Regulated microbe vaccines: from concept to (pre-clinical) reduction to practice

Asthma and respiratory syncytial virus infection in infancy: is there a link?

Role of respiratory pathogens in infants hospitalized for a first episode of wheezing and their impact on recurrences

Intranasal DNA Vaccination Induces Potent Mucosal and Systemic Immune Responses and Cross-protective Immunity Against Influenza Viruses

Respiratory syncytial virus (RSV) is a major cause of serious acute lower respiratory tract infection in infants and the elderly. The lack of a licensed RSV vaccine calls for the development of vaccines with other targets and vaccination strategies. Here, we construct a recombinant protein, designated P-KFD1, comprising RSV phosphoprotein (P) and the E.-coli-K12-strain-derived flagellin variant KFD1. Intranasal immunization with P-KFD1 inhibits RSV replication in the upper and lower respiratory tract and protects mice against lung disease without vaccine-enhanced disease (VED). The P-specific CD4+ Tcells provoked by P-KFD1 intranasal (i.n.) immunization either reside in or migrate to the respiratory tract and mediate protection against RSV infection. Single-cell RNA sequencing (scRNA-seq) and carboxyfluorescein succinimidyl ester (CFSE)-labeled cell transfer further characterize the Th1 and Th17 responses induced by P-KFD1. Finally, we find that anti-viral protection depends on either interferon-γ (IFN-γ) or interleukin-17A (IL-17A). Collectively, P-KFD1 is a promising safe and effective mucosal vaccine candidate for the prevention of RSV infection.

Antiviral biomaterials

Detection of viral, Chlamydia pneumoniae and Mycoplasma pneumoniae infections in exacerbations of asthma in children.

Infections with influenza and respiratory syncytial virus (RSV) rank high among the most common human respiratory diseases worldwide. Previously, we developed a replication-incompetent influenza virus by replacing the coding sequence of the PB2 gene, which encodes one of the viral RNA polymerase subunits, with that of a reporter gene. Here, we generated a PB2-knockout recombinant influenza virus expressing the F protein of RSV (PB2-RSVF virus) and tested its potential as a bivalent vaccine. In mice intranasally immunized with the PB2-RSVF virus, we detected high levels of antibodies against influenza virus, but not RSV. PB2-RSVF virus-immunized mice were protected from a lethal challenge with influenza virus but experienced severe body weight loss when challenged with RSV, indicating that PB2-RSVF vaccination enhanced RSV-associated disease. These results highlight one of the difficulties of developing an effective bivalent vaccine against influenza virus and RSV infections.

The long-term control strategy of SARS-CoV-2 and other major respiratory viruses needs to include antivirals to treat acute infections, in addition to the judicious use of effective vaccines. Whilst COVID-19 vaccines are being rolled out for mass vaccination, the modest number of antivirals in use or development for any disease bears testament to the challenges of antiviral development. We recently showed that non-cytotoxic levels of thapsigargin (TG), an inhibitor of the sarcoplasmic/endoplasmic reticulum (ER) Ca2+ ATPase pump, induces a potent host innate immune antiviral response that blocks influenza A virus replication. Here we show that TG is also highly effective in blocking the replication of respiratory syncytial virus (RSV), common cold coronavirus OC43, SARS-CoV-2 and influenza A virus in immortalized or primary human cells. TG’s antiviral performance was significantly better than remdesivir and ribavirin in their respective inhibition of OC43 and RSV. Notably, TG was just as inhibitory to coronaviruses (OC43 and SARS-CoV-2) and influenza viruses (USSR H1N1 and pdm 2009 H1N1) in separate infections as in co-infections. Post-infection oral gavage of acid-stable TG protected mice against a lethal influenza virus challenge. Together with its ability to inhibit the different viruses before or during active infection, and with an antiviral duration of at least 48 h post-TG exposure, we propose that TG (or its derivatives) is a promising broad-spectrum inhibitor against SARS-CoV-2, OC43, RSV and influenza virus.

