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Message-Locked Encryption (MLE) is a useful tool to enable deduplication over encrypted data in cloud storage. It can significantly improve the cloud service quality by eliminating redundancy to save storage resources, and hence user cost, and also providing defense against different types of attacks, such as duplicate faking attack and brute-force attack. A typical MLE scheme only focuses on deduplication. On the other hand, supporting search operations on stored content is another essential requirement for cloud storage. In this paper, we present a message-locked searchable encryption (MLSE) scheme in a dual-server setting, which achieves simultaneously the desirable features of supporting deduplication and enabling users to perform search operations over encrypted data. In addition, it supports both multi-keyword and negative keyword searches. We formulate the security notions of MLSE and prove our scheme satisfies all the security requirements. Moreover, we provide an interesting extension of our construction to support Proof of Storage (PoS). Compared with the existing solutions, MLSE achieves better functionalities and efficiency, and hence enables more versatile and efficient cloud storage service.

The pediatric live-attenuated bovine/human parainfluenza virus type 3 (B/HPIV3)-vectored vaccine expressing the prefusion-stabilized SARS-CoV-2 spike (S) protein (B/HPIV3/S-2P) was previously evaluatedin vitroand in hamsters. To improve its immunogenicity, we generated B/HPIV3/S-6P, expressing S further stabilized with 6 proline mutations (S-6P). Intranasal immunization of hamsters with B/HPIV3/S-6P reproducibly elicited significantly higher serum anti-S IgA/IgG titers than B/HPIV3/S-2P; hamster sera efficiently neutralized variants of concern (VoCs), including Omicron variants. B/HPIV3/S-2P and B/HPIV3/S-6P immunization protected hamsters against weight loss and lung inflammation following SARS-CoV-2 challenge with the vaccine-matched strain WA1/2020 or VoCs B.1.1.7/Alpha or B.1.351/Beta and induced near-sterilizing immunity. Three weeks post-challenge, B/HPIV3/S-2P- and B/HPIV3/S-6P-immunized hamsters exhibited a robust anamnestic serum antibody response with increased neutralizing potency to VoCs, including Omicron sublineages. B/HPIV3/S-6P primed for stronger anamnestic antibody responses after challenge with WA1/2020 than B/HPIV3/S-2P. B/HPIV3/S-6P will be evaluated as an intranasal vaccine to protect infants against both HPIV3 and SARS-CoV-2.AUTHOR SUMMARYSARS-CoV-2 infects and causes disease in all age groups. While injectable SARS-CoV-2 vaccines are effective against severe COVID-19, they do not fully prevent SARS-CoV-2 replication and transmission. This study describes the preclinical comparison in hamsters of B/HPIV3/S-2P and B/HPIV3/S-6P, live-attenuated pediatric vector vaccine candidates expressing the “2P” prefusion stabilized version of the SARS-CoV-2 spike protein, or the further-stabilized “6P” version. B/HPIV3/S-6P induced significantly stronger anti-S serum IgA and IgG responses than B/HPIV3/S-2P. A single intranasal immunization with B/HPIV3/S-6P elicited broad systemic antibody responses in hamsters that efficiently neutralized the vaccine-matched isolate as well as variants of concern, including Omicron. B/HPIV3/S-6P immunization induced near-complete airway protection against the vaccine-matched SARS-CoV-2 isolate as well as two variants. Furthermore, following SARS-CoV-2 challenge, immunized hamsters exhibited strong anamnestic serum antibody responses. Based on these data, B/HPIV3/S-6P will be further evaluated in a phase I study.

Significance Pediatric SARS-CoV-2 infections, though generally mild, are associated with substantial morbidity and contribute to transmission dynamics. No SARS-CoV-2 vaccines are available for young children. Bovine/human parainfluenza virus 3 (B/HPIV3) vectors for intranasal immunization of children were evaluated previously in phase 1/2 studies and were well-tolerated in children as young as 2 mo of age. This manuscript describes a B/HPIV3 vector expressing a prefusion-stabilized version of the SARS-CoV-2 S protein (S-2P), and shows that a single intranasal dose is highly immunogenic and protective against SARS-CoV-2 challenge in the hamster model, the most robust SARS-CoV-2 challenge model available. Based on these results, B/HPIV3/S-2P represents a promising vaccine candidate for clinical evaluation as a pediatric vaccine for intranasal immunization against HPIV3 and SARS-CoV-2., 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 104.5 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 106.6 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.

In a cloud data storage system, symmetric key encryption is usually used to encrypt files due to its high efficiency. In order allow the untrusted/semi-trusted cloud storage server to perform searching over encrypted data while maintaining data confidentiality, searchable symmetric encryption (SSE) has been proposed. In a typical SSE scheme, a users stores encrypted files on a cloud storage server and later can retrieve the encrypted files containing specific keywords. The basic security requirement of SSE is that the cloud server learns no information about the files or the keywords during the searching process. Some SSE schemes also offer additional functionalities such as detecting cheating behavior of a malicious server (i.e., verifiability) and allowing update (e.g., modifying, deleting and adding) of documents on the server. However, the previous (verifiable) SSE schemes were designed for single users, which means the searching can only be done by the data owner, whereas in reality people often use cloud storage to share files with other users. In this paper we present a multi-user verifiable searchable symmetric encryption (MVSSE) scheme that achieves all the desirable features of a verifiable SSE and allows multiple users to perform searching. We then define an ideal functionality for MVSSE under the Universally Composable (UC-) security framework and prove that our ideal functionality implies the security requirements of a secure MVSSE, and our multi-user verifiable SSE scheme is UC-secure. We also implement our scheme to verify its high performance based on some real dataset.

