Severe Respiratory Tract Disease Research Articles

Faktor-Faktor yang Berhubungan dengan Kelengkapan Vaksinasi Covid-19 pada Masyarakat di Wilayah Kerja Puskesmas Kenali Besar Kota Jambi

The Covid-19 pandemic caused by the Sars Cov-2 virus has the effect of triggering mild to severe acute respiratory tract disease with symptoms namely fever, shortness of breath, cough, fatigue, runny nose, sore throat, diarrhea, loss of the ability to smell and taste on the tongue and can cause death. Indonesia is the country with the most new cases and deaths in Southeast Asia as of 24-30 October 2022 with 19,661 new cases or 7.2 new cases per 100,000 population, even though the previous week there were 14,093 cases. This means that the addition occurred as much as 40%. This type of research is quantitative with a cross sectional design. The population of this study was all people aged > 17 years in the working area of the Puskesmas Iknew Besar, totaling 42,796 people with a sample of 172 respondents. The sampling technique used multistage random sampling. Data were collected using questionnaires and observation sheets, then univariate data analysis using the chi-square test. The results of the multivariate analysis show that there is a relationship between the completeness of the Covid-19 vaccination and attitudes, history of Covid-19, history of non-communicable diseases, perception of Covid-19 risk, family support and health information among the community in the working area of the Puskesmas Kenali Besar, Jambi City in 2022.

Intranasal Vaccination with a Respiratory-Syncytial-Virus-Based Virus-like Particle Displaying the G Protein Conserved Region Induces Severe Weight Loss and Pathology upon Challenge with Wildtype Respiratory Syncytial Virus.

Respiratory syncytial virus (RSV) is a major cause of severe respiratory tract disease worldwide, and a pediatric vaccine is not available. We generated a filamentous RSV-based virus-like particle (VLP) that presents the central conserved region of the attachment protein G. This was achieved by co-expressing the matrix protein, phosphoprotein, nucleoprotein, and a hybrid fusion protein in which the F ectodomain was replaced with the G central region (GCR). The latter is relatively conserved and contains a receptor binding site and hence is a logical vaccine target. The immunogenicity and efficacy of the resulting VLP, termed VLP-GCR, were examined in mice using intranasal application without adjuvant. VLP-GCR induced substantial anti-N antibody levels but very low anti-G antibody levels, even after three vaccinations. In contrast, a VLP presenting prefusion-stabilized fusion (preF) protein instead of GCR induced both high anti-F and anti-nucleoprotein antibody levels, suggesting that our GCR antigen was poorly immunogenic. Challenge of VLP-GCR-vaccinated mice caused increased weight loss and lung pathology, and both VLPs induced mucus in the lungs. Thus, neither VLP is suitable as a vaccine for RSV-naive individuals. However, VLP-preF enhanced the proportion of preF antibodies and could serve as a multi-antigen mucosal booster vaccine in the RSV-experienced population.

Mucosal immunity in upper and lower respiratory tract to MERS-CoV.

Middle East respiratory syndrome coronavirus (MERS-CoV) has emerged as a deadly pathogen with a mortality rate of up to 36.2%. MERS-CoV can cause severe respiratory tract disease and multiorgan failure. Therefore, therapeutic vaccines are urgently needed. This intensive review explores the human immune responses and their immunological mechanisms during MERS-CoV infection in the mucosa of the upper and lower respiratory tracts (URT and LRT, respectively). The aim of this study is to provide a valuable, informative, and critical summary of the protective immune mechanisms against MERS-CoV infection in the URT/LRT for the purpose of preventing and controlling MERS-CoV disease and designing effective therapeutic vaccines. In this review, we focus on the immune potential of the respiratory tract following MERS-CoV infection. We searched PubMed, Embase, Web of Science, Cochrane, Scopus, and Google Scholar using the following terms: "MERS-CoV", "B cells", "T cells", "cytokines", "chemokines", "cytotoxic", and "upper and lower respiratory tracts". We found and included 152 studies in this review. We report that the cellular innate immune response, including macrophages, dendritic cells, and natural killer cells, produces antiviral substances such as interferons and interleukins to prevent the virus from spreading. In the adaptive and humoral immune responses, CD4+ helper T cells, CD8+ cytotoxic T cells, B cells, and plasma cells protect against MERS-CoV infection in URT and LRT. The human nasopharynx-associated lymphoid tissue (NALT) and bronchus-associated lymphoid tissue (BALT) could successfully limit the spread of several respiratory pathogens. However, in the case of MERS-CoV infection, limited research has been conducted in humans with regard to immunopathogenesis and mucosal immune responses due to the lack of relevant tissues. A better understanding of the immune mechanisms of the URT and LRT is vital for the design and development of effective MERS-CoV vaccines.

