IL-10 suppresses T cell expansion while promoting tissue-resident memory cell formation during SARS-CoV-2 infection in rhesus macaques.

The regulation of inflammatory responses and pulmonary disease during SARS-CoV-2 infection is incompletely understood. Here we examine the roles of the prototypic pro- and anti-inflammatory cytokines IFNγ and IL-10 using the rhesus macaque model of mild COVID-19. We find that IFNγ drives the development of 18fluorodeoxyglucose (FDG)-avid lesions in the lungs as measured by PET/CT imaging but is not required for suppression of viral replication. In contrast, IL-10 limits the duration of acute pulmonary lesions, serum markers of inflammation and the magnitude of virus-specific T cell expansion but does not impair viral clearance. We also show that IL-10 induces the subsequent differentiation of virus-specific effector T cells into CD69+CD103+ tissue resident memory cells (Trm) in the airways and maintains Trm cells in nasal mucosal surfaces, highlighting an unexpected role for IL-10 in promoting airway memory T cells during SARS-CoV-2 infection of macaques.

Human parainfluenza virus 3 vaccine candidates attenuated by codon-pair deoptimization are immunogenic and protective in hamsters.

Human parainfluenza virus type 3 (HPIV3) is a major pediatric respiratory pathogen lacking available vaccines or antiviral drugs. We generated live-attenuated HPIV3 vaccine candidates by codon-pair deoptimization (CPD). HPIV3 open reading frames (ORFs) encoding the nucleoprotein (N), phosphoprotein (P), matrix (M), fusion (F), hemagglutinin-neuraminidase (HN), and polymerase (L) were modified singly or in combination to generate 12 viruses designated Min-N, Min-P, Min-M, Min-FHN, Min-L, Min-NP, Min-NPM, Min-NPL, Min-PM, Min-PFHN, Min-MFHN, and Min-PMFHN. CPD of N or L severely reduced growth in vitro and was not further evaluated. CPD of P or M was associated with increased and decreased interferon (IFN) response in vitro, respectively, but had little effect on virus replication. In Vero cells, CPD of F and HN delayed virus replication, but final titers were comparable to wild-type (wt) HPIV3. In human lung epithelial A549 cells, CPD F and HN induced a stronger IFN response, viral titers were reduced 100-fold, and the expression of F and HN proteins was significantly reduced without affecting N or P or the relative packaging of proteins into virions. Following intranasal infection in hamsters, replication in the nasal turbinates and lungs tended to be the most reduced for viruses bearing CPD F and HN, with maximum reductions of approximately 10-fold. Despite decreased in vivo replication (and lower expression of CPD F and HN in vitro), all viruses induced titers of serum HPIV3-neutralizing antibodies similar to wt and provided complete protection against HPIV3 challenge. In summary, CPD of HPIV3 yielded promising vaccine candidates suitable for further development.

Intranasal respiratory syncytial virus vaccine attenuated by codon-pair deoptimization of seven open reading frames is genetically stable and elicits mucosal and systemic immunity and protection against challenge virus replication in hamsters.

Respiratory syncytial virus (RSV) is the most important viral agent of severe pediatric respiratory illness worldwide, but there is no approved pediatric vaccine. Here, we describe the development of the live-attenuated RSV vaccine candidate Min AL as well as engineered derivatives. Min AL was attenuated by codon-pair deoptimization (CPD) of seven of the 11 RSV open reading frames (ORFs) (NS1, NS2, N, P, M, SH and L; 2,073 silent nucleotide substitutions in total). Min AL replicated efficiently in vitro at the permissive temperature of 32°C but was highly temperature sensitive (shut-off temperature of 36°C). When serially passaged at increasing temperatures, Min AL retained greater temperature sensitivity compared to previous candidates with fewer CPD ORFs. However, whole-genome deep-sequencing of passaged Min AL revealed mutations throughout its genome, most commonly missense mutations in the polymerase cofactor P and anti-termination transcription factor M2-1 (the latter was not CPD). Reintroduction of selected mutations into Min AL partially rescued its replication in vitro at temperatures up to 40°C, confirming their compensatory effect. These mutations restored the accumulation of positive-sense RNAs to wild-type (wt) RSV levels, suggesting increased activity by the viral transcriptase, whereas viral protein expression, RNA replication, and virus production were only partly rescued. In hamsters, Min AL and derivatives remained highly restricted in replication in the upper and lower airways, but induced serum IgG and IgA responses to the prefusion form of F (pre F) that were comparable to those induced by wt RSV, as well as robust mucosal and systemic IgG and IgA responses against RSV G. Min AL and derivatives were fully protective against challenge virus replication. The derivatives had increased genetic stability compared to Min AL. Thus, Min AL and derivatives with selected mutations are stable, attenuated, yet highly-immunogenic RSV vaccine candidates that are available for further evaluation.