Influenza virus, respiratory syncytial virus, parainfluenza virus, and metapneumovirus are the most common viruses associated with acute lower respiratory infections in young children (<5 years) and older people (≥65 years). A global report of the monthly activity of these viruses is needed to inform public health strategies and programmes for their control. In this systematic analysis, we compiled data from a systematic literature review of studies published between Jan 1, 2000, and Dec 31, 2017; online datasets; and unpublished research data. Studies were eligible for inclusion if they reported laboratory-confirmed incidence data of human infection of influenza virus, respiratory syncytial virus, parainfluenza virus, or metapneumovirus, or a combination of these, for at least 12 consecutive months (or 52 weeks equivalent); stable testing practice throughout all years reported; virus results among residents in well-defined geographical locations; and aggregated virus results at least on a monthly basis. Data were extracted through a three-stage process, from which we calculated monthly annual average percentage (AAP) as the relative strength of virus activity. We defined duration of epidemics as the minimum number of months to account for 75% of annual positive samples, with each component month defined as an epidemic month. Furthermore, we modelled monthly AAP of influenza virus and respiratory syncytial virus using site-specific temperature and relative humidity for the prediction of local average epidemic months. We also predicted global epidemic months of influenza virus and respiratory syncytial virus on a 5° by 5° grid. The systematic review in this study is registered with PROSPERO, number CRD42018091628. We initally identified 37 335 eligible studies. Of 21 065 studies remaining after exclusion of duplicates, 1081 full-text articles were assessed for eligibility, of which 185 were identified as eligible. We included 246 sites for influenza virus, 183 sites for respiratory syncytial virus, 83 sites for parainfluenza virus, and 65 sites for metapneumovirus. Influenza virus had clear seasonal epidemics in winter months in most temperate sites but timing of epidemics was more variable and less seasonal with decreasing distance from the equator. Unlike influenza virus, respiratory syncytial virus had clear seasonal epidemics in both temperate and tropical regions, starting in late summer months in the tropics of each hemisphere, reaching most temperate sites in winter months. In most temperate sites, influenza virus epidemics occurred later than respiratory syncytial virus (by 0·3 months [95% CI -0·3 to 0·9]) while no clear temporal order was observed in the tropics. Parainfluenza virus epidemics were found mostly in spring and early summer months in each hemisphere. Metapneumovirus epidemics occurred in late winter and spring in most temperate sites but the timing of epidemics was more diverse in the tropics. Influenza virus epidemics had shorter duration (3·8 months [3·6 to 4·0]) in temperate sites and longer duration (5·2 months [4·9 to 5·5]) in the tropics. Duration of epidemics was similar across all sites for respiratory syncytial virus (4·6 months [4·3 to 4·8]), as it was for metapneumovirus (4·8 months [4·4 to 5·1]). By comparison, parainfluenza virus had longer duration of epidemics (6·3 months [6·0 to 6·7]). Our model had good predictability in the average epidemic months of influenza virus in temperate regions and respiratory syncytial virus in both temperate and tropical regions. Through leave-one-out cross validation, the overall prediction error in the onset of epidemics was within 1 month (influenza virus -0·2 months [-0·6 to 0·1]; respiratory syncytial virus 0·1 months [-0·2 to 0·4]). This study is the first to provide global representations of month-by-month activity of influenza virus, respiratory syncytial virus, parainfluenza virus, and metapneumovirus. Our model is helpful in predicting the local onset month of influenza virus and respiratory syncytial virus epidemics. The seasonality information has important implications for health services planning, the timing of respiratory syncytial virus passive prophylaxis, and the strategy of influenza virus and future respiratory syncytial virus vaccination. European Union Innovative Medicines Initiative Respiratory Syncytial Virus Consortium in Europe (RESCEU).