Clinical characterization of human bocavirus 1 infection in infants hospitalized in an intensive care unit for severe acute respiratory tract disease

Acute respiratory infections represent the leading cause of morbimortality in children and viruses are the main etiological agents. Here we describe the clinical characteristics and evolution of infants admitted to intensive care unit with severe acute respiratory infection (SARI) due to Human Bocavirus 1 mono-infection in patients without previous comorbidity. We also compared them with respiratory syncytial virus (RSV) cases. Of 141 cases included (age 5.43 ± 4.54 months, 52% male), 80% had at least 1 virus detected. RSV was the most frequent in the series (71.6%) followed by HBoV1 (28%). Five cases of HBoV1 mono-detection were identified. Pediatric acute respiratory distress syndrome was present in both groups, HBoV1 and RSV. The clinical presentation and evolution of HBoV1 single infection was similar to RSV. HBoV1 should be included among the agents investigated in cases of SARI in infants.

Feline Calicivirus P39 Inhibits Innate Immune Responses by Autophagic Degradation of Retinoic Acid Inducible Gene I.

Feline calicivirus (FCV) is a feline pathogen that can cause severe upper respiratory tract disease in cats, thus posing a major threat to their health. The exact pathogenic mechanism of FCV is still unclear, although it has been identified as having the ability to induce immune depression. In this study, we discovered that FCV infection triggers autophagy and that its non-structural proteins, P30, P32, and P39, are responsible for initiating this process. Additionally, we observed that altering autophagy levels via chemical modulation resulted in different influences on FCV replication. Moreover, our findings indicate that autophagy can modify the innate immunity induced by FCV infection, with increased autophagy further suppressing FCV-induced RIG-I signal transduction. This research provides insights into the mechanism of FCV replication and has the potential to aid in the development of autophagy-targeted drugs to inhibit or prevent FCV infection.

Enhancing Anti-G Antibody Induction by a Live Single-Cycle Prefusion F-Expressing RSV Vaccine Improves In Vitro and In Vivo Efficacy.

The human respiratory syncytial virus (RSV) is a major cause of severe respiratory tract disease, and a vaccine is not available. We previously reported a novel live vaccine expressing prefusion-stabilized fusion protein (preF) in place of the native F protein (RSV-preFΔCT). As preF is non-functional, RSV-preFΔCT was amplified in a production line expressing a functional substitute, and exhibited a single-cycle replication phenotype, which holds several unique potential advantages. RSV-preFΔCT prevented shedding and lung pathology after viral challenge in mice, but induced low levels of anti-attachment protein (G) antibodies (Abs). Given the significant contributions of anti-G Abs toward disease prevention, we generated modifications to RSV-preFΔCT in an effort to induce higher anti-G Ab levels. The Ab levels were monitored after the prime-boost vaccination of mice with modified vaccines. The most successful modification for enhancing induced anti-G Abs was seen with the placement of G in the first genome position. This vaccine also reduced the pathology after challenge with a high dose of wt RSV, and outperformed the sera from wt RSV-vaccinated mice in in vitro neutralization. Thus, raising the anti-G Ab levels induced by RSV-preFΔCT enhanced efficacy in vitro and in vivo, and constitutes an important next step in developing a live, single-cycle, efficacious vaccine for the human population.

