PbRC, respectively (7), serve as a PT pathway toward the distal Q_B O site (8). As D(L213)N (9) and S(L223)A (10, 11) mutations decrease the k_AB⁽²⁾ significantly, these residues are likely involved in the PT pathway toward the distal Q_B O site in the PbRC. Although Asp-H124, His-H126, and His-H128 are likely to form the entry point of the major PT pathway (12), it also seems plausible that the PT pathway is delocalized toward the protein bulk surface (13–15). The delocalization of the PT pathway is also observed in PSII; specifically, the PT pathway is branched as it proceeds from the oxygen-evolving complex via D1-Asp61 toward the protein bulk surface (16).Glu-L212 is the titratable residue that is nearest to and a candidate for the proton donor to the proximal Q_B O site. Indeed, the uptake of 0.3 to 0.8 H⁺ by Glu-L212 has been reported upon Q_B^•− formation (17–21). The protonation of Glu-L212 plays a role in electron transfer, increasing the redox potential E_m(Q_B) with respect to E_m(Q_A) (22). However, the PT pathway from Glu-L212 to Q_BH⁻ is unclear (4), because the crystal structure shows that Glu-L212 cannot form an H-bond with the proximal Q_B O site (Glu-L212…Q_B = 5.7 Å) (23). PT occurs most efficiently along H-bonds (24). Okamura et al. (4) proposed that the movement of Q_BH⁻ toward Glu-L212 and the formation of an H-bond might be required for PT. Alternatively, His-L190, which forms an H-bond with the proximal Q_B O site (His-L190…Q_B = 2.81 Å) (23), might serve as a proton donor for Q_BH⁻, as proposed by Wraight (25). However, Wraight also argued that the pK_a value for the deprotonation of singly protonated His-L190 might be too high even in the presence of the cationic nonheme Fe.Notably, the crystal structures of PSII and PbRC show large structural similarity (7). His-L190 is conserved as D1-His215 at the nonheme Fe complex in PSII (7). In PSII, D1-His215 can form a low-barrier H-bond with Q_BH⁻, which facilitates Q_BH₂ formation (8, 26, 27). Low-barrier H-bonds can form when the pK_a values of the H-bond donor and acceptor moieties are nearly equal (28, 29). The shape of the potential energy curve of a low-barrier H-bond is symmetric, while that of a standard H-bond is asymmetric because pK_a(donor) > pK_a(acceptor) (30) (Fig. 1). In addition, Glu-L212 in PbRCs is not conserved as a titratable residue in PSII. These findings for PSII might provide an opportunity to revisit the mechanism of PT toward the proximal Q_B O site in PbRCs. Here, we report how PT pathways form in response to electron transfer in the PbRC protein environment, by adopting a large-scale quantum mechanical/molecular mechanical (QM/MM) approach based on the PbRC crystal structure (23).Open in a separate windowFig. 1.Typical potential-energy profiles of H-bonds. (A) Standard H-bond. (B) Low-barrier H-bond (LBHB). In low-barrier H-bonds, the H-bond acceptor (O_acceptor) and donor (O_donor) cannot be distinguishable due to the same pK_a values.     

Molecular recognition of human ephrinB2 cell surface receptor by an emergent African henipavirus

The discovery of African henipaviruses (HNVs) related to pathogenic Hendra virus (HeV) and Nipah virus (NiV) from Southeast Asia and Australia presents an open-ended health risk. Cell receptor use by emerging African HNVs at the stage of host-cell entry is a key parameter when considering the potential for spillover and infection of human populations. The attachment glycoprotein from a Ghanaian bat isolate (GhV-G) exhibits <30% sequence identity with Asiatic NiV-G/HeV-G. Here, through functional and structural analysis of GhV-G, we show how this African HNV targets the same human cell-surface receptor (ephrinB2) as the Asiatic HNVs. We first characterized this virus−receptor interaction crystallographically. Compared with extant HNV-G–ephrinB2 structures, there was significant structural variation in the six-bladed β-propeller scaffold of the GhV-G receptor-binding domain, but not the Greek key fold of the bound ephrinB2. Analysis revealed a surprisingly conserved mode of ephrinB2 interaction that reflects an ongoing evolutionary constraint among geographically distal and phylogenetically divergent HNVs to maintain the functionality of ephrinB2 recognition during virus–host entry. Interestingly, unlike NiV-G/HeV-G, we could not detect binding of GhV-G to ephrinB3. Comparative structure–function analysis further revealed several distinguishing features of HNV-G function: a secondary ephrinB2 interaction site that contributes to more efficient ephrinB2-mediated entry in NiV-G relative to GhV-G and cognate residues at the very C terminus of GhV-G (absent in Asiatic HNV-Gs) that are vital for efficient receptor-induced fusion, but not receptor binding per se. These data provide molecular-level details for evaluating the likelihood of African HNVs to spill over into human populations.The emergence of negative-sense, single-stranded RNA viruses belonging to the genus Henipavirus, family Paramyxoviridae, epitomizes the increasing threat of zoonotic viruses to human health (1). Since the discovery of the highly pathogenic Nipah virus (NiV) and Hendra virus (HeV) in the 1990s, >20 henipaviruses (HNVs) have been detected throughout Africa, Asia, Australia, and Central America (2, 3). NiV is the prototypic member of this group and enzoonitically resides (>50% seroprevalence in some instances) in Old World fruit bat populations throughout Australasia (4). Zoonotic transfer of NiV to human populations from these natural reservoirs (5), sometimes through an animal intermediary such as pigs (6), leads to rapid-onset encephalitis with case-fatality rates >90% (1). Following zoonosis, some cases of person-to-person transmission have been observed (7, 8). Although bats are the predominant host reservoir, a putative henipa-like virus (HNLV), Mojiang virus (MojV), associated with severe pneumonia and three case fatalities, has also been isolated from rats in China (9). The extreme pathogenicity and potential for misuse has led to the designation of NiV and HeV as high-priority agents that require handling under biosafety-level-four conditions.The detection of 19 distinct clades of HNV in Africa (2, 10, 11) correlates with the broad geographic distribution and wide-ranging migrational patterns of the fruit bat host reservoir, Eidolon helvum (11). The remarkably high seroprevalence (∼40%) of HNV cross-reactive antibodies and the localization of many E. helvum communities near African towns and cities underscores the potential risk of spillover events into human populations (11, 12). Indeed, NiV cross-neutralizing antibodies have been detected in the sera of humans living in Cameroon (12). That these antibodies were found exclusively in individuals at high risk for zoonotic transmission, such as those that slaughter bats for bushmeat consumption and sale, suggests that such spillover events can occur. Whether or not African HNVs are as pathogenic to humans as NiV or HeV remains to be determined. Although it has also been suggested that these viruses may be the causative agent of misdiagnosed encephalitis-associated malaria (2, 13, 14), it is likely that the divergent clades of African HNVs are also diverse in their pathogenic potential.HNV entry into a host cell is a pH-independent process orchestrated by two membrane-anchored glycoproteins, HNV-G and -F (15). These viral glycoproteins interdependently facilitate cellular attachment and fusion, whereby receptor recognition by HNV-G at the cell surface triggers rearrangements in the HNV-F fusion glycoprotein (16). HNV-G is an oligomeric membrane protein, consisting of a short N-terminal cytoplasmic tail, a transmembrane region, an oligomerization-inducing stalk region, and a receptor-binding C-terminal six-bladed β-propeller. Identification of the ubiquitously expressed cell-surface signaling glycoproteins, ephrinB2 and ephrinB3, as functional receptors used during viral attachment by NiV and HeV has been key to understanding the broad tissue tropism of these viruses (16–21). Structural investigations of these ephrins in complex with NiV- and HeV-G have revealed the molecular determinants for host-cell recognition and zoonosis (22–26).In contrast to the wealth of available NiV and HeV genome sequences, only one African HNV has been sequenced to entirety, but it has not yet been isolated (2, 27). The sequence of this putative HNV (Gh-M74a; termed here as GhV) was derived from a bat in Ghana and is genetically distinct from Asiatic HeV and NiV (2). In contrast to NiV- and HeV-G, which are genetically quite similar (80% sequence identity), the putative GhV attachment glycoprotein from this virus, GhV-G, exhibits very limited sequence identity (<30%) with its Asiatic counterparts. Despite this genetic distance, ephrinB2 has been suggested as a functional interaction partner for this virus (27, 28). The conserved use of this receptor by GhV-G and Asiatic HNVs supports a general mechanism for HNV zoonosis in human populations. The likelihood of zoonotic transmission and the pathogenicity of such zoonotic viruses may depend, at least in part, on what adaptations are necessary for efficient use of the host receptor(s).Here, we determined the molecular basis for the interaction between GhV-G and ephrinB2 by X-ray crystallographic analysis. Despite the varied architecture of the henipaviral β-propeller scaffold between GhV-G and Asiatic HNV-Gs, we observed a highly conserved mode of ephrinB2 engagement. However, we also identify a secondary ephrinB2 interaction site that contributes to the more efficient receptor-mediated entry exhibited by NiV-G relative to GhV-G. These data verify a conserved HNV cell-attachment strategy for African and pathogenic Asiatic HNVs and establish a mechanism by which humans may be susceptible to African HNV infection.     