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

Pediatric SARS-CoV-2 vaccines are needed that elicit immunity directly in the airways as well as systemically. Building on pediatric parainfluenza virus vaccines in clinical development, we generated a live-attenuated parainfluenza-virus-vectored vaccine candidate expressing SARS-CoV-2 prefusion-stabilized spike (S) protein (B/HPIV3/S-6P) and evaluated its immunogenicity and protective efficacy in rhesus macaques. A single intranasal/intratracheal dose of B/HPIV3/S-6P induced strong S-specific airway mucosal immunoglobulin A (IgA) and IgG responses. High levels of S-specific antibodies were also induced in serum, which efficiently neutralized SARS-CoV-2 variants of concern of alpha, beta, and delta lineages, while their ability to neutralize Omicron sub-lineages was lower. Furthermore, B/HPIV3/S-6P induced robust systemic and pulmonary S-specific CD4

In an identity-based broadcast encryption (IBBE) scheme, the ciphertext is usually appended with a set of user identities to specify intended recipients. However, as IBBE is adopted in extensive industries, the demand of anonymity for specific scenarios such as military applications is urgent and ought no more to be ignored. On the contrary, how to optimize computation and communication is an unavoidable challenge in the IBBE scheme construction, especially in the large-scaled resource-limited wireless networks such as the Internet of Things (IoT), where the cost of computation and communication should be mitigated as much as possible since other functions including connectivity and privacy should be given the top priority. Thus, we present an IBBE scheme from the lattice, in which we employ the Chinese remainder theorem and lattice basis delegation in fixed dimensions to obtain several desirable characteristics, such as constant-size public parameter, private key, and ciphertext. In addition, our encryption and decryption algorithms are more efficient than broadcast encryption (BE) schemes based on number-theoretic problems. To be noticed, our scheme can simultaneously achieve confidentiality and outsider anonymity against the chosen-plaintext attack under the hardness of the learning with error (LWE) problem.

Live-attenuated pediatric vaccines for intranasal administration are being developed for human respiratory syncytial virus (RSV), an important worldwide pediatric respiratory pathogen that lacks a licensed vaccine or suitable antiviral drug. We evaluated a prime-boost strategy in which primary immunization with RSV was boosted by secondary immunization with RSV or with a chimeric recombinant bovine/human parainfluenza virus type 3 (rB/HPIV3) vector expressing the RSV fusion F protein. The vector-expressed F protein had been engineered (DS-Cav1 mutations) for increased stability in the highly immunogenic prefusion (pre-F) conformation, with or without replacement of its transmembrane and cytoplasmic tail domains with their counterparts from bovine parainfluenza virus type 3 (BPIV3) F protein to direct incorporation into the vector virion for increased immunogenicity. In hamsters that received a primary infection with RSV, a booster infection with RSV ∼6 weeks later was completely restricted for producing infectious virus but induced a significant increase in the serum RSV-plaque-reduction neutralizing antibody titer (RSV-PRNT). Boosting instead with the rB/HPIV3-RSV-pre-F vectors resulted in efficient replication and induced significantly higher RSV-PRNTs than RSV. In African green monkeys that received a primary infection with RSV, a booster infection with RSV ∼2, ∼6, or ∼15 months later was highly restricted, whereas booster infections with the vectors had robust replication. Compared with RSV, boosts with the vectors induced 7- to 15-fold higher titers of RSV-specific serum antibodies with high neutralizing activity, as well as significantly higher titers of RSV-specific mucosal IgA antibodies. These findings support further development of this heterologous prime-boost strategy. IMPORTANCE Immune responses to RSV in infants can be reduced due to immunological immaturity and immunosuppression by RSV-specific maternal antibodies. In infants and young children, two infections with wild-type RSV typically are needed to achieve the titers of RSV-specific serum antibodies and protection against illness that are observed in adults. Therefore, a boost might substantially improve the performance of live pediatric RSV vaccines presently being developed. Hamsters and African green monkeys received a primary intranasal infection with RSV and were given a boost with RSV or a parainfluenza virus (PIV) vector expressing RSV fusion protein engineered for enhanced immunogenicity. The RSV boost was highly restricted but induced a significant increase in serum RSV-neutralizing antibodies. The PIV vectors replicated efficiently and induced significantly higher antibody responses. The use of an attenuated PIV vector expressing RSV antigen to boost a primary immunization with an attenuated RSV warrants further evaluation.