SARS-CoV-2 VIRUS: ISOLATION, GROWTH, THERMOSTABILITY, INACTIVATION AND PASSAGES

Coronaviruses are a large family of RNA-containing viruses capable of infecting humans and a number of animal species. Several species of coronaviruses are now known to circulate in the population and generally cause moderate to severe upper respiratory tract and lung disease. A new coronavirus infection, COVID-19 (SARS-CoV-2), has been added to this list. The virus has spread rapidly around the world. Recently in Kazakhstan, this infection has been observed with pronounced pneumonia among the population. In developing the vaccine, we had to work with live SARS-CoV-2 virus. It should be noted that when working with live virus, we faced some issues concerning the biological and physicochemical properties of the virus, which have remained outside the attention of scientists of the world. In this connection, in these studies we studied some physicochemical and biological properties of Kazakh strain of SARS-CoV-2 virus isolated from a patient with new coronavirus infection COVID-19, such as thermal stability (persistence), virus inactivation by different concentrations of formaldehyde at different temperature and time regimes and virus passivation in cell culture. Studies were conducted to find additional biological models sensitive to the SARS-CoV-2 virus and used in vitro. Of the 11 cell culture species tested, monkey and pig cell cultures were found to be the most sensitive to the SARS-CoV-2 virus, in which the virus was actively reproducing. We studied the effect of storage conditions of SARS-CoV-2 nucleic acid samples as well as the effect of environmental conditions on the SARS-CoV-2 virus itself. The results of our studies showed that freezing at minus 70 оС and thawing at room temperature (20-22 оС) had no appreciable effect on the qualitative character of cytopathogenicity but had a negative effect on the preservation of virus titer. Data on the effects of chemical factors when working with pathogens are necessary to create conditions of biological safety for personnel and the environment, as well as the design of technologies for the manufacture of biological products for diagnostic and prophylactic purposes. In this study, we studied the effect of formaldehyde to the virus. The results obtained show that inactivation of Sars-CoV-2 virus with formalin depends on formaldehyde concentration, reaction temperature, incubation time.

Treatment of psoriasis with biologics in the early COVID-19 pandemic: A study examining patient attitudes toward the treatment and disease course.

BackgroundSince March 2020, the coronavirus disease 2019 (COVID‐19) pandemic has been ongoing all around the world with a wide range of clinical course including asymptomatic cases to severe and fatal respiratory tract disease. Patients on immunosuppressive treatments were predicted to be more susceptible to COVID‐19.AimsIt was aimed to assess treatment continuity, the course of psoriasis and the course and clinical features of COVID‐19 in patients treated with biological agents for psoriasis at the early initial period of COVID‐19 pandemic.Patients/MethodsPatients treated with biological agents for psoriasis at our institute were contacted by phone between 1 and 10 July 2020 and fulfilled a questionnaire about their continuity to psoriasis treatments, clinical course of psoriasis, and any suspicion/diagnosis of COVID‐19.ResultsA total of 106 patients, 41 females and 65 males, were enrolled. Mean age of the patients was 46.1 ± 12.1 years (range: 19–77). Median duration of psoriasis was 18 years (min–max: 1 month–51 years). Twenty‐four patients (22.6%) were using tumor necrosis alpha inhibitors (ETA:1, IFX:19, ADA:4), whereas 82 patients (77.4%) were using interleukin (IL) 12/23 or IL‐17 inhibitors (UST:48, SECU:30, IXE:4). Seventy‐six patients (71.7%) continued the treatment, whereas 30 patients (28.3%) interrupted the treatment voluntarily. Twenty out of 30 patients (66.6%) who interrupted the treatment had an exacerbation of psoriasis. None of the patients were diagnosed with COVID‐19 in the study period.ConclusionPatients with psoriasis who received biological therapy continued their treatment at a high rate during the early period of the COVID‐19 pandemic. No COVID‐19 diagnosis was made among patients whether they continued or discontinued treatment. Recurrence and exacerbation of psoriasis in a significant proportion of patients who interrupted treatment and absence of COVID‐19 diagnosis in each group support the importance and safety of continuity of biological treatments for psoriasis in COVID‐19 era.