Heterodimeric coiled-coil interactions of human GABAB receptor

Metabotropic GABA_B receptor is a G protein-coupled receptor that mediates inhibitory neurotransmission in the CNS. It functions as an obligatory heterodimer of GABA_B receptor 1 (GBR1) and GABA_B receptor 2 (GBR2) subunits. The association between GBR1 and GBR2 masks an endoplasmic reticulum (ER) retention signal in the cytoplasmic region of GBR1 and facilitates cell surface expression of both subunits. Here, we present, to our knowledge, the first crystal structure of an intracellular coiled-coil heterodimer of human GABA_B receptor. We found that polar interactions buried within the hydrophobic core determine the specificity of heterodimer pairing. Disruption of the hydrophobic coiled-coil interface with single mutations in either subunit impairs surface expression of GBR1, confirming that the coiled-coil interaction is required to inactivate the adjacent ER retention signal of GBR1. The coiled-coil assembly buries an internalization motif of GBR1 at the heterodimer interface. The ER retention signal of GBR1 is not part of the core coiled-coil structure, suggesting that it is sterically shielded by GBR2 upon heterodimer formation.The major inhibitory neurotransmitter in the CNS is GABA. Metabotropic GABA_B receptor is a G protein-coupled receptor (GPCR) that mediates slow synaptic inhibition (1, 2). It constitutes an important drug target for many neurological disorders, including epilepsy, spasticity, anxiety, and nociception (1, 2).Formation of a functional GABA_B receptor requires the heterodimeric assembly of GABA_B receptor 1 (GBR1) and GABA_B receptor 2 (GBR2) subunits (3–7). Both consist of an N-terminal extracellular domain, a seven-helix transmembrane domain, and a C-terminal intracellular domain. The intracellular domain of each subunit contains a stretch of coiled-coil sequence, and interaction between the coiled-coil helices is partly responsible for GABA_B receptor heterodimerization (5, 8).The intracellular region of GABA_B receptor hosts elements that control receptor trafficking (9). Specifically, GBR1 has a di-leucine internalization signal (EKSRLL) (9) and an endoplasmic reticulum (ER) retention signal (RSRR) (9–11) located within or near its coiled-coil domain (9). GBR1 is trapped within the ER when expressed alone (12) but can reach the cell surface upon association with GBR2 (9, 11). Mutation or removal of the ER retention signal in GBR1 results in plasma membrane expression of GBR1 (9–11). Furthermore, interaction between the coiled-coil domains of GBR1 and GBR2 masks this ER retention signal to facilitate the cell surface expression of both subunits (9–11). Although mutation of the di-leucine motif itself is not sufficient to release GBR1 from intracellular retention, it enhances cell surface expression of various GBR1 mutants that lack the ER retention signal (9).The coiled-coil domain of GBR1 associates with a number of intracellular proteins involved in trafficking, including the coat protein complex I (COPI) (13), the scaffolding protein 14-3-3 (13, 14), the GPCR interacting scaffolding protein GISP (15), and the guanidine exchange factor msec7-1 (16). In particular, COPI specifically recognizes the ER retention signal sequence of GBR1 and is involved in the intracellular retention of GBR1 (13). The msec7-1 protein increases the cell surface expression of GABA_B receptor by binding to the di-leucine internalization motif (16).Despite its important role in GABA_B receptor assembly and trafficking, the atomic details of the coiled-coil interaction between subunits are not known. In this study, we present the crystal structure of a GBR1/GBR2 coiled-coil heterodimer and identify specific contacts at the heterodimer interface that control the surface expression of GBR1.     

Immunogenicity of rVSVΔG-ZEBOV-GP Ebola vaccination in exposed and potentially exposed persons in the Democratic Republic of the Congo

Despite more than 300,000 rVSVΔG-ZEBOV-glycoprotein (GP) vaccine doses having been administered during Ebola virus disease (EVD) outbreaks in the Democratic Republic of the Congo (DRC) between 2018 and 2020, seroepidemiologic studies of vaccinated Congolese populations are lacking. This study examines the antibody response at 21 d and 6 mo postvaccination after single-dose rVSVΔG-ZEBOV-GP vaccination among EVD-exposed and potentially exposed populations in the DRC. We conducted a longitudinal cohort study of 608 rVSVΔG-ZEBOV-GP–vaccinated individuals during an EVD outbreak in North Kivu Province, DRC. Participants provided questionnaires and blood samples at three study visits (day 0, visit 1; day 21, visit 2; and month 6, visit 3). Anti-GP immunoglobulin G (IgG) antibody titers were measured in serum by the Filovirus Animal Nonclinical Group anti-Ebola virus GP IgG enzyme-linked immunosorbent assay. Antibody response was defined as an antibody titer that had increased fourfold from visit 1 to visit 2 and was above four times the lower limit of quantification at visit 2; antibody persistence was defined as a similar increase from visit 1 to visit 3. We then examined demographics for associations with follow-up antibody titers using generalized linear mixed models. A majority of the sample, 87.2%, had an antibody response at visit 2, and 95.6% demonstrated antibody persistence at visit 3. Being female and of young age was predictive of a higher antibody titer postvaccination. Antibody response and persistence after Ebola vaccination was robust in this cohort, confirming findings from outside of the DRC.