Mucosal prime-boost immunization with live murine pneumonia virus-vectored SARS-CoV-2 vaccine is protective in macaques.

Immunization via the respiratory route is predicted to increase the effectiveness of a SARS-CoV-2 vaccine. Here, we evaluate the immunogenicity and protective efficacy of one or two doses of a live-attenuated murine pneumonia virus vector expressing SARS-CoV-2 prefusion-stabilized spike protein (MPV/S-2P), delivered intranasally/intratracheally to male rhesus macaques. A single dose of MPV/S-2P is highly immunogenic, and a second dose increases the magnitude and breadth of the mucosal and systemic anti-S antibody responses and increases levels of dimeric anti-S IgA in the airways. MPV/S-2P also induces S-specific CD4+ and CD8+ T-cells in the airways that differentiate into large populations of tissue-resident memory cells within a month after the boost. One dose induces substantial protection against SARS-CoV-2 challenge, and two doses of MPV/S-2P are fully protective against SARS-CoV-2 challenge virus replication in the airways. A prime/boost immunization with a mucosally-administered live-attenuated MPV vector could thus be highly effective in preventing SARS-CoV-2 infection and replication.

Investigation of the antibacterial activity of benziothiazolinone against Xanthomonas oryzae pv. oryzae.

Plant pathogenic bacteria can cause numerous diseases for higher plants and result in severe reduction of crop yield. Introduction of new bactericides can always effectively control these plant diseases. Benziothiazolinone (BIT) is a novel fungicide registered in China for the control of plant fungal diseases, however, its anti-bacterial activity is not well studied. The results of activity tests showed that BIT exhibited stronger inhibitory activity against bacteria, particularly for Xanthomonas oryzae pv. oryzae (Xoo) (EC50 = 0.17 µg/mL), which was superior than that of the tested fungi in vitro. BIT also exhibited excellent protective and curative activity against rice bacterial leaf blight (BLB) caused by Xoo with the control efficacies of 71.37% and 91.64% at 600 µg/mL, respectively. After treatment with BIT, Xoo cell surface became wrinkled and the cell shape was distorted with extruding cellular content. It was also found that BIT decreased DNA synthesis and affected the biofilm formation and motility of Xoo cells. However, no significant change in the protein content was observed. Moreover, the results of quantitative real-time PCR also showed that expressions of several genes related to DNA synthesis, biofilm formation and motility of Xoo cells were down- or up-regulated, which further proved the anti-bacterial activity of BIT in influencing the biological properties of Xoo. Additionally, BIT also enhanced the activity of phenylalanine ammonia lyase (PAL), a plant defense enzyme. Taken together, benziothiazolinone might be served as an alternative candidate for the control of BLB.

Intranasal murine pneumonia virus-vectored SARS-CoV-2 vaccine induces mucosal and serum antibodies in macaques.

Next-generation SARS-CoV-2 vaccines are needed that induce systemic and mucosal immunity. Murine pneumonia virus (MPV), a murine homolog of respiratory syncytial virus, is attenuated by host-range restriction in nonhuman primates and has a tropism for the respiratory tract. We generated MPV vectors expressing the wild-type SARS-CoV-2 spike protein (MPV/S) or its prefusion-stabilized form (MPV/S-2P). Both vectors replicated similarly in cell culture and stably expressed S. However, only S-2P was associated with MPV particles. After intranasal/intratracheal immunization of rhesus macaques, MPV/S and MPV/S-2P replicated to low levels in the airways. Despite its low-level replication, MPV/S-2P induced high levels of mucosal and serum IgG and IgA to SARS-CoV-2 S or its receptor-binding domain. Serum antibodies from MPV/S-2P-immunized animals efficiently inhibited ACE2 receptor binding to S proteins of variants of concern. Based on its attenuation and immunogenicity in macaques, MPV/S-2P will be further evaluated as a live-attenuated vaccine for intranasal immunization against SARS-CoV-2.