A Review on Potentialities of Selenium Nanoparticles and Its Application Using Air Borne Fungus

Airborne fungal pathogens are known as pathogens and cause number of diseases including infections of skin and severe respiratory tract diseases. The presence of mycotoxins in fungi are found responsible for causing infections and these mycotoxins degrade substances also. Keeping in view of this property, a number of researchers explored different fungal species to synthesize nanoparticles which exhibit promising therapeutic properties. Some of the examples of fungi used for nanoparticles include Aspergillus and Trichoderma. The biosynthesis of fungi based nanoparticles is safe, eco-friendly, biocompatible and low cost. Present review deals with the synthesis of selenium nanoparticles using air borne fungus. Selenium is one of the micronutrient required by plants in trace amounts also has therapeutic properties. But large amount of selenium is toxic and may be hazadorous when enters via food chain. Nanoselenium has similar bioactivity like other forms of selenium in humans and has many biological applications in the field of medical and pharmaceutical research to combat threats to number of diseases and for human health. Biogenic SeNPs have antimicrobial, anticancer (cytotoxic), antioxidant activity. The present review emphasizes on myconanotechnology and its application, synthesis of myconanoparticles. Application of selenium and its therapeutic properties as antimicrobial, anticancer and antiviral, whereas can be used as remedy for number of diseases. Collectively, self-assembly of SeNPs-fungal complexes affects their (patho) biological identity, which may impact human health and ecology.

Comparative Study of the Pathogenesis of Rhinopneumonitis Induced by Intranasal Inoculation of Hamsters with Equine Herpesvirus-9, Equine Herpesvirus-1 strain Ab4p and Zebra-borne Equine Herpesvirus-1

Equine herpesvirus-9 (EHV-9), equine herpesvirus-1 (EHV-1) and zebra-borne EHV-1 are members of the family Herpesviridae and cause encephalitis and rhinopneumonitis in a range of animal species. The aim of this study was to characterize and compare the rhinopneumonitis induced by experimental intranasal inoculation of groups of hamsters with EHV-9, EHV-1 strain Ab4p or zebra-borne EHV-1 viruses. Animals inoculated with EHV-9 had earlier and more severe neurological and respiratory signs than those inoculated with EHV-1 strain Ab4p or zebra-borne EHV-1. At 4-5 days post inoculation (dpi), hamsters inoculated with EHV-9 had significantly increased expression of open reading fame (ORF) 30, the viral gene encoding the DNA polymerase, in lung tissue. ORF 30 expression at these time points was higher in the hamsters infected with EHV-9 than in those inoculated with the other two viruses. Severe, mild or very mild rhinitis was seen in animals inoculated with EHV-1 strain Ab4p, EHV-9 and zebra-borne EHV-1, respectively. Viral antigen was detected in olfactory receptor neurons, inflammatory cells and desquamated epithelial cells in animals in all groups until 5 dpi. Tracheitis was also seen in all three virus-infected groups with viral antigen detected in tracheal epithelium. Inoculated hamsters developed interstitial pneumonia of increasing severity over the course of the experiment. Bronchopneumonia and vasculitis were also seen in all three infected groups. These results confirm that, in addition to their neurotropism, EHV-9 and zebra-borne EHV-1 are pneumotropic viruses. EHV-1 strain Ab4p caused more severe upper respiratory tract disease, but no significant differences were detected in the severity of pneumonia induced by each virus.