Interferon alpha (IFN-α) and IFN-β are type I IFNs that are induced by virus infection and are important in the host’s innate antiviral response. EBV infection activates multiple cell signaling pathways, resulting in the production of type I IFN which inhibits EBV infection and virus-induced B-cell transformation. We reported previously that EBV tegument protein BGLF2 activates p38 and enhances EBV reactivation. To further understand the role of BGLF2 in EBV infection, we used mass spectrometry to identify cellular proteins that interact with BGLF2. We found that BGLF2 binds to Tyk2 and confirmed this interaction by coimmunoprecipitation. BGLF2 blocked type I IFN-induced Tyk2, STAT1, and STAT3 phosphorylation and the expression of IFN-stimulated genes (ISGs) IRF1, IRF7, and MxA. In contrast, BGLF2 did not inhibit STAT1 phosphorylation induced by IFN-γ. Deletion of the carboxyl-terminal 66 amino acids of BGLF2 reduced the ability of the protein to repress type I IFN signaling. Treatment of gastric carcinoma and Raji cells with IFN-α blocked BZLF1 expression and EBV reactivation; however, expression of BGLF2 reduced the ability of IFN-α to inhibit BZLF1 expression and enhanced EBV reactivation. In summary, EBV BGLF2 interacts with Tyk2, inhibiting Tyk2, STAT1, and STAT3 phosphorylation and impairs type I IFN signaling; BGLF2 also counteracts the ability of IFN-α to suppress EBV reactivation. IMPORTANCE Type I interferons are important for controlling virus infection. We have found that the Epstein-Barr virus (EBV) BGLF2 tegument protein binds to a protein in the type I interferon signaling pathway Tyk2 and inhibits the expression of genes induced by type I interferons. Treatment of EBV-infected cells with type I interferon inhibits reactivation of the virus, while expression of EBV BGLF2 reduces the ability of type I interferon to inhibit virus reactivation. Thus, a tegument protein delivered to cells during virus infection inhibits the host’s antiviral response and promotes virus reactivation of latently infected cells. Therefore, EBV BGLF2 might protect virus-infected cells from the type I interferon response in cells undergoing lytic virus replication.

Human respiratory syncytial virus (RSV) and parainfluenza virus type 3 (HPIV3) are among the most common viral causes of childhood bronchiolitis and pneumonia worldwide, and lack effective antiviral drugs or vaccines. Recombinant (r) HPIV3 was modified to express the RSV fusion (F) glycoprotein, the major RSV neutralization and protective antigen, providing a live intranasal bivalent HPIV3/RSV vaccine candidate. This extends previous studies using a chimeric bovine-human PIV3 vector (rB/HPIV3). One advantage is that rHPIV3 expresses all of the HPIV3 antigens compared to only two for rB/HPIV3. In addition, the use of rHPIV3 as vector should avoid excessive attenuation following addition of the modified RSV F gene, which may occur with rB/HPIV3. To enhance its immunogenicity, RSV F was modified (i) to increase the stability of the prefusion (pre-F) conformation and (ii) by replacement of its transmembrane (TM) and cytoplasmic tail (CT) domains with those of HPIV3 F (H3TMCT) to increase incorporation in the vector virion. RSV F (+/- H3TMCT) was expressed from the first (F/preN) or the second (F/N-P) gene position of rHPIV3. The H3TMCT modification dramatically increased packaging of RSV F into the vector virion and, in hamsters, resulted in significant increases in the titer of high-quality serum RSV-neutralizing antibodies, in addition to the increase conferred by pre-F stabilization. Only F-H3TMCT/preN replication was significantly attenuated in the nasal turbinates by the RSV F insert. F-H3TMCT/preN, F/N-P, and F-H3TMCT/N-P provided complete protection against wt RSV challenge. F-H3TMCT/N-P exhibited the most stable and highest expression of RSV F, providing impetus for its further development.