Mucosal prime-boost immunization with live murine pneumonia virus-vectored SARS-CoV-2 vaccine is protective in macaques.

Immunization via the respiratory route is predicted to increase the effectiveness of a SARS-CoV-2 vaccine. We evaluated the immunogenicity and protective efficacy of one or two doses of a live-attenuated murine pneumonia virus vector expressing SARS-CoV-2 prefusion-stabilized spike protein (MPV/S-2P), delivered intranasally/intratracheally to rhesus macaques. A single dose of MPV/S-2P was highly immunogenic, and a second dose increased the magnitude and breadth of the mucosal and systemic anti-S antibody responses and increased levels of dimeric anti-S IgA in the airways. MPV/S-2P also induced S-specific CD4+ and CD8+ T-cells in the airways that differentiated into large populations of tissue-resident memory cells within a month after the boost. One dose induced substantial protection against SARS-CoV-2 challenge, and two doses of MPV/S-2P were fully protective against SARS-CoV-2 challenge virus replication in the airways. A prime/boost immunization with a mucosally-administered live-attenuated MPV vector could thus be highly effective in preventing SARS-CoV-2 infection and replication.

Increased risk of dementia among people with a history of fractures: a systematic review and meta-analysis of population-based studies.

Background: Emerging evidence suggests that there may be an association between a history of fractures and dementia risk, but the epidemiological findings are inconsistent. We, therefore, conducted a meta-analysis to systematically assess the risk of dementia among people with a history of fractures. Methods: We comprehensively searched four electronic databases (PubMed, Web of Science, Embase, and Cochrane Library) for relevant literature published from inception to 10 January 2023. Longitudinal observational studies that investigated the association between any type of fracture occurrence and the subsequent risk of dementia were included for qualitative and quantitative analysis. Risk estimates were pooled using fixed-effects or random-effects models according to the level of heterogeneity. The Newcastle-Ottawa scale was used to evaluate the risk of bias in the included studies. Results: A total of seven population-based studies involving 3,658,108 participants (136,179 with a history of fractures) were eventually included. Pooled results showed a significant association between fracture and subsequent risk of dementia [hazard ratio (HR) = 1.28, 95% confidence interval (CI): 1.11-1.48] in cohort studies. Patients with fractures at different sites showed a similar trend toward increased risk of subsequent dementia. No gender, age, region, duration of follow-up, study quality, or study design specificity were observed. Sensitivity analysis indicates that the current results are robust. No publication bias existed. The results were similar in the cohort study with the standardized incidence ratio (SIR) as the statistical measure (SIR = 1.58, 95% CI: 1.25-2.00) and in the case-control study (OR = 1.38, 95% CI: 1.18-1.61). Of note, the causal relationship between fracture and dementia was not demonstrated in this meta-analysis. Conclusion: People with a history of fractures are at increased risk of developing dementia. Enhanced screening and preventive management of dementia in people with a history of fractures may be beneficial.

Live-attenuated pediatric parainfluenza vaccine expressing 6P-stabilized SARS-CoV-2 spike protein is protective against SARS-CoV-2 variants in hamsters.

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 evaluated in vitro and 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.

Live-attenuated pediatric parainfluenza vaccine expressing 6P-stabilized SARS-CoV-2 spike protein is protective against SARS-CoV-2 variants in hamsters.

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 evaluated in vitro and 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 SUMMARY: SARS-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.

Intranasal pediatric parainfluenza virus-vectored SARS-CoV-2 vaccine is protective in monkeys.

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+ and CD8+ T cell responses, including tissue-resident memory cells in the lungs. Following challenge, SARS-CoV-2 replication was undetectable in airways and lung tissues of immunized macaques. B/HPIV3/S-6P will be evaluated clinically as pediatric intranasal SARS-CoV-2/parainfluenza virus type 3 vaccine.

IL-10 suppresses T cell expansion while promoting tissue-resident memory cell formation during SARS-CoV-2 infection in rhesus macaques.