Since the rVSVΔG-ZEBOV-glycoprotein (GP) vaccine completed clinical trials in West Africa, over 300,000 doses of the vaccine have been deployed in response to the multiple Ebola virus disease (EVD) outbreaks in the Democratic Republic of the Congo (DRC). While initially deployed under a “compassionate use/expanded access” protocol (1, 2), as of December 19, 2019, the vaccine was officially licensed by both the American (Food and Drug Administration, FDA) and European (European Medicines Agency) regulatory agencies (3, 4). Wide use of this vaccine was supported by evidence gathered in clinical trials and other studies, including those postlicensure conducted in North America and West Africa, which demonstrated short-term vaccine efficacy (5–16). In addition to short-term protection, clinical trials and other studies have provided evidence of Ebolavirus Zaire (EBOV)–specific antibody persistence up to 2 y postvaccination, suggesting that the vaccine may continue to offer protective immunity over time (5, 7, 8, 14, 15). While promising, observations of successful rVSVΔG-ZEBOV-GP vaccine performance in outbreak settings have mostly come from studies conducted at the end of the 2014 to 2016 West African EVD outbreak (7, 13, 14, 17). Such studies in the DRC are lacking.Furthermore, recent evidence of breakthrough infections within the DRC has highlighted the need for DRC-specific vaccine research, including magnitude and durability of serological response after rVSVΔG-ZEBOV-GP vaccination in Congolese populations. In April 2019, the World Health Organization (WHO) released a preliminary report of rVSVΔG-ZEBOV-GP efficacy in the 2018 to 2020 Beni outbreak. Among 93,965 people at risk who were vaccinated, there were 15 confirmed EVD cases with onset of symptoms 10 d or more postvaccination (18). Another report describes an individual who presented with EVD 6 mo after vaccination, initiating a chain of transmission resulting in 91 subsequent infections (19), prompting questions around the duration of protection. These recent events highlight both the consequences of breakthrough infections and the possibility of waning immunity postvaccination.When considering rVSVΔG-ZEBOV-GP performance in the DRC, there are several factors that may impact the effect of vaccination in Congolese populations. First, an increase in vaccination dose could have resulted in increased immunogenicity in the DRC. Vaccination deployment during the EVD outbreak of 2018 initially included double the plaque-forming units (PFUs) in the vaccine dosage compared to what was used in West Africa (20 million PFU/mL versus 10 million PFU/mL, respectively) (20). As previous studies have identified varying immunogenicity after different vaccine doses in different locations, this variation in vaccine dose could lead to differing antibody responses from previously studied cohorts (15). Second, an important component of the vaccine deployment was the requirement for an ultracold chain (storage of vaccine at −70 °C), which poses extreme logistical challenges in resource-constrained environments. Despite considerable efforts to avoid cold chain failures, it is plausible that fluctuations could have occurred and caused changes in vaccine effectiveness (21). Third, populations in this region may have a baseline level of filovirus seroreactivity that may enable a more robust response to Ebola vaccination (15, 22). Previous serologic studies in the DRC have indicated that Congolese populations may not be naive to filovirus exposures, with individuals presenting evidence of robust antibody responses to various filoviruses in the absence of a known history of EVD (23–26). While there had never been a reported EVD outbreak in North Kivu prior to 2018, this province is known for highly mobile populations; proximity to large forested areas, which may harbor filovirus or filovirus-like pathogens; and access to cross-border populations, including those from Uganda, which have had previous filovirus outbreaks (27–29). Finally, the underlying prevalence of immunosuppressive conditions, such as HIV infection and poor nutritional status, could hinder vaccine immunogenicity in Congolese populations (30).Given the DRC’s unique landscape, which includes evidence of breakthrough infections, a more thorough region-specific understanding of serologic response to Ebola vaccination is needed. Various factors such as vaccine dose, storage conditions, current infections, and previous exposure may alter the magnitude and durability of antibody response after vaccination in Congolese populations (7, 31, 32). To better understand rVSVΔG-ZEBOV-GP performance in the DRC, we conducted a seroepidemiologic study of postvaccination antibody persistence in Congolese populations, who may have meaningfully different experiences than those in West Africa. Here, we provide a preliminary report of antibody response and persistence, along with potential predictors, after single-dose rVSVΔG-ZEBOV-GP vaccination among EVD-exposed and potentially exposed populations in the DRC.     

A highly efficacious live attenuated mumps virus–based SARS-CoV-2 vaccine candidate expressing a six-proline stabilized prefusion spike

With the rapid increase in SARS-CoV-2 cases in children, a safe and effective vaccine for this population is urgently needed. The MMR (measles/mumps/rubella) vaccine has been one of the safest and most effective human vaccines used in infants and children since the 1960s. Here, we developed live attenuated recombinant mumps virus (rMuV)–based SARS-CoV-2 vaccine candidates using the MuV Jeryl Lynn (JL2) vaccine strain backbone. The soluble prefusion SARS-CoV-2 spike protein (preS) gene, stablized by two prolines (preS-2P) or six prolines (preS-6P), was inserted into the MuV genome at the P–M or F–SH gene junctions in the MuV genome. preS-6P was more efficiently expressed than preS-2P, and preS-6P expression from the P–M gene junction was more efficient than from the F–SH gene junction. In mice, the rMuV-preS-6P vaccine was more immunogenic than the rMuV-preS-2P vaccine, eliciting stronger neutralizing antibodies and mucosal immunity. Sera raised in response to the rMuV-preS-6P vaccine neutralized SARS-CoV-2 variants of concern, including the Delta variant equivalently. Intranasal and/or subcutaneous immunization of IFNAR1^−/− mice and golden Syrian hamsters with the rMuV-preS-6P vaccine induced high levels of neutralizing antibodies, mucosal immunoglobulin A antibody, and T cell immune responses, and were completely protected from challenge by both SARS-CoV-2 USA-WA1/2020 and Delta variants. Therefore, rMuV-preS-6P is a highly promising COVID-19 vaccine candidate, warranting further development as a tetravalent MMR vaccine, which may include protection against SARS-CoV-2.

The current pandemic of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused tremendous damage to all aspects of our society (1–3). As of 1 June 2022, nearly 528 million cases have been reported worldwide, with nearly 6.3 million deaths (∼1.20% mortality). Symptoms of SARS-CoV-2 infection are primarily respiratory although increasing numbers of other syndromes such as cognitive deficits are being reported. As of June 2022, several SARS-CoV-2 vaccines based on messenger RNA (mRNA), inactivated virus, and adenovirus vectors (Ad26.COV2.S and ChAdOx1) have been approved for vaccination in humans over the age of 5 (4). These vaccines are highly efficacious, reaching 70 to 95% effectiveness against SARS-CoV-2 infection (4).Despite the high success of the current SARS-CoV-2 vaccines, there are several limitations. Protection provided by current vaccines begins to decline after 3 mo (5), which has required a third or fourth dose to boost the immune response. Current vaccines are less effective against recently emergent SARS-CoV-2 variants of concern (VoCs) (6–9). More and more evidence has shown that vaccine-induced neutralizing antibodies were significantly weakened or insufficient to neutralize VoCs such as the Delta variant (7–9), which spreads much faster and causes more severe illness than the earlier strains. In addition, the current vaccines neutralize the most recently emerged variant, Omicron, ∼40 times less efficiently compared with early SARS-CoV-2 isolates (10, 11). The mRNA vaccines are expensive to produce, hard to transport internationally, and difficult to store in many countries because of the requirement for expensive −80 °C freezers.A safe and efficacious pediatric SARS-CoV-2 vaccine is needed to halt the current pandemic. Pfizer’s mRNA vaccine is 90.7% effective in preventing COVID-19 symptoms in children 5 to 11 y old (12, 13). On 17 June 2022, Food and Drug Administration (FDA) authorized emergency use of the Moderna and Pfizer mRNA vaccines for children down to 6 mo of age. As of 23 June 2022, a total of 13.7 million COVID-19 cases have occurred in children, representing 18.8% of the total COVID-19 cases in the United States. Notably, COVID-19 cases in children have increased significantly after the reopening of schools. Therefore, development of other vaccine platforms and strategies to enhance durability, reduce cost, and enhance stability are essential for terminating the pandemic.Historically, the MMR (measles/mumps/rubella) vaccine has been one of the safest and most effective human vaccines ever developed (14–16). The application in children started in the 1960s and provides long-lasting protection against these three viruses (14, 16). Among the three MMR components, measles virus (MeV) and mumps virus (MuV) are nonsegmented negative-sense (NNS) RNA viruses, belonging to the family Paramyxoviridae in the order Mononegavirales. The MuV genome is 15,384 nt in length, and it encodes seven structural proteins arranged in the order 3′-leader-N-P-M-F-SH-HN-L-trailer-5′ (17). The limited number of discrete genes of the NNS RNA genome and the intergenic regions available for inserting additional genes facilitates the development of live vectored vaccines. MuV is an excellent viral vector for delivery of vaccines against other highly pathogenic viruses, primarily because of its high safety and efficacy, well-established good manufacturing practices, induction of long-lived immunity, and the potential for the development of a quadrivalent vaccine against four major pediatric diseases (18, 19).In this study, we developed a suite of safe and highly efficacious recombinant MuV (rMuV)–based SARS-CoV-2 vaccine candidates expressing a stabilized prefusion spike with two prolines (preS-2P) or six prolines (preS-6P) at different gene junctions in the MuV genome. Among them, the rMuV-based preS-6P vaccine induces a broad neutralizing antibody against VoCs and T cell immunity, and provides complete protection against SARS-CoV-2 WA1 and the Delta variant challenge in animal models.     