Human respiratory syncytial virus (RSV) is the leading viral cause of acute pediatric lower respiratory tract infections worldwide, with no available vaccine or effective antiviral drug. To gain insight into virus-host interactions, we performed a genome-wide siRNA screen. The expression of over 20,000 cellular genes was individually knocked down in human airway epithelial A549 cells, followed by infection with RSV expressing green fluorescent protein (GFP). Knockdown of expression of the cellular ATP1A1 protein, which is the major subunit of the Na+,K+-ATPase of the plasma membrane, had one of the strongest inhibitory effects on GFP expression and viral titer. Inhibition was not observed for vesicular stomatitis virus, indicating that it was RSV-specific rather than a general effect. ATP1A1 formed clusters in the plasma membrane very early following RSV infection, which was independent of replication but dependent on the attachment glycoprotein G. RSV also triggered activation of ATP1A1, resulting in signaling by c-Src-kinase activity that transactivated epidermal growth factor receptor (EGFR) by Tyr845 phosphorylation. ATP1A1 signaling and activation of both c-Src and EGFR were found to be required for efficient RSV uptake. Signaling events downstream of EGFR culminated in the formation of macropinosomes. There was extensive uptake of RSV virions into macropinosomes at the beginning of infection, suggesting that this is a major route of RSV uptake, with fusion presumably occurring in the macropinosomes rather than at the plasma membrane. Important findings were validated in primary human small airway epithelial cells (HSAEC). In A549 cells and HSAEC, RSV uptake could be inhibited by the cardiotonic steroid ouabain and the digitoxigenin derivative PST2238 (rostafuroxin) that bind specifically to the ATP1A1 extracellular domain and block RSV-triggered EGFR Tyr845 phosphorylation. In conclusion, we identified ATP1A1 as a host protein essential for macropinocytic entry of RSV into respiratory epithelial cells, and identified PST2238 as a potential anti-RSV drug., Author summary RSV continues to be the most important viral cause of severe bronchiolitis and pneumonia in infants and young children, and also has a substantial impact in the elderly. It is estimated to claim the lives of ~118,000 children under five years of age annually. No vaccine or antiviral drug suitable for general use is available. The involvement of host factors in RSV infection and replication is not well understood, but this knowledge might lead to intervention strategies to prevent infection. Using a genome-wide siRNA screen to knock down the expression of over 20,000 individual cellular genes, we identified ATP1A1, the major subunit of the Na+,K+-ATPase, as an important host protein for RSV entry. We showed that ATP1A1 activation by RSV resulted in transactivation of EGFR by Src-kinase activity, resulting in the uptake of RSV particles into the host cell through macropinocytosis. We also showed that the cardiotonic steroid ouabain and the synthetic digitoxigenin derivative PST2238, which bind specifically to the extracellular domain of ATP1A1, significantly reduced RSV entry. Taken together, we describe a novel ATP1A1-enabled mechanism used by RSV to enter the host cell, and describe candidate antiviral drugs that block this entry.

Human respiratory syncytial virus (RSV) is a major pediatric respiratory pathogen. The attachment (G) and fusion (F) glycoproteins are major neutralization and protective antigens. RSV G is expressed as membrane-anchored (mG) and -secreted (sG) forms, both containing a central fractalkine-like CX3C motif. The CX3C motif and sG are thought to interfere with host immune responses and have been suggested to be omitted from a vaccine. We used a chimeric bovine/human parainfluenza virus type 3 (rB/HPIV3) vector to express RSV wild-type (wt) G and modified forms, including sG alone, mG alone, mutants with ablated CX3C, and G with enhanced packaging into vector virions. In hamsters, these viruses replicated to similar titers. When assayed with a complement-enhanced neutralization assay in Vero cells, sG did not reduce the serum RSV- or PIV3-neutralizing antibody (NAb) responses, whereas ablating CX3C drastically reduced the RSV NAb response. Protective efficacy against RSV challenge was not reduced by sG but was strongly dependent on the CX3C motif. In ciliated human airway epithelial (HAE) cells, NAbs induced by wt G, but not by wt F, completely blocked RSV infection in the absence of added complement. This activity was dependent on the integrity of the CX3C motif. In hamsters, the rB/HPIV3 expressing wt G conferred better protection against RSV challenge than that expressing wt F. Codon optimization of the wt G further increased its immunogenicity and protective efficacy. This study showed that ablation of the CX3C motif or sG in an RSV vaccine, as has been suggested previously, would be ill advised. IMPORTANCE Human RSV is the leading viral cause of severe pediatric respiratory illness. An RSV vaccine is not yet available. The RSV attachment protein G is an important protective and neutralization antigen. G contains a conserved fractalkine-like CX3C motif and is expressed in mG and sG forms. sG and the CX3C motif are thought to interfere with host immune responses, but this remains poorly characterized. Here, we used an attenuated chimeric bovine/human parainfluenza virus type 3 (rB/HPIV3) vector to express various modified forms of RSV G. We demonstrated that strong antibody and protective responses could be induced by G alone, and that this was highly dependent on the integrity of the CX3C motif. There was no evidence that sG or the CX3C motif impaired immune responses against RSV G or the rB/HPIV3 vector. rB/HPIV3 expressing wt RSV G provides a bivalent vaccine against RSV and HPIV3.