The pro- and anti-inflammatory pathways that determine the balance of inflammation and viral control during SARS-CoV-2 infection are not well understood. Here we examine the roles of IFNγ and IL-10 in regulating inflammation, immune cell responses and viral replication during SARS-CoV-2 infection of rhesus macaques. IFNγ blockade tended to decrease lung inflammation based on 18 FDG-PET/CT imaging but had no major impact on innate lymphocytes, neutralizing antibodies, or antigen-specific T cells. In contrast, IL-10 blockade transiently increased lung inflammation and enhanced accumulation of virus-specific T cells in the lower airways. However, IL-10 blockade also inhibited the differentiation of virus-specific T cells into airway CD69 + CD103 + T RM cells. While virus-specific T cells were undetectable in the nasal mucosa of all groups, IL-10 blockade similarly reduced the frequency of total T RM cells in the nasal mucosa. Neither cytokine blockade substantially affected viral load and infection ultimately resolved. Thus, in the macaque model of mild COVID-19, the pro- and anti-inflammatory effects of IFNγ and IL-10 have no major role in control of viral replication. However, IL-10 has a key role in suppressing the accumulation of SARS-CoV-2-specific T cells in the lower airways, while also promoting T RM at respiratory mucosal surfaces.

Intranasal pediatric parainfluenza virus-vectored SARS-CoV-2 vaccine candidate is protective in macaques.

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 IgA and IgG responses. High levels of S-specific antibodies were also induced in serum, which efficiently neutralized SARS-CoV-2 variants of concern. Furthermore, B/HPIV3/S-6P induced robust systemic and pulmonary S-specific CD4+ and CD8+ T-cell responses, including tissue-resident memory cells in lungs. Following challenge, SARS-CoV-2 replication was undetectable in airways and lung tissues of immunized macaques. B/HPIV3/S-6P will be evaluated clinically as pediatric intranasal SARS-CoV-2/parainfluenza virus type 3 vaccine.

Intranasal immunization with avian paramyxovirus type 3 expressing SARS-CoV-2 spike protein protects hamsters against SARS-CoV-2.

Current vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are administered parenterally and appear to be more protective in the lower versus the upper respiratory tract. Vaccines are needed that directly stimulate immunity in the respiratory tract, as well as systemic immunity. We used avian paramyxovirus type 3 (APMV3) as an intranasal vaccine vector to express the SARS-CoV-2 spike (S) protein. A lack of pre-existing immunity in humans and attenuation by host-range restriction make APMV3 a vector of interest. The SARS-CoV-2 S protein was stabilized in its prefusion conformation by six proline substitutions (S-6P) rather than the two that are used in most vaccine candidates, providing increased stability. APMV3 expressing S-6P (APMV3/S-6P) replicated to high titers in embryonated chicken eggs and was genetically stable, whereas APMV3 expressing non-stabilized S or S-2P were unstable. In hamsters, a single intranasal dose of APMV3/S-6P induced strong serum IgG and IgA responses to the S protein and its receptor-binding domain, and strong serum neutralizing antibody responses to SARS-CoV-2 isolate WA1/2020 (lineage A). Sera from APMV3/S-6P-immunized hamsters also efficiently neutralized Alpha and Beta variants of concern. Immunized hamsters challenged with WA1/2020 did not exhibit the weight loss and lung inflammation observed in empty-vector-immunized controls; SARS-CoV-2 replication in the upper and lower respiratory tract of immunized animals was low or undetectable compared to the substantial replication in controls. Thus, a single intranasal dose of APMV3/S-6P was highly immunogenic and protective against SARS-CoV-2 challenge, suggesting that APMV3/S-6P is suitable for clinical development.

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 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.

A Parainfluenza Virus Vector Expressing the Respiratory Syncytial Virus (RSV) Prefusion F Protein Is More Effective than RSV for Boosting a Primary Immunization with RSV.

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.

Epstein-Barr Virus (EBV) Tegument Protein BGLF2 Suppresses Type I Interferon Signaling To Promote EBV Reactivation.

Interferon alpha (IFN-α) and IFN-ß 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 parainfluenza virus type 3 expressing the respiratory syncytial virus pre-fusion F protein modified for virion packaging yields protective intranasal vaccine candidates.

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.

The alpha-1 subunit of the Na+,K+-ATPase (ATP1A1) is required for macropinocytic entry of respiratory syncytial virus (RSV) in human respiratory epithelial cells.

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.

Effects of Alterations to the CX3C Motif and Secreted Form of Human Respiratory Syncytial Virus (RSV) G Protein on Immune Responses to a Parainfluenza Virus Vector Expressing the RSV G Protein.

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.