Aims:To develop experimental bivalent vaccines against influenza and RSV using a cold-adapted LAIV backbone.Background:Respiratory syncytial virus (RSV) is a causative agent of bronchiolitis and pneumonia in young children, elderly and immunocompromised adults. No vaccine against RSV has been licensed to date for various reasons. One of the promising platforms for designing RSV vaccine is the use of live attenuated influenza vaccine (LAIV) viruses to deliver RSV epitopes to the respiratory mucosa.Objective:To generate recombinant LAIV viruses encoding a neutralizing epitope of the RSV fusion protein and assess their protective potential against both influenza and RSV infections in a mouse model.Methods:Reverse genetics methods were used to rescue recombinant LAIV+HA/RSV viruses expressing chimeric hemagglutinins encoding the RSV-F epitope at its N-terminus using two different flexible linkers. BALB/c mice were intranasally immunized with two doses of the recombinant viruses and then challenged with the influenza virus or RSV. The LAIV viral vector and formalin-inactivated RSV (FI-RSV) were included as control vaccines. Protection was assessed by the reduction of virus pulmonary titers. In addition, RSV-induced lung pathology was evaluated by histopathology studies.Results:Two rescued chimeric LAIV+HA/RSV viruses were identical to the LAIV vector in terms of replication capacityin vitroandin vivo. The RSV-F neutralizing epitope was successfully expressed only if inserted into the HA molecule via G-linker, but not A-linker. Both chimeric viruses induced high influenza-specific antibody levels and fully protected mice against a lethal influenza challenge virus. However, they induced weak anti-RSV antibody responses which did not prevent RS virus replication upon challenge, and only LAIV-HA+G-RSV variant protected mice against RSV-induced lung pathology.Conclusion:Although the designed LAIV-RSV chimeric viruses were unable to neutralize the RS virus pulmonary replication, the LAIV-HA+G-RSV reduced RSV-induced lung pathology and can be considered a promising bivalent vaccine against influenza and RSV infections and warrants its further development.

Cytotoxic T lymphocyte (CTL)-mediated death of virus-infected cells requires prior recognition of short viral peptide antigens that are presented by human leukocyte antigen (HLA) class I molecules on the surface of infected cells. The CTL response is critical for the clearance of human respiratory syncytial virus (HRSV) infection. Using mass spectrometry analysis of complex HLA-bound peptide pools isolated from large amounts of HRSV-infected cells, we identified nine naturally processed HLA-B27 ligands. The isolated peptides are derived from six internal, not envelope, proteins of the infective virus. The sequences of most of these ligands are not conserved between different HRSV strains, suggesting a mechanism to explain recurrent infection with virus of different HRSV antigenic subgroups. In addition, these nine ligands represent a significant fraction of the proteome of this virus, which is monitored by the same HLA class I allele. These data have implications for vaccine development as well as for analysis of the CTL response.

Community-acquired respiratory viruses

Objective To investigate the status of influenza virus (IFV) in children with acute lower respiratory tract infection (ALRTI) in Beijing, and to compare the clinical characteristics of ALRTI with single IFV and respiratory syncytial virus(RSV) in children. Methods Throat swab of nasopharyngeal aspirate specimens were collec-ted from children with ALRTI and tested for the presence of respiratory viruses (such as RSV, parainfluenza virus, rhinovirus and IFV) by reverse transcription-polymerase chain reaction.The epidemiological features of patients with influenza A, B or C virus (IFA, IFB, IFC) infection were analyzed.Clinical features of patients with single IFV and single RSV infection were compared. Results (1) During March 2007 and March 2014, at least one virus was detected in 2 697 (67.2%)of 4 012 children.There were 256 samples positive for IFV (6.4%), including 154 samples for IFA (3.8%), 97 samples for IFB (2.4%), and 6 samples for IFC (0.1%). (2) The IFV positive rate of 6 years groups were 4.1%, 7.6%, 12.0%, 7.8% and 4.8%, respectively.The 1-3 years-old group showed the highest positive rate of IFV.The positive rate of IFV showed no difference between males and females(6.7% vs 5.9%, χ2=2.63, P>0.05) (3) There were 16, 15 and 175 hospitalized children in single IFV, single IFB and single RSV infection group, respectively.The rate of <1-year children in single IFA(43.8% vs 84.0%, χ2 =12.74, P<0.001 or IFB (33.3% vs 84.0%, χ2 =19.11, P<0.001)infection group were both higher than it in single RSV infection group, and there was a significant difference.The incident rate of severe pneumonia in single IFA or IFB infection groups were both higher than that in single RSV infection group significant difference (31.2%, 26.7% vs 4.0%; χ2=18.49, 13.01, all P<0.001). The incident rate of trachea cannula in single IFA and IFB infection groups were higher than in single RSV infection group, and the difference was significant(25.0%, 13.3% vs 1.7%; χ2=22.51, 7.28, P<0.001, P=0.001). There was no significant difference in other clinical features, diagnosis and mechanical ventilation between single IFA infection group and single RSV infection group or single IFB infection group and single RSV infection group. Conclusions IFV is one of the major viral pathogens in children in Beijing.Peaks of influenza infection activity in children were in spring and winter.Children, especially 1-6 years old group, were more susceptible to IFV.The ALRTI children infected with IFV or RSV showed the similar clinical characteristics and severity of disease, but the incident rate of severe pneumonia was higher in children infection with IFV. Key words: Acute lower respiratory tract infection; Influenza virus; Respiratory syncytial virus; Clinical cha-racteristics; Child