Efficacy and breadth of adjuvanted SARS-CoV-2 receptor-binding domain nanoparticle vaccine in macaques

Emergence of novel variants of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) underscores the need for next-generation vaccines able to elicit broad and durable immunity. Here we report the evaluation of a ferritin nanoparticle vaccine displaying the receptor-binding domain of the SARS-CoV-2 spike protein (RFN) adjuvanted with Army Liposomal Formulation QS-21 (ALFQ). RFN vaccination of macaques using a two-dose regimen resulted in robust, predominantly Th1 CD4+ T cell responses and reciprocal peak mean serum neutralizing antibody titers of 14,000 to 21,000. Rapid control of viral replication was achieved in the upper and lower airways of animals after high-dose SARS-CoV-2 respiratory challenge, with undetectable replication within 4 d in seven of eight animals receiving 50 µg of RFN. Cross-neutralization activity against SARS-CoV-2 variant B.1.351 decreased only approximately twofold relative to WA1/2020. In addition, neutralizing, effector antibody and cellular responses targeted the heterotypic SARS-CoV-1, highlighting the broad immunogenicity of RFN-ALFQ for SARS-CoV−like Sarbecovirus vaccine development.

The COVID-19 pandemic, precipitated by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), continues to threaten global public health and economies. Threats of future outbreaks also loom, as evidenced by three emergent SARS-like diseases caused by zoonotic Betacoronaviruses in the last two decades. While several emergency use authorized (EUA) vaccines currently in use are expected to curb both disease and transmission of SARS-CoV-2 (1–6), the emergence of circulating variants of concern (VOCs) less sensitive to vaccine-elicited immunity has raised concerns for sustained vaccine efficacy (7). Logistical challenges of vaccine production, distribution, storage, and access for these vaccines must be resolved to achieve resolution to the pandemic (8, 9). The rapid and unparalleled spread of SARS-CoV-2 has driven an urgent need to deploy scalable vaccine platforms to combat the ongoing pandemic and mitigate future outbreaks.Current vaccines primarily focus the immune response on the spike glycoprotein (S) as it mediates host cell viral fusion and entry. The receptor-binding domain (RBD) of S engages the primary host cell receptor, angiotensin-converting enzyme 2 (ACE2), for both SARS-CoV-2 and SARS-CoV-1, making RBD a promising domain for vaccine-elicited immune focus (10–12). Moreover, many of the potently neutralizing monoclonal antibodies isolated against SARS-CoV-2 target the RBD (13, 14). Vaccination of nonhuman primates (NHPs) with RBD-encoding RNA or DNA protects against respiratory tract challenge, indicating that immune responses to the RBD can prevent viral replication (15, 16). RBD vaccination also elicits cross-reactive responses to circulating SARS-CoV-2 VOCs in both animals and humans (17, 18), with decrements against the B.1.351 variant similar to that seen with S immunogens (19). The breadth of RBD immunogenicity is further supported by the ability of RBD-specific monoclonal antibodies isolated from SARS-CoV-1 convalescent individuals to cross-neutralize SARS-CoV-2 (20, 21). These findings suggest potential for RBD-based vaccines being efficacious against SARS-CoV-2 variants and other related coronavirus species.Approaches to improve immunogenicity of S or RBD protein vaccines include optimizing antigen presentation and coformulating with adjuvants to enhance the protective immunity. One common approach to enhance the induction of adaptive immune responses is the multimeric presentation of antigen, for example, on the surface of nanoparticles or virus-like particles (22). Presenting RBD in ordered, multivalent arrays on the surface of self-assembling protein nanoparticles is immunogenic and efficacious in animals (23–28), with improved immunogenicity relative to monomeric soluble RBD and cross-reactive responses to variants (17, 24, 26). However, it is unknown whether RBD nanoparticle vaccines protect against infection in primates, which have become a standard model for benchmarking performance of vaccine candidates by virological and immunologic endpoints. Liposomal adjuvants incorporating QS-21, such as that used in the efficacious varicella zoster vaccine, SHINGRIX, may augment protective immunity to SARS-CoV-2 vaccines. Such adjuvants have superior humoral and cellular immunogenicity relative to conventional adjuvants (29, 30).Here, we evaluate the use of a ferritin nanoparticle vaccine presenting the SARS-CoV-2 RBD (RFN) adjuvanted with the Army Liposomal Formulation QS-21 (ALFQ) (31). Both ferritin nanoparticles and ALFQ have been evaluated for vaccination against multiple pathogens in humans in phase 1 clinical trials (32–34). We demonstrate, in an NHP model, that immunization with RFN induces robust and broad antibody and T cell responses, as well as protection against viral replication and lung pathology following high-dose respiratory tract challenge with SARS-CoV-2.     

Phospholipase A2 inhibitor and LY6/PLAUR domain-containing protein PINLYP regulates type I interferon innate immunity

Type I interferons (IFNs) are the first frontline of the host innate immune response against invading pathogens. Herein, we characterized an unknown protein encoded by phospholipase A2 inhibitor and LY6/PLAUR domain-containing (PINLYP) gene that interacted with TBK1 and induced type I IFN in a TBK1- and IRF3-dependent manner. Loss of PINLYP impaired the activation of IRF3 and production of IFN-β induced by DNA virus, RNA virus, and various Toll-like receptor ligands in multiple cell types. Because PINLYP deficiency in mice engendered an early embryonic lethality in mice, we generated a conditional mouse in which PINLYP was depleted in dendritic cells. Mice lacking PINLYP in dendritic cells were defective in type I IFN induction and more susceptible to lethal virus infection. Thus, PINLYP is a positive regulator of type I IFN innate immunity and important for effective host defense against viral infection.