Human respiratory syncytial virus (RSV) continues to be the leading viral cause of severe acute lower respiratory tract disease in infants and children worldwide. A licensed vaccine or antiviral drug suitable for routine use remains unavailable. Like RSV, Murine pneumonia virus (MPV) is a member of the genus Orthopneumovirus, family Pneumoviridae Humans are not normally exposed to MPV, and MPV is not cross-protective with RSV. We evaluated MPV as an RSV vaccine vector expressing the RSV fusion (F) glycoprotein. The RSV F open reading frame (ORF) was codon optimized, and the encoded RSV F protein was made identical to an early passage of RSV strain A2. The RSV F ORF was placed under the control of MPV transcription signals and inserted at the first (rMPV-F1), third (rMPV-F3), or fourth (rMPV-F4) gene position of a version of the MPV genome that contained a codon-pair-optimized polymerase (L) gene. The recovered viruses replicated in vitro as efficiently as the empty vector, with stable expression of RSV F protein. Replication and immunogenicity of rMPV-F1 and rMPV-F3 were evaluated in rhesus macaques following intranasal and intratracheal administration. Both viruses replicated at low levels in the upper and lower respiratory tracts, maintained stable RSV F expression, and induced RSV-neutralizing serum antibodies at high levels similar to those induced by wild-type RSV replicating to a 5- to 25-fold-higher titer. In conclusion, this study demonstrated that rMPV provides a highly attenuated yet immunogenic vector for the expression of RSV F protein, with potential application in RSV-naive and RSV-experienced populations.IMPORTANCE Human respiratory syncytial virus (RSV) is an important human pathogen that lacks a licensed vaccine or antiviral drug suitable for routine use. We describe here the evaluation of recombinant murine pneumonia virus (rMPV) as a live-attenuated vector that expresses the RSV F protein, the major RSV neutralization antigen, as an experimental RSV vaccine. The rMPV-RSV-F vectors expressing RSV F from the first, third, or fourth gene position were genetically stable and were not restricted for replication in vitro In contrast, the vectors exhibited highly attenuated replication in the respiratory tract of rhesus macaques, maintained stable RSV F expression, and induced RSV-neutralizing serum antibodies at high titers similar to those conferred by wild-type RSV. Given the lack of preexisting immunity to MPV in humans and the lack of cross-neutralization and cross-protection between MPV and RSV, an rMPV-vectored RSV vaccine should be immunogenic in both RSV-naive children and RSV-experienced adults.

A collection of genomic DNA sequences of herpes simplex virus (HSV) strains has been defined and analyzed, and some information is available about genomic stability upon limited passage of viruses in culture. The nature of genomic change upon extensive laboratory passage remains to be determined. In this report we review the history of the HSV-1 KOS laboratory strain and the related KOS1.1 laboratory sub-strain, also called KOS (M), and determine the complete genomic sequence of an early passage stock of the KOS laboratory sub-strain and a laboratory stock of the KOS1.1 sub-strain. The genomes of the two sub-strains are highly similar with only five coding changes, 20 non-coding changes, and about twenty non-ORF sequence changes. The coding changes could potentially explain the KOS1.1 phenotypic properties of increased replication at high temperature and reduced neuroinvasiveness. The study also provides sequence markers to define the provenance of specific laboratory KOS virus stocks.

Human respiratory syncytial virus (RSV) is the most prevalent worldwide cause of severe respiratory tract infection in infants and young children. Human parainfluenza virus type 1 (HPIV1) also causes severe pediatric respiratory illness, especially croup. Both viruses lack vaccines. Here, we describe the preclinical development of a bivalent RSV/HPIV1 vaccine based on a recombinant HPIV1 vector, attenuated by a stabilized mutation, that expresses RSV F protein modified for increased stability in the prefusion (pre-F) conformation by previously described disulfide bond (DS) and hydrophobic cavity-filling (Cav1) mutations. RSV F was expressed from the first or second gene position as the full-length protein or as a chimeric protein with its transmembrane and cytoplasmic tail (TMCT) domains substituted with those of HPIV1 F in an effort to direct packaging in the vector particles. All constructs were recovered by reverse genetics. The TMCT versions of RSV F were packaged in the rHPIV1 particles much more efficiently than their full-length counterparts. In hamsters, the presence of the RSV F gene, and in particular the TMCT versions, was attenuating and resulted in reduced immunogenicity. However, the vector expressing full-length RSV F from the pre-N position was immunogenic for RSV and HPIV1. It conferred complement-independent high-quality RSV-neutralizing antibodies at titers similar to those of wild-type RSV and provided protection against RSV challenge. The vectors exhibited stable RSV F expression in vitro and in vivo . In conclusion, an attenuated rHPIV1 vector expressing a pre-F-stabilized form of RSV F demonstrated promising immunogenicity and should be further developed as an intranasal pediatric vaccine. IMPORTANCE RSV and HPIV1 are major viral causes of acute pediatric respiratory illness for which no vaccines or suitable antiviral drugs are available. The RSV F glycoprotein is the major RSV neutralization antigen. We used a rHPIV1 vector, bearing a stabilized attenuating mutation, to express the RSV F glycoprotein bearing amino acid substitutions that increase its stability in the pre-F form, the most immunogenic form that elicits highly functional virus-neutralizing antibodies. RSV F was expressed from the pre-N or N-P gene position of the rHPIV1 vector as a full-length protein or as a chimeric form with its TMCT domain derived from HPIV1 F. TMCT modification greatly increased packaging of RSV F into the vector particles but also increased vector attenuation in vivo , resulting in reduced immunogenicity. In contrast, full-length RSV F expressed from the pre-N position was immunogenic, eliciting complement-independent RSV-neutralizing antibodies and providing protection against RSV challenge.