WITHDRAWN: Nonglycosylated RSV F protein elicited protective immunity in mice

Abstract Human respiratory syncytial virus (RSV) is the primary cause of severe respiratory tract disease in infants and young children, but no vaccine is yet on the market. The fusion (F) protein of RSV, especially the prefusion F protein (pre-F), is the best antigen for eliciting a protective immunity and neutralizing antibody response in the host. In this study, we constructed and purified the nonglycosylated pre-F protein using Escherichia coli BL21, called pre-RBF. We found that pre-RBF could be produced at high levels with stable trimer conformations. Importantly, pre-RBF was shown to bind 5C4, a monoclonal antibody specific for the pre-F. Compared to mice immunized with formalin-inactivated RSV (FI-RSV), mice immunized with different doses of pre-RBF all showed superior protective immunity, which was confirmed by serum-neutralizing activity and viral clearance after challenge. Pre-RBF induced a balanced Th1-biased immunity response, whereas FI-RSV induced Th2-biased immune responses. So pre-RBF represents a potential vaccine candidate for the prevention of human infection with RSV.

Human respiratory syncytial virus F protein expressed in Pichia pastoris or Escherichia coli induces protective immunity without inducing enhanced respiratory disease in mice.

Human respiratory syncytial virus (hRSV) is the primary cause of severe respiratory tract disease in children and infants as well as in elderly and immunocompromised adults. The fusion protein (F) of hRSV is the major antigen eliciting a neutralizing antibody response and protective immunity in the host, especially those recognizing the prefusion F protein (pre-F). In this study, we made genetic constructs for expression of a recombinant prefusion F protein in Pichia pastoris GS115, called RGF. Using Escherichia coli BL21, we expressed the pre-F and postfusion F protein (Post-F), called RBF and Post-RBF, respectively. RGF and RBF showed high affinity for 5C4, a highly potent monoclonal antibody specific for pre-F. We studied the immunogenicity of RGF and RBF in mice. Compared to mice immunized with formalin-inactivated RSV (FI-RSV), mice immunized with RGF or RBF exhibited superior protective immunity, which was confirmed by serum neutralizing activity and viral clearance after challenge. As judged from the IgG1/IgG2a ratios and numbers of IFN-γ- and IL-4-secreting cells, RGF or RBF with alum adjuvant induced a balanced Th1-biased immune response and produced no signs of enhanced respiratory disease (ERD) upon hRSV challenge. In addition, the immunogenicity and protective efficacy of RGF were superior to those of RBF in mice. Therefore, RGF represents a potential vaccine candidate for the prevention of human infection with hRSV.

Expanding Diversity of Susceptible Hosts in Peste Des Petits Ruminants Virus Infection and Its Potential Mechanism Beyond.

Peste des petits ruminants (PPR) is a severe respiratory and digestive tract disease of domestic small ruminants caused by PPR virus (PPRV) of the genus Morbillivirus. Although the primary hosts of PPRV are goats and sheep, the host range of PPRV has been continuously expanding and reported to infect various animal hosts over the last decades, which could bring a potential challenge to effectively control and eradicate PPR globally. In this review, we focused on current knowledge about host expansion and interspecies infection of PPRV and discussed the potential mechanisms involved.

In vivo comparison of a laboratory-adapted and clinical-isolate-based recombinant human respiratory syncytial virus.

Human respiratory syncytial virus (HRSV) is the leading cause of severe respiratory tract disease in infants. Most HRSV infections remain restricted to the upper respiratory tract (URT), but in a small percentage of patients the infection spreads to the lower respiratory tract, resulting in bronchiolitis or pneumonia. We have a limited understanding of HRSV pathogenesis and what factors determine disease severity, partly due to the widespread use of tissue-culture-adapted viruses. Here, we studied early viral dissemination and tropism of HRSV in cotton rats, BALB/cJ mice and C57BL/6 mice. We used a novel recombinant (r) strain based on a subgroup A clinical isolate (A11) expressing EGFP [rHRSVA11EGFP(5)]. A recombinant laboratory-adapted HRSV strain [rHRSVA2EGFP(5)] was used as a direct comparison. Our results show that rHRSVA11EGFP(5) replicated to higher viral titres than laboratory-adapted rHRSVA2EGFP(5) in the URT of cotton rats and mice. HRSV-infected cells were detected as early as 2 days post-inoculation in both species in the nasal septa and lungs. Infection was predominantly present in ciliated epithelial cells in cotton rats and in the olfactory mucosa of mice. In our opinion, this study highlights that the choice of virus strain is important when studying HRSV pathogenesis in vivo and demonstrates that A11 is a representative clinical-based virus. Additionally, we show critical differences in tropism and inflammation when comparing HRSV infection of cotton rats and mice.