Interferon (IFN)-mediated antiviral responses serve as the first line of the host innate immune defense against viral infection. IFNs are divided into three families based on sequence homology: type I, type II, and type III (1, 2). The type I IFN family encodes 13 subtypes of IFN-α in humans (14 in mice), a single IFN-β subtype, and several poorly defined subtypes (3, 4). Type I IFNs were originally identified based on their ability to interfere with viral replication, restrain virus dissemination, and activate adaptive immune responses (5–7). They can be induced in most cell types by microbial pathogen-associated and damage-associated molecular patterns recognized by pattern recognition receptors (PRRs) (3). By inducing the expression of IFN-stimulated genes (ISGs), type I IFNs elicit antiviral innate immunity and mediate adaptive immune responses (8, 9).The induction of antiviral type I IFN response is elicited in response to the stimulation of PRRs that detect pathogen-associated molecular patterns, such as viral nucleic acids, viral replicative intermediates, and surface glycoproteins (10, 11). There are four major subfamilies of PRRs: the Toll-like receptors (TLRs), nucleotide-binding oligomerization domain/leucine-rich repeat-containing receptors, RIG-1-like receptors (RLRs), and the C-type lectin receptors, which are located at the cell surface, in the cytosol, or endosomal compartments (11–14). Among the TLR family members, TLR3, TLR7, TLR8, and TLR9 are involved in the recognition of viral nucleotides. Viral DNA enriched in CpG-DNA motifs is recognized by TLR9, single-stranded RNA is recognized by TLR7 and TLR8, and double-stranded RNA and its synthetic analog polyinosinic-polycytidylic acid (poly I:C) are recognized by TLR3 (15, 16). Some viral envelope proteins can be recognized by TLR4 or TLR2 (16, 17).Following viral infection, cytosolic DNA can be sensed by cyclic guanosine monophosphate (GMP)–adenosine monophosphate (AMP) synthase (cGAS) that induces the production of cyclic GMP-AMP (cGAMP) (18, 19). cGAMP functions as a second messenger that binds and activates the endoplasmic reticulum (ER) adaptor STING (19–22). Translocation of activated STING from the ER to the Golgi apparatus leads to the activation of kinase TBK1, which subsequently phosphorylates IRF3 and triggers the production of type I IFN (22–24). Cytosolic RNA can be recognized by the RLRs like RIG-1 and MDA5, which signal via mitochondrial antiviral signaling protein (MAVS; also known as CARDIF, IPS1, and VISA) and subsequently activate TBK1 and IRF3–IRF7, leading to the induction of type I IFNs and other antiviral genes (25–27).The lymphocyte antigen-6 (Ly6)/urokinase-type plasminogen activator receptor (uPAR) superfamily is characterized by the LU domain and a domain containing 10 cysteines that form distinct disulfide bridges, which create the three-fingered structural motif. The Ly6/uPAR family members regulate a wide range of functions in various cell types (28). Here, we uncovered the previously uncharacterized role of the Ly6/uPAR family member PINLYP in the induction of type I IFNs in response to DNA virus, RNA virus, and other TLR ligands. This study further defined the pivotal function of PINLYP in the effective host defense against virus infection.     

A single intranasal dose of a live-attenuated parainfluenza virus-vectored SARS-CoV-2 vaccine is protective in hamsters

Single-dose vaccines with the ability to restrict SARS-CoV-2 replication in the respiratory tract are needed for all age groups, aiding efforts toward control of COVID-19. We developed a live intranasal vector vaccine for infants and children against COVID-19 based on replication-competent chimeric bovine/human parainfluenza virus type 3 (B/HPIV3) that express the native (S) or prefusion-stabilized (S-2P) SARS-CoV-2 S spike protein, the major protective and neutralization antigen of SARS-CoV-2. B/HPIV3/S and B/HPIV3/S-2P replicated as efficiently as B/HPIV3 in vitro and stably expressed SARS-CoV-2 S. Prefusion stabilization increased S expression by B/HPIV3 in vitro. In hamsters, a single intranasal dose of B/HPIV3/S-2P induced significantly higher titers compared to B/HPIV3/S of serum SARS-CoV-2–neutralizing antibodies (12-fold higher), serum IgA and IgG to SARS-CoV-2 S protein (5-fold and 13-fold), and IgG to the receptor binding domain (10-fold). Antibodies exhibited broad neutralizing activity against SARS-CoV-2 of lineages A, B.1.1.7, and B.1.351. Four weeks after immunization, hamsters were challenged intranasally with 10^4.5 50% tissue-culture infectious-dose (TCID₅₀) of SARS-CoV-2. In B/HPIV3 empty vector-immunized hamsters, SARS-CoV-2 replicated to mean titers of 10^6.6 TCID₅₀/g in lungs and 10⁷ TCID₅₀/g in nasal tissues and induced moderate weight loss. In B/HPIV3/S-immunized hamsters, SARS-CoV-2 challenge virus was reduced 20-fold in nasal tissues and undetectable in lungs. In B/HPIV3/S-2P–immunized hamsters, infectious challenge virus was undetectable in nasal tissues and lungs; B/HPIV3/S and B/HPIV3/S-2P completely protected against weight loss after SARS-CoV-2 challenge. B/HPIV3/S-2P is a promising vaccine candidate to protect infants and young children against HPIV3 and SARS-CoV-2.

The betacoronavirus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in 2019 and rapidly spread globally (1). In the first year of the pandemic, over 105 million infections and 2.3 million deaths have been reported worldwide, including over 27 million cases and 500,000 deaths in the United States (https://covid19.who.int/). Vaccines are being rapidly deployed in a race to control ongoing infections and the emergence of variants of concern (2), with increased virulence and altered antigenicity.SARS-CoV-2 infects and spreads primarily via the respiratory route (3, 4), and mucosal surfaces of the respiratory tract represent the primary site of infection. COVID-19, the disease caused by SARS-CoV-2, is characterized by upper and lower respiratory tract symptoms, fever, chills, body aches, and fatigue, and in some cases gastrointestinal and other symptoms with involvement of additional tissues (5, 6).SARS-CoV-2 infection is initiated by the spike (S) surface glycoprotein, the main target for SARS-CoV-2–neutralizing antibodies. The S protein is a trimeric class I fusion glycoprotein. Each protomer consists of two functionally distinct subunits, S1 and S2, linked by a furin cleavage site; S2 contains an additional proteolytic cleavage site S2′. S2/S2′ cleavage is mediated by the transmembrane protease serine 2 (TMPRSS2) (1, 7–9). The S1 subunit contains the receptor-binding domain (RBD). The S2 subunit contains the membrane fusion machinery, including the hydrophobic fusion peptide and α-helical heptad repeats (7, 9).Binding of the S RBD to its receptor, human angiotensin converting enzyme 2, triggers a change, from the closed and metastable prefusion conformation to the open and stable postfusion form that drives membrane fusion enabling viral entry (1). Stabilization of the S protein in its native prefusion state should preserve antibody epitopes, including immunodominant sites of the RBD, required to elicit high-quality neutralizing antibody responses (9–13). Thus, a prefusion-stabilized version of the S protein is the optimal vaccine immunogen (13–15).Vaccines for SARS-CoV-2 are available, but currently are limited to individuals 12 y of age or older. They are administered intramuscularly, which does not directly stimulate mucosal immunity in the respiratory tract, the primary site of SARS-CoV-2 infection and shedding. While the major burden of COVID-19 disease is in adults, infection and disease also occurs in infants and young children, contributing to viral transmission. Therefore, the development of safe and effective pediatric COVID-19 vaccines is critical for worldwide control of COVID-19. The ideal vaccine should be effective at a single dose, inducing durable and broad systemic immunity, as well as T and B cell respiratory mucosal immunity that completely blocks SARS-CoV-2 infection and transmission.Here we describe a vectored SARS-CoV-2 vaccine candidate for intranasal immunization of infants and young children. The vaccine is based on an attenuated, replication-competent parainfluenza virus type 3 (PIV3) vector called B/HPIV3 (16) expressing the SARS-CoV-2 S protein. B/HPIV3 consists of bovine PIV3 (BPIV3) strain Kansas in which the BPIV3 hemagglutinin-neuraminidase (HN) and fusion (F) glycoproteins (the two PIV3 neutralization antigens) have been replaced by those of human PIV3 strain JS (16, 17). The BPIV3 backbone provides host range restriction of replication in humans, serving as the basis for strong and stable attenuation (17, 18). B/HPIV3 originally was developed as a live vaccine candidate against HPIV3, and was well-tolerated in young children (17). Moreover, B/HPIV3 has been used to express the F glycoprotein of another human respiratory pathogen, human respiratory syncytial virus (HRSV), as a bivalent HPIV3/HRSV vaccine candidate. This vaccine candidate was well-tolerated in children >2 mo of age (18) (Clinicaltrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00686075","term_id":"NCT00686075"}}NCT00686075), and optimized versions are in further clinical development as pediatric vaccines (19, 20). In the present study, we used B/HPIV3 to express wild-type (S) or prefusion-stabilized (S-2P) versions of the SARS-CoV-2 S protein, creating the vaccine candidates B/HPIV3/S and B/HPIV3/S-2P. These were evaluated in vitro and in hamsters as live-attenuated SARS-CoV-2 intranasal vaccine candidates.     