Respiratory syncytial virus (RSV) is the most important viral agent of severe pediatric respiratory tract disease worldwide, but it lacks a licensed vaccine or suitable antiviral drug. A live attenuated chimeric bovine/human parainfluenza virus type 3 (rB/HPIV3) was developed previously as a vector expressing RSV fusion (F) protein to confer bivalent protection against RSV and HPIV3. In a previous clinical trial in virus-naive children, rB/HPIV3 was well tolerated but the immunogenicity of wild-type RSV F was unsatisfactory. We previously modified RSV F with a designed disulfide bond (DS) to increase stability in the prefusion (pre-F) conformation and to be efficiently packaged in the vector virion. Here, we further stabilized pre-F by adding both disulfide and cavity-filling mutations (DS-Cav1), and we also modified RSV F codon usage to have a lower CpG content and a higher level of expression. This RSV F open reading frame was evaluated in rB/HPIV3 in three forms: (i) pre-F without vector-packaging signal, (ii) pre-F with vector-packaging signal, and (iii) secreted pre-F ectodomain trimer. Despite being efficiently expressed, the secreted pre-F was poorly immunogenic. DS-Cav1 stabilized pre-F, with or without packaging, induced higher titers of pre-F specific antibodies in hamsters, and improved the quality of RSV-neutralizing serum antibodies. Codon-optimized RSV F containing fewer CpG dinucleotides had higher F expression, replicated more efficiently in vivo , and was more immunogenic. The combination of DS-Cav1 pre-F stabilization, optimized codon usage, reduced CpG content, and vector packaging significantly improved vector immunogenicity and protective efficacy against RSV. This provides an improved vectored RSV vaccine candidate suitable for pediatric clinical evaluation. IMPORTANCE RSV and HPIV3 are the first and second leading viral causes of severe pediatric respiratory disease worldwide. Licensed vaccines or suitable antiviral drugs are not available. We are developing a chimeric rB/HPIV3 vector expressing RSV F as a bivalent RSV/HPIV3 vaccine and have been evaluating means to increase RSV F immunogenicity. In this study, we evaluated the effects of improved stabilization of F in the pre-F conformation and of codon optimization resulting in reduced CpG content and greater pre-F expression. Reduced CpG content dampened the interferon response to infection, promoting higher replication and increased F expression. We demonstrate that improved pre-F stabilization and strategic manipulation of codon usage, together with efficient pre-F packaging into vector virions, significantly increased F immunogenicity in the bivalent RSV/HPIV3 vaccine. The improved immunogenicity included induction of increased titers of high-quality complement-independent antibodies with greater pre-F site Ø binding and greater protection against RSV challenge.

The recent 2014-2016 Ebola virus (EBOV) outbreak prompted increased efforts to develop vaccines against EBOV disease. We describe the development and preclinical evaluation of an attenuated recombinant human parainfluenza virus type 1 (rHPIV1) expressing the membrane-anchored form of EBOV glycoprotein GP, as an intranasal (i.n.) EBOV vaccine. GP was codon optimized and expressed either as a full-length protein or as an engineered chimeric form in which its transmembrane and cytoplasmic tail (TMCT) domains were replaced with those of the HPIV1 F protein in an effort to enhance packaging into the vector particle and immunogenicity. GP was inserted either preceding the N gene (pre-N) or between the N and P genes (N-P) of rHPIV1 bearing a stabilized attenuating mutation in the P/C gene (C Δ170 ). The constructs grew to high titers and efficiently and stably expressed GP. Viruses were attenuated, replicating at low titers over several days, in the respiratory tract of African green monkeys (AGMs). Two doses of candidates expressing GP from the pre-N position elicited higher GP neutralizing serum antibody titers than the N-P viruses, and unmodified GP induced higher levels than its TMCT counterpart. Unmodified EBOV GP was packaged into the HPIV1 particle, and the TMCT modification did not increase packaging or immunogenicity but rather reduced the stability of GP expression during in vivo replication. In conclusion, we identified an attenuated and immunogenic i.n. vaccine candidate expressing GP from the pre-N position. It is expected to be well tolerated in humans and is available for clinical evaluation. IMPORTANCE EBOV hemorrhagic fever is one of the most lethal viral infections and lacks a licensed vaccine. Contact of fluids from infected individuals, including droplets or aerosols, with mucosal surfaces is an important route of EBOV spread during a natural outbreak, and aerosols also might be exploited for intentional virus spread. Therefore, vaccines that protect against mucosal as well as systemic inoculation are needed. We evaluated a version of human parainfluenza virus type 1 (HPIV1) bearing a stabilized attenuating mutation in the P/C gene (C Δ170 ) as an intranasal vaccine vector to express the EBOV glycoprotein GP. We evaluated expression from two different genome positions (pre-N and N-P) and investigated the use of vector packaging signals. African green monkeys immunized with two doses of the vector expressing GP from the pre-N position developed high titers of GP neutralizing serum antibodies. The attenuated vaccine candidate is expected to be safe and immunogenic and is available for clinical development.