Development of two antigen-binding fragments to a conserved linear epitope of human adenovirus and their application in immunofluorescence

Detection of human adenoviruses (HAdVs) in nasopharyngeal swab samples by immunofluorescence assay (IFA) will be valuable for diagnosing HAdV infection, which is a leading cause of severe respiratory tract disease, and will help in curbing the spread of HAdV. Monoclonal antibodies employed in IFA for HAdV detection should ideally target highly conserved epitope types. Here, we describe the development of two antigen-binding fragments (Fabs) with specific reactivity to HAdV using phage antibody library technology. When tested with IFA, both Fabs recognized cells infected with several types of HAdV, some of which have been identified in epidemics globally, or associated with outbreaks of severe or fatal acute respiratory diseases. The specificity and cross-reactivity of both Fabs to HAdVs indicated that the generated Fabs could be applied in the development of IFAs to detect HAdVs. Both Fabs bound to the knob proteins, as shown by chemiluminescence enzyme immunoassay and western blot. In addition, epitope mapping showed that both Fabs recognized a conserved linear epitope among several types of HAdV. Two different Fabs recognized the same epitope, suggesting that the epitope triggered the production of at least two kinds of antibodies in the body. The generated Fabs exerted no neutralization against HAdVs. The results demonstrate that both Fabs bind to an epitope that plays no role in neutralization of HAdV.

Involvement of Actin-Regulating Factor Cofilin in the Inclusion Body Formation and RNA Synthesis of Human Parainfluenza Virus Type 3 via Interaction With the Nucleoprotein.

Human parainfluenza virus type 3 (HPIV3) is one of the primary pathogens that causing severe respiratory tract diseases in newborns and infants. It could induce inclusion bodies (IBs) in infected cells. Comprised of viral nucleoprotein (N) and phosphoprotein (P), as well as some cellular factors, HPIV3 IBs are unique platform for efficient viral synthesis. Although several studies have demonstrated the formation of IBs, little is known about cellular proteins involved in HPIV3 IBs formation. By quantitative real-time PCR assays after cytochalasin D treatment, we found actin microfilaments of the cytoskeleton were indispensible for HPIV3 RNA synthesis. Using co-immunoprecipitation and immunofluorescence assays, an actin-modulating protein, cofilin was found to involve in the IBs formation through interaction with the N protein in N–P induced IBs complex. Viral IBs formation reduced upon RNA interference knockdown of cellular cofilin, thus viral RNA synthesis and protein expression level were also suppressed. What’s more, the inactive form of cofilin, p-cofilin was increased after HPIV3 infection, and phosphorylation of cofilin was required for interacting with N–P complex and IBs formation. We further identified that the regions in cofilin interacting with N protein lies in the C-terminus. Our findings for the first time to state that cellular cofilin involves in HPIV3 IBs and interaction with N is critical for cofilin to aid IBs formation and enhancing viral RNA synthesis.

A Dual-Replicon Shuttle Vector System for Heterologous Gene Expression in a Broad Range of Gram-Positive and Gram-Negative Bacteria.