Competitive exclusion by autologous antibodies can prevent broad HIV-1 antibodies from arising

The past decade has seen the discovery of numerous broad and potent monoclonal antibodies against HIV type 1 (HIV-1). Eliciting these antibodies via vaccination appears to be remarkably difficult, not least because they arise late in infection and are highly mutated relative to germline antibody sequences. Here, using a computational model, we show that broad antibodies could in fact emerge earlier and be less mutated, but that they may be prevented from doing so as a result of competitive exclusion by the autologous antibody response. We further find that this competitive exclusion is weaker in infections founded by multiple distinct strains, with broadly neutralizing antibodies emerging earlier than in infections founded by a single strain. Our computational model simulates coevolving multitype virus and antibody populations. Broadly neutralizing antibodies may therefore be easier for the adaptive immune system to generate than previously thought. If less mutated broad antibodies exist, it may be possible to elicit them with a vaccine containing a mixture of diverse virus strains.A major challenge to developing an HIV type 1 (HIV-1) vaccine is the difficulty of eliciting an immune response that can neutralize the large diversity of viral strains circulating in the human population. The discovery of broad and potent neutralizing monoclonal antibodies against HIV-1 in humans has renewed hopes for an effective HIV vaccine (1–4). Termed broadly neutralizing antibodies (BnAbs), they are found some years into chronic infection and typically neutralize a large fraction of a diverse panel of HIV-1 strains in vitro (5). The B-cell lineages that produce these antibodies constitute on the order of 0.1% of the host’s B-cell population (6–8), and their presence does not seem to reduce viral load or decrease transmission in the individuals in which they are found (9). However, passive immunization with BnAbs has been shown to block infections of simian immunodeficiency virus (SIV) and simian-HIV-1 in nonhuman primates (10; see ref. 11 for a review). In addition, passive infusion with BnAbs leads to rapid, although transient, suppression of viral load in macaques chronically infected with simian-HIV-1 (12, 13). These studies suggest that if a vaccine can elicit BnAbs to a sufficiently high level before exposure to HIV-1, the transmitted virus may be neutralized before it becomes established in the host.Attempts to elicit BnAbs have so far been unsuccessful (1–3, 14–16). One reason is the antibodies are highly mutated relative to their germline ancestor (Fig. 1A), and it is a challenge to develop a vaccine that can induce antibodies mutated to such an extent (3). However, several recent lines of research indicate that high levels of mutation may not be necessary for breadth. First, the extent of mutation is correlated with the length of infection (Fig. 1B), and as all broad antibodies to date have been found late into chronic infection, the high level of mutation may be a result of the passage of time. This is supported by a study that found high levels of somatic mutation in antibodies associated with chronic infections, regardless of whether the antibodies were broadly neutralizing (17). Second, B cells with long heavy chain complementarity determining region 3 loops, a property associated with neutralizing breadth, have been found to exist in the naive B-cell population (18). This suggests the potential for broad HIV-1 antibodies to be generated with few somatic mutations. Last, cross-clade neutralizing responses have been found in HIV-1-infected infants as early as 11–15 mo postinfection (19). That an infant immune response is capable of producing antibodies de novo that neutralize HIV-1 strains distinct from the founder strain within a relatively short period further supports the idea that B cells need not undergo substantial amounts of affinity maturation and mutation to produce BnAbs.Open in a separate windowFig. 1.Levels of somatic mutation in HIV-1 antibodies. (A) Number of mutations in the heavy chain variable gene (V_H) in antibodies developed against pandemic H1N1 influenza (pH1N1; blue, data from ref. 40) compared with BnAbs against HIV-1 (green, data from ref. 4). (B) The number of mutations on V_H for antibodies in the CH103 BnAb lineage in a patient over three years (data from ref. 8). The length of V_H is ∼300 base pairs, and the number of mutations is relative to the closest germline gene, as determined by the international ImMunoGeneTics (IMGT) information system alignments (41).Here we demonstrate with simulations of coevolving virus and antibody populations a possible reason why less-mutated broad antibodies have not been observed and suggest a vaccine strategy to elicit them. We find that broad antibodies have a low chance of emerging early in infection, even if we assume they require only moderately more mutations than specific antibodies. This is because antibodies that target the founder strain competitively exclude broad antibodies in the initial stages of chronic infection. This competition is strongest when there is a single founder strain, as is often the case in natural HIV-1 infections (20, 21). When we remove this competition between broad and specific antibodies from our model, we find that broad antibodies arise earlier. We also demonstrate that this competitive exclusion is reduced in infections initialized with multiple strains.     

Herpesvirus-mediated stabilization of ICP0 expression neutralizes restriction by TRIM23

Herpes simplex virus (HSV) infection relies on immediate early proteins that initiate viral replication. Among them, ICP0 is known, for many years, to facilitate the onset of viral gene expression and reactivation from latency. However, how ICP0 itself is regulated remains elusive. Through genetic analyses, we identify that the viral γ₁34.5 protein, an HSV virulence factor, interacts with and prevents ICP0 from proteasomal degradation. Furthermore, we show that the host E3 ligase TRIM23, recently shown to restrict the replication of HSV-1 (and certain other viruses) by inducing autophagy, triggers the proteasomal degradation of ICP0 via K11- and K48-linked ubiquitination. Functional analyses reveal that the γ₁34.5 protein binds to and inactivates TRIM23 through blockade of K27-linked TRIM23 autoubiquitination. Deletion of γ₁34.5 or ICP0 in a recombinant HSV-1 impairs viral replication, whereas ablation of TRIM23 markedly rescues viral growth. Herein, we show that TRIM23, apart from its role in autophagy-mediated HSV-1 restriction, down-regulates ICP0, whereas viral γ₁34.5 functions to disable TRIM23. Together, these results demonstrate that posttranslational regulation of ICP0 by virus and host factors determines the outcome of HSV-1 infection.

Herpes simplex viruses (HSV) are human pathogens that switch between lytic and latent infections intermittently (1, 2). This is a lifelong source of infectious viruses (1, 2), in which immediate early proteins drive the onset of HSV replication. Among them, ICP0 enables viral gene expression or reactivation from latency (2–4), which involves chromatin remodeling of the HSV genome, resulting in de novo virus production. In this process, the accessory factor γ₁34.5 of HSV is thought to govern viral protein synthesis (5, 6). It has long been known that γ₁34.5 precludes translation arrest mediated by double-stranded RNA–dependent protein kinase PKR (7–9). The γ₁34.5 protein has also been shown to dampen intracellular nucleic acid sensing, inhibit autophagy, and facilitate virus nuclear egress (10–17). In experimental animal models, wild-type HSV, but not HSV that lacks the γ₁34.5 gene, replicates competently, penetrates from the peripheral tissues to the nervous system and reactivates from latency (18–23). Despite these observations, active HSV replication or reactivation from latency is not readily reconciled by the currently known functions of the γ₁34.5 protein (8–13, 16, 17).Several lines of work demonstrate that tripartite motif (TRIM) proteins regulate innate immune signaling and cell intrinsic resistance to virus infections (24, 25). These host factors typically work as E3 ubiquitin ligases that can synthesize degradative or nondegradative ubiquitination on viral or host proteins. A number of TRIM proteins, for example TRIM5α, TRIM19, TRIM21, TRIM22, and TRIM43, act at different steps of virus replication and subsequently inhibit viral production (26–32). Recent evidence indicates that TRIM23 limits the replication of certain RNA viruses and DNA viruses, including HSV-1 (33). In doing so, TRIM23 recruits TANK-binding kinase 1 (TBK1) to autophagosomes, thus promoting TBK1-mediated phosphorylation and activation of the autophagy receptor p62 and ultimately leading to autophagy. It is unknown whether TRIM23 plays an additional role(s) in HSV infection.Here, we report that ICP0 expression is regulated by the γ₁34.5 protein and TRIM23 during HSV-1 infection. We show that TRIM23 facilitates the proteasomal degradation of ICP0, whereas viral γ₁34.5 maintains steady-state ICP0 expression by preventing K27-linked TRIM23 autoubiquitination that is required for TRIM23 activation. The γ₁34.5 protein also interacts with and stabilizes ICP0, enabling productive infection. Furthermore, we provide evidence that TRIM23 binds to ICP0 and induces its K11-linked polyubiquitination, which triggers K48-linked polyubiquitin-dependent proteasomal degradation of ICP0. These insights establish a model of posttranslational networks in which virus- and host-mediated mechanisms regulate immediate early protein ICP0 stability and thereby lytic HSV replication.     