Mammalian cells activate DNA damage response pathways in response to virus infections. Activation of these pathways can enhance replication of many viruses, including herpesviruses. Activation of cellular ATM results in phosphorylation of H2AX and recruits proteins to sites of DNA damage. We found that varicella-zoster (VZV) infected cells had elevated levels of phosphorylated H2AX and phosphorylated ATM and that these levels increased in cells infected with VZV deleted for ORF61 or ORF63, but not deleted for ORF67. Expression of VZV ORF61, ORF62, or ORF63 alone did not result in phosphorylation of H2AX. While BGLF4, the Epstein-Barr virus homolog of VZV ORF47 protein kinase, phosphorylates H2AX and ATM, neither VZV ORF47 nor ORF66 protein kinase phosphorylated H2AX or ATM. Cells lacking ATM had no reduction in VZV replication. Thus, VZV induces phosphorylation of H2AX and ATM and this effect is associated with the presence of specific VZV genes in virus-infected cells.

Programmed cell death (apoptosis) is an important host defense mechanism against intracellular pathogens, such as viruses. Accordingly, viruses have evolved multiple mechanisms to modulate apoptosis to enhance replication. Varicella-zoster virus (VZV) induces apoptosis in human fibroblasts and melanoma cells. We found that VZV triggered the phosphorylation of the proapoptotic proteins Bim and BAD but had little or no effect on other Bcl-2 family members. Since phosphorylation of Bim and BAD reduces their proapoptotic activity, this may prevent or delay apoptosis in VZV-infected cells. Phosphorylation of Bim but not BAD in VZV-infected cells was dependent on activation of the MEK/extracellular signal-regulated kinase (ERK) pathway. Cells knocked down for Bim showed delayed VZV plaque formation, resulting in longer survival of VZV-infected cells and increased replication of virus, compared with wild-type cells infected with virus. Conversely, overexpression of Bim resulted in earlier plaque formation, smaller plaques, reduced virus replication, and increased caspase 3 activity. Inhibition of caspase activity in VZV-infected cells overexpressing Bim restored levels of virus production similar to those seen with virus-infected wild-type cells. Previously we showed that VZV ORF12 activates ERK and inhibits apoptosis in virus-infected cells. Here we found that VZV ORF12 contributes to Bim and BAD phosphorylation. In summary, VZV triggers Bim phosphorylation; reduction of Bim levels results in longer survival of VZV-infected cells and increased VZV replication.

Herpes simplex virus (HSV) has evolved multiple strategies to modulate host immune responses. In a screen of HSV open reading frames to identify additional HSV-encoded proteins that affect NF-κB signaling, we identified the viral US3 tegument protein as an inhibitor of NF-κB signaling. We found that the US3 protein is required for inhibition of TLR2 signaling induced by viral infection and that this inhibition occurs at very early times post-infection. Expression of US3 in transfected cells inhibits TLR2 signaling induced by Zymosan, and this inhibition occurs at or downstream of MyD88 and upstream of p65. Polyubiquitination of TRAF6 is critical for its function in TLR2 signaling. Using US3-null and US3 kinase-defective mutant viruses, we demonstrate that HSV US3 reduces TRAF6 polyubiquitination and that the kinase activity of US3 is necessary for this effect. Therefore, US3 is necessary and sufficient for inhibiting TLR2 signaling at or before the stage of TRAF6 ubiquitination.

Mitogen-activated protein kinases (MAPKs) are a family of serine-threonine protein kinases involved in many cellular processes, including cell proliferation, differentiation, inflammation, and cell death. Activation of several MAPKs, including extracellular signal-regulated kinase 1 and 2 (ERK1/2), p38, and c-Jun N-terminal kinase (JNK), results in stimulation of activator protein 1 (AP-1), which promotes gene transcription. Previous studies have demonstrated that varicella-zoster virus (VZV) infection activates ERK1/2, p38, and JNK to promote viral replication, but the underlying mechanism(s) is unclear. To identify viral proteins responsible for the activation of MAPK, we used a proteomic approach to screen viral proteins for AP-1 promoter activation by an AP-1–luciferase reporter assay. We found that VZV ORF12 protein, located in the tegument of virions, enhances AP-1 reporter activity. This effect of ORF12 protein was markedly inhibited by a MAPK/ERK kinase 1 and 2 (MEK1/2) inhibitor (U0126), partially blocked by a p38 inhibitor (SB202190), but not inhibited by a JNK inhibitor (SP600125). Expression of VZV ORF12 protein in cells resulted in phosphorylation of ERK1/2 and p38 but not JNK. Infection of cells with a VZV ORF12 deletion mutant resulted in reduced levels of phosphorylated ERK1/2 (p-ERK1/2) compared to infection with wild-type VZV. Furthermore, deletion of ORF12 rendered VZV-infected cells more susceptible to staurosporine-induced apoptosis. In conclusion, VZV ORF12 protein activates the AP-1 pathway by selectively triggering the phosphorylation of ERK1/2 and p38. Cells infected with a VZV ORF12 deletion mutant have reduced levels of p-ERK1/2 and are more susceptible to apoptosis than cells infected with wild-type VZV.