Origin of replication (ori in theta-replicating plasmids or dso in rolling circle replicating plasmids) initiates plasmid replication in a broad range of bacteria. These two kinds of plasmids were both identified in Streptococcus, a genus composed of both human commensal bacteria and pathogens with the ability to cause severe community-acquired infections, including meningitides, septicemia, and respiratory tract diseases. Given the important roles of Streptococcus in the exchange of genetic elements with other symbiotic microbes, the genotypes and phenotypes of both Streptococcus spp. and other symbiotic species could be changed during colonization of the host. Therefore, an improved plasmid system is required to study the functional, complicated, and changeable genomes of Streptococcus. In this study, a dual-replicon shuttle vector system named pDRE was constructed to achieve heterologous gene expression. The vector system contained theta replicon for Escherichia coli. The origin of rolling circle replicon was synthesized according to pMV158 in Gram-positive bacteria. By measuring the products of inserted genes at multiple cloning sites, the ability of this vector system in the replication and expression of heterologous genes was assessed in four Streptococcus and three other Gram-positive bacteria: Bacillus subtilis, Lactococcus lactis, and Staphylococcus aureus. The results showed that the newly constructed vector could simultaneously replicate and express heterologous genes in a broad range of Gram-positive and Gram-negative bacteria, thus providing a potentially powerful genetic tool for further functional analysis.

Nanoparticle decoration impacts airborne fungal pathobiology

Airborne fungal pathogens, predominantly Aspergillus fumigatus, can cause severe respiratory tract diseases. Here we show that in environments, fungal spores can already be decorated with nanoparticles. Using representative controlled nanoparticle models, we demonstrate that various nanoparticles, but not microparticles, rapidly and stably associate with spores, without specific functionalization. Nanoparticle-spore complex formation was enhanced by small nanoparticle size rather than by material, charge, or "stealth" modifications and was concentration-dependently reduced by the formation of environmental or physiological biomolecule coronas. Assembly of nanoparticle-spore surface hybrid structures affected their pathobiology, including reduced sensitivity against defensins, uptake into phagocytes, lung cell toxicity, and TLR/cytokine-mediated inflammatory responses. Following infection of mice, nanoparticle-spore complexes were detectable in the lung and less efficiently eliminated by the pulmonary immune defense, thereby enhancing A. fumigatus infections in immunocompromised animals. Collectively, self-assembly of nanoparticle-fungal complexes affects their (patho)biological identity, which may impact human health and ecology.

Improved Prefusion Stability, Optimized Codon Usage, and Augmented Virion Packaging Enhance the Immunogenicity of Respiratory Syncytial Virus Fusion Protein in a Vectored-Vaccine Candidate

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.

Ferrets as a Novel Animal Model for Studying Human Respiratory Syncytial Virus Infections in Immunocompetent and Immunocompromised Hosts.

Human respiratory syncytial virus (HRSV) is an important cause of severe respiratory tract disease in immunocompromised patients. Animal models are indispensable for evaluating novel intervention strategies in this complex patient population. To complement existing models in rodents and non-human primates, we have evaluated the potential benefits of an HRSV infection model in ferrets (Mustela putorius furo). Nine- to 12-month-old HRSV-seronegative immunocompetent or immunocompromised ferrets were infected with a low-passage wild-type strain of HRSV subgroup A (105 TCID50) administered by intra-tracheal or intra-nasal inoculation. Immune suppression was achieved by bi-daily oral administration of tacrolimus, mycophenolate mofetil, and prednisolone. Throat and nose swabs were collected daily and animals were euthanized four, seven, or 21 days post-infection (DPI). Virus loads were determined by quantitative virus culture and qPCR. We observed efficient HRSV replication in both the upper and lower respiratory tract. In immunocompromised ferrets, virus loads reached higher levels and showed delayed clearance as compared to those in immunocompetent animals. Histopathological evaluation of animals euthanized 4 DPI demonstrated that the virus replicated in the respiratory epithelial cells of the trachea, bronchi, and bronchioles. These animal models can contribute to an assessment of the efficacy and safety of novel HRSV intervention strategies.