Near-germline human monoclonal antibodies neutralize and protect against multiple arthritogenic alphaviruses

Arthritogenic alphaviruses are globally distributed, mosquito-transmitted viruses that cause rheumatological disease in humans and include Chikungunya virus (CHIKV), Mayaro virus (MAYV), and others. Although serological evidence suggests that some antibody-mediated heterologous immunity may be afforded by alphavirus infection, the extent to which broadly neutralizing antibodies that protect against multiple arthritogenic alphaviruses are elicited during natural infection remains unknown. Here, we describe the isolation and characterization of MAYV-reactive alphavirus monoclonal antibodies (mAbs) from a CHIKV-convalescent donor. We characterized 33 human mAbs that cross-reacted with CHIKV and MAYV and engaged multiple epitopes on the E1 and E2 glycoproteins. We identified five mAbs that target distinct regions of the B domain of E2 and potently neutralize multiple alphaviruses with differential breadth of inhibition. These broadly neutralizing mAbs (bNAbs) contain few somatic mutations and inferred germline–revertants retained neutralizing capacity. Two bNAbs, DC2.M16 and DC2.M357, protected against both CHIKV- and MAYV-induced musculoskeletal disease in mice. These findings enhance our understanding of the cross-reactive and cross-protective antibody response to human alphavirus infections.

Alphaviruses are enveloped, positive sense single-stranded RNA viruses that can cause significant human diseases ranging from arthritis to encephalitis (1–3). Alphaviruses that are associated with musculoskeletal disease (arthritogenic alphaviruses) include Chikungunya virus (CHIKV), Mayaro virus (MAYV), Ross River virus (RRV), O’nyong-nyong virus (ONNV), and others; these viruses are globally distributed and transmitted by mosquitos. Symptomatic infection by arthritogenic alphaviruses is characterized by fever, rash, myalgia, as well as both acute and chronic peripheral polyarthralgia (4, 5). The arthropathy can be debilitating and persist for months to years after infection. More severe manifestations of alphavirus disease—including encephalopathy and mortality—have been reported (6, 7). These viruses cause endemic disease as well as large, sporadic global outbreaks (8–10). Currently, there are no approved vaccines or antiviral therapies for the prevention or treatment of alphavirus infection.MAYV is an arthritogenic alphavirus that was first isolated in 1954 in Trinidad, and recent outbreaks have been reported in numerous areas of Central and South America (11, 12). The primary vectors for MAYV are Haemagogus spp. mosquitoes, which transmit the virus to primates in a sylvatic cycle. However, MAYV vector competence studies have demonstrated transmission potential in multiple Aedes and Anopheles mosquitoes (13–17). The wide range and distribution of MAYV-competent vectors underscores the risk of potential urban transmission (18) and global spread (19).The alphavirus glycoprotein is composed of heterodimers of two transmembrane subunits, E2 and E1, which mediate viral attachment and membrane fusion, respectively (20–22). The prefusion E2/E1 heterodimer forms a trimeric spike that is arranged in an icosahedral lattice on the viral particle. E2 is initially expressed as a precursor polypeptide known as p62. During virus biogenesis, p62 is processed by cellular furin to generate E2 and the peripheral E3 polypeptide. E3 remains bound to the E2/E1 heterodimer during exocytic transport and prevents premature conformational changes and membrane fusion (23, 24). The release of E3 is the final step of virus maturation and primes the glycoprotein for membrane fusion.Both E2 and E1 proteins are targets of the neutralizing antibody response. Antibody-mediated protection by neutralizing monoclonal antibodies (mAbs) has been shown against several alphaviruses (25–31). We and others have reported the isolation of potent and protective neutralizing CHIKV mAbs targeting regions of E2, such as the β-connector region and the A domain (25–27). These mAbs neutralize viral particles via multiple mechanisms, including the prevention of attachment and membrane fusion. The alphavirus receptor Mxra8 binds to regions spanning the A and B domains of E2 protein (32), and neutralizing mAbs targeting these regions can effectively disrupt virus interaction with the host receptor (33).Many of the identified, neutralizing human mAbs against alphaviruses are virus-specific and do not inhibit heterologous alphaviruses. Notably, most of these mAbs target CHIKV, and there are few examples of MAYV-reactive human mAbs. Recent work has demonstrated the cross-reactivity and cross-neutralization of human polyclonal sera to heterologous alphaviruses (34–36), suggesting that broadly reactive and/or broadly neutralizing monoclonal antibodies (bNAbs) may be elicited by alphavirus infection in humans. While a number of murine bNAbs have been characterized (30, 37, 38), few human bNAbs that engage multiple alphaviruses have been described (33). For example, the murine mAb CHK-265 can protect against CHIKV, MAYV, and RRV challenge in mice (38). More recently, a human mAb, RRV-12, was shown to protect mice against RRV and MAYV infection (33). Both CHK-265 and RRV-12 broadly neutralize infection by engaging the B domain of E2, but whether such protective alphavirus bNAbs are elicited commonly during the course of human CHIKV infection is unknown.Here, we describe the isolation and characterization of cross-reactive alphavirus mAbs from a CHIKV-convalescent donor. We employed a single B cell sorting strategy using a heterologous MAYV antigen to isolate 33 cross-reactive mAbs and found that they target multiple epitopes on the E1 and E2 proteins. We identified five human bNAbs that neutralize CHIKV, MAYV, and other alphaviruses with differing potencies. Epitope binning and viral escape studies suggest that human bNAbs target related but distinct regions of the B domain of E2. Remarkably, the sequence analysis of human bNAbs showed few somatic mutations, and inferred germline variants largely retained neutralizing function. Two bNAbs demonstrated protection against both CHIKV- and MAYV-induced musculoskeletal disease in mice. Together, these studies further define heterologous humoral immunity among related alphaviruses in humans as well as the determinants of antibody-mediated cross-protection.     