The herpes simplex virus 1 (HSV-1) d106 mutant virus is a multiple immediate-early gene deletion mutant virus that has been effective as an AIDS vaccine vector in rhesus macaques (Kaur A, Sanford HB, Garry D, Lang S, Klumpp SA, Watanabe D, et al. Ability of herpes simplex virus vectors to boost immune responses to DNA vectors and to protect against challenge by simian immunodeficiency virus. Virology 2007;357:199-214). Further analysis of this vector is needed to advance development into clinical trials. In this study we have defined the precise nature of the multiple IE gene mutations in the d106 viral genome and have used this information to construct a new transfer plasmid for gene transfer into d106. We tested the effect of an additional mutation in the U(L)41 gene on d106 immunogenicity and found that it did not improve the efficacy of the d106 vector, in contrast with results from other studies with U(L)41 gene mutants. The safety profile of d106 was improved by generating a new vector strain, d106S, with increased sensitivity to acyclovir. Finally, we have constructed a d106S recombinant vector that expresses the HIV clade C envelope protein. The d106S HIVenvC recombinant has retained the sensitivity to acyclovir, indicating that this phenotype is a stable property of the d106S vector.

Viruses remodel the host cell to optimize their replication both by delivery of virion proteins into the cell and by de novo expression of viral proteins. The HSV particle contains several proteins that function to prepare the host cell for viral replication, including the VP16 transcriptional activator protein and virion host shutoff protein. HSV infection activates NF-κB pathways through Toll-like receptor (TLR) 2 and non-TLR pathways, and NF-κB activity is required for efficient viral replication. In a screen of the HSV proteome, we observed that the HSV U L 37 tegument protein activates NF-κB signaling in a TLR2-independent manner. Expression of U L 37 in transfected cells leads to IκB degradation and activation of both reporter genes and the endogenous IL-8 gene. This activation requires TNF receptor–associated factor 6 (TRAF6), and U L 37 contains a TRAF6-binding domain that is required for interaction with TRAF6 and activation of NF-κB. A mutant virus encoding U L 37 with an altered TRAF6-binding site shows reduced NF-κB activation in the early phase of infection. Therefore, the HSV U L 37 virion structural protein can activate NF-κB through TRAF6. Activation of NF-κB by a virion tegument protein that is delivered into the host cell cytoplasm during viral entry represents a mechanism for activation of this pathway by a virus.

Epstein-Barr virus (EBV) is a ubiquitous gammaherpesvirus associated with both B cell and epithelial cell malignancies. EBV infection of B cells triggers activation of several signaling pathways that are critical for cell survival, virus latency, and growth transformation. To identify EBV proteins important for regulating cell signaling, we used a proteomic approach to screen viral proteins for AP-1 and NF-κB promoter activity in AP-1– and NF-κB–luciferase reporter assays. We found that EBV BGLF2 activated AP-1 but not NF-κB reporter activity. Expression of EBV BGLF2 in cells activated p38 and c-Jun N-terminal kinase (JNK), both of which are important for mitogen-activated protein kinase (MAPK) signaling. Deletion of the carboxyl-terminal 66 amino acids of BGLF2 reduced the ability of BGLF2 to activate JNK and p38. Expression of BGLF2 enhanced BZLF1 expression in latently EBV-infected lymphoblastoid cell lines, and knockdown of BGLF2 reduced EBV reactivation induced by IgG cross-linking. Expression of BGLF2 induced BZLF1 expression and virus production in EBV-infected gastric carcinoma cells. BGLF2 enhanced BZLF1 expression and EBV production by activating p38; chemical inhibition of p38 and MAPK/ERK kinases 1 and 2 (MEK1/2) reduced expression of BZLF1 and virus production induced by BGLF2. In summary, the EBV tegument protein BGLF2, which is delivered to the cell at the onset of virus infection, activates the AP-1 pathway and enhances EBV reactivation and virus production. IMPORTANCE Epstein-Barr virus (EBV) is associated with both B cell and epithelial cell malignancies, and the virus activates multiple signaling pathways important for its persistence in latently infected cells. We identified a viral tegument protein, BGLF2, which activates members of the mitogen-activated protein kinase signaling pathway. Expression of BGLF2 increased expression of EBV BZLF1, which activates a switch from latent to lytic virus infection, and increased production of EBV. Inhibition of BGFL2 expression or inhibition of p38/MAPK, which is activated by BGLF2, reduced virus reactivation from latency. These results indicate that a viral tegument protein which is delivered to cells upon infection activates signaling pathways to enhance virus production and facilitate virus reactivation from latency.

Micro lens arrays are widely used in optical communication and laser-fiber coupling applications. In this paper, a technique to fabricate concave micro lens arrays on glass substrate using a third harmonic Nd:YAG laser direct patterning and followed by chemical wet isotropic etching is presented. The patterning process was done on gold film, which was coated on a glass substrate by using a NC controlled laser ablation tool paths. The glass substrate is then etched by using hydrofluoric acid (HF) solutions whereby the exposed area will be dissolved away by chemical reaction with HF. The type of etching process is an isotropic etching which the etching rate is equal at all direction thus produce hemispherical concave profile on glass. The optimum laser patterning parameters is obtained and the effect of different types of HF solutions on etching efficiency is studied. The surface morphology, 2D and 3D profiles are also measured. Various micro lens diameters are fabricated with different values of lens sag.