Structure and stabilization of the Hendra virus F glycoprotein in its prefusion form

Hendra virus (HeV) is one of the two prototypical members of the Henipavirus genus of paramyxoviruses, which are designated biosafety level 4 (BSL-4) organisms due to the high mortality rate of Nipah virus (NiV) and HeV in humans. Paramyxovirus cell entry is mediated by the fusion protein, F, in response to binding of a host receptor by the attachment protein. During posttranslational processing, the fusion peptide of F is released and, upon receptor-induced triggering, inserts into the host cell membrane. As F undergoes a dramatic refolding from its prefusion to postfusion conformation, the fusion peptide brings the host and viral membranes together, allowing entry of the viral RNA. Here, we present the crystal structure of the prefusion form of the HeV F ectodomain. The structure shows very high similarity to the structure of prefusion parainfluenza virus 5 (PIV5) F, with the main structural differences in the membrane distal apical loops and the fusion peptide cleavage loop. Functional assays of mutants show that the apical loop can tolerate perturbation in length and surface residues without loss of function, except for residues involved in the stability and conservation of the F protein fold. Structure-based disulfide mutants were designed to anchor the fusion peptide to conformationally invariant residues of the F head. Two mutants were identified that inhibit F-mediated fusion by stabilizing F in its prefusion conformation.The Paramyxoviridae family of viruses includes many species known to cause human and animal disease, including Nipah virus (NiV) and Hendra virus (HeV) of the genus Henipavirus (1). This emergent genus was first described in 1994 with a disease outbreak of HeV, followed by a disease outbreak of NiV in 1999 (2), and was recently expanded by the discovery of the Cedar virus (3) and evidence for 19 new species of African henipaviruses (4). Both NiV and HeV have caused outbreaks of encephalitic and respiratory illness in humans in Malaysia, Bangladesh, Australia, and several neighboring countries, with high morbidity and mortality, and are designated biosafety level 4 (BSL-4) organisms (5, 6). The animal reservoir for NiV and HeV is Pteropus spp. fruit bats. These viruses are transmitted to humans from an intermediate animal vector, pigs in the case of NiV and horses in the case of HeV. Serological and genetic evidence for henipaviruses has been discovered in Pteropus far from known locations of disease incidence (6).Like other paramyxoviruses, the henipaviruses are enveloped viruses that are densely studded on their outer surfaces with the two transmembrane-anchored glycoproteins involved in entry of the virion into host cells via membrane fusion (7). These glycoproteins are the fusion glycoprotein, F, which mediates fusion of the viral lipid envelope with the host cell plasma membrane, and the attachment glycoprotein, G, in henipaviruses, which acts as a trigger for fusion upon specific recognition of the host cell receptors ephrinB2 and ephrinB3 (8, 9). Triggering is thought to occur via sequential conformational changes in G that are communicated to F while they associate in F–G complexes (10, 11). Exposure of stalk residues in G, which are thought to be occluded by its receptor-binding head domains before triggering, appear key to initiating F refolding and membrane fusion.The trimeric F protein in its prefusion form has a globular conformation consisting of three domains (DI, DII, and DIII), followed by a C-terminal stalk, transmembrane domain, and cytoplasmic tail (12). DI and the Ig-like fold DII are implicated in interactions with the attachment protein (13, 14). F also contains two heptad repeats, HRA in DIII and HRB in the stalk. During posttranslational processing, the F₀ precursor is cleaved by the cellular protease cathepsin L at a defined site following a basic residue, K109 in HeV F, resulting in release of the fusion peptide segment located C-terminal to the cleavage site. Cleavage results in two disulfide-linked fragments, F₁ and F₂ (15, 16). Upon activation, the F protein undergoes large-scale refolding, mostly in DIII. HRA is extended into a long α-helix, which forms a six-helix bundle with the HRB region of the stalk. This refolded F forms a golf tee-shaped postfusion conformation (17), which is modeled to bring the host and virus membranes together as the fusion peptides inserted into the host-membrane oligomerize with the virion-embedded portion of the HRB stalk (12).The atomic resolution structures of the prefusion forms of the F protein have been solved for two paramyxoviruses, parainfluenza virus 5 (PIV5) and respiratory syncytial virus (RSV) (12, 18). These two proteins exhibit fairly low sequence identity and significant differences in their structures, although several key domain features are conserved (18). HeV F shares low sequence similarity with PIV5 and lower sequence similarity with RSV (27.9% and 21.3% identity, respectively, calculated with ClustalX). The fusion (F)-attachment (HN) protein pair of PIV5, along with the fusion-attachment protein pair of Newcastle disease virus and human parainfluenza virus type 3, behave according to the “association” model of fusion activation. Although interaction is required for fusion, the F-HN pairs of these proteins have relatively low affinity for each other and host receptor binding is thought to bring the proteins into greater association at initiation of fusion. The F protein of RSV does not require its cognate attachment protein for virus fusion and replication. In contrast, the fusion-attachment protein pairs of the Morbillivirus genus (measles and canine distemper virus) and henipaviruses, NiV and HeV, behave more consistently with the “dissociation” model of fusion activation. Biochemical evidence suggests a relatively higher affinity for the fusion-attachment proteins and preassociation in complexes where dissociation only occurs upon receptor binding (8, 9, 19). To date, no high-resolution structural information has been available for fusion proteins in the dissociation class.Here, we present the crystal structure of the HeV F ectodomain. Its structure has a high degree of structural similarity to the structure of PIV5 F despite low sequence similarity. The areas of greatest structural deviation are at the pair of loops at the apical region of the trimer and at the fusion peptide cleavage site. Site-directed mutagenesis within the apical loops showed that residues that have a role in maintaining tertiary structural integrity of the region have effects on fusion and cell surface expression. Based on the crystal structure, amino acid pairs with a likelihood of forming disulfide bonds were predicted computationally. Double-Cys mutants of HeV F were generated with the intent of preventing refolding of the HRA region that is crucial for fusion. Two of the mutants show loss of fusion activity, while being expressed at the cell surface and recognized by a mAb (5B3) specific for the prefusion conformational state.     

Vaccination of monoglycosylated hemagglutinin induces cross-strain protection against influenza virus infections

The 2009 H1N1 pandemic and recent human cases of H5N1, H7N9, and H6N1 in Asia highlight the need for a universal influenza vaccine that can provide cross-strain or even cross-subtype protection. Here, we show that recombinant monoglycosylated hemagglutinin (HA_mg) with an intact protein structure from either seasonal or pandemic H1N1 can be used as a vaccine for cross-strain protection against various H1N1 viruses in circulation from 1933 to 2009 in mice and ferrets. In the HA_mg vaccine, highly conserved sequences that were originally covered by glycans in the fully glycosylated HA (HA_fg) are exposed and thus, are better engulfed by dendritic cells (DCs), stimulated better DC maturation, and induced more CD8+ memory T cells and IgG-secreting plasma cells. Single B-cell RT-PCR followed by sequence analysis revealed that the HA_mg vaccine activated more diverse B-cell repertoires than the HA_fg vaccine and produced antibodies with cross-strain binding ability. In summary, the HA_mg vaccine elicits cross-strain immune responses that may mitigate the current need for yearly reformulation of strain-specific inactivated vaccines. This strategy may also map a new direction for universal vaccine design.HA glycoprotein on the surface of influenza virus is a major target for infectivity-neutralizing antibodies. However, the antigenic drift and shift of this protein mean that influenza vaccines must be reformulated annually to include HA proteins of the viral strains predicted for the upcoming flu season (1). This time-consuming annual reconfiguration process has led to efforts to develop new strategies and identify conserved epitopes recognized by broadly neutralizing antibodies as the basis for designing universal vaccines to elicit antibodies with a broad protection against various strains of influenza infection (2–6). Previous studies have shown that the stem region of HA is more conserved and able to induce cross-reactive and broadly neutralizing antibodies (7–9) to prevent the critical fusion of viral and endosomal membranes in the influenza lifecycle (10–14). Other broadly neutralizing antibodies have been found to bind regions near the receptor binding site of the globular domain, although these antibodies are fewer in number (15, 16).Posttranslational glycosylation of HA plays an important role in the lifecycle of the influenza virus and also contributes to the structural integrity of HA and the poor immune response of the infected hosts. Previously, we trimmed down the size of glycans on avian influenza H5N1 HA with enzymes and showed that H5N1 HA with a single N-linked GlcNAc at each glycosylation site [monoglycosylated HA (HA_mg)] produces a superior vaccine with more enhanced antibody response and neutralization activity against the homologous influenza virus than the fully glycosylated HA (HA_fg) (17). Here, to test whether the removal of glycans from HA contributes to better immune responses and possibly protects against heterologous strains of influenza viruses, we compared and evaluated the efficacy of HA glycoproteins with various lengths of glycans as potential vaccine candidates.