Globally, it is estimated that respiratory syncytial virus (RSV) causes 33 million new episodes of acute lower respiratory tract infection (LRTI) in children <5 years of age and ≈120,000 deaths annually. In infants, RSV represents the leading cause of hospitalization worldwide and the second commonest cause of mortality in low- and middle-income countries.1,2 RSV also causes significant disease in immunocompromised hosts and the elderly and has been associated with the development of asthma.3 The increasingly recognized burden of RSV disease has made the development of a vaccine(s) a global health priority. The World Health Organization recently released a roadmap to facilitate the development and implementation of vaccines and monoclonal antibodies (mAbs) and estimated that RSV vaccination will be available in the next 5–10 years.4 This review summarizes the strategies and challenges associated with RSV vaccine development and the vaccine candidates undergoing clinical evaluation, with a focus on those geared toward the pediatric population. THE STRUCTURE OF RSV RSV has a negative sense nonsegmented RNA genome that encodes 11 proteins: 3 are nonstructural (NS1/NS2—that counteract interferon responses—and M2-2), and 8 are structural proteins. Of those 8 proteins, 5 are internal [N, P, M, M2-1, L]), and 3 are embedded in the virion membrane: the small hydrophobic (SH), fusion (F) and attachment (G) glycoproteins. RSV G and F carry antigenic determinants that elicit neutralizing antibodies. However, F is the preferred target for vaccine, mAb and antiviral development because it plays an essential role in host cell viral entry, is highly conserved within and among RSV A and B subtypes and because of its 6 antigenic sites that elicit the production of high-potency neutralizing antibodies (≥90% of neutralizing antibodies are directed against this protein).5 Most of the G protein is covered in glycans, leaving the central conserved domain available for neutralizing antibody binding. Except for this domain, G is not well conserved and it is recognized by few neutralizing antibodies, which has reduced enthusiasm for it as a vaccine target. Our understanding of the F protein in its 2 conformations, prefusion (pre-F) and postfusion (post-F), has revolutionized the field of RSV biology. Pre-F, the active form of F on the virion, is metastable and switches unpredictably to the stable post-F conformation that once it is folded cannot return to the pre-F form. Antibodies that bind to pre-F are more efficient at neutralizing RSV than those against post-F. As examples, antibodies against site ϕ, a pre-F-specific epitope, are 150 times more potent than palivizumab that binds to site-II, present in both F conformations, while antibodies against site I, exclusively present in post-F, show weak or no neutralization.5 In addition, non-neutralizing antibodies to F, G and also SH, may inhibit infection by complement-mediated neutralization or antibody-dependent cell-mediated cytotoxicity. Furthermore, all viral antigens have the potential to induce protection by T-cell-mediated immunity. CHALLENGES FOR RSV VACCINE DEVELOPMENT Despite the burden associated with RSV, and after 60 years of active research, there is no licensed vaccine due in part of our incomplete understanding of the pathogenesis of the disease. In general, primary RSV infections are more severe; however, reinfections are common throughout life as immunity is neither complete nor long-lasting. The ideal vaccine should induce a more durable and improved immune response than natural infection. Legacy of the Formalin-inactivated Vaccine RSV vaccine development has been hindered after the safety concerns of the first RSV vaccine that was developed in the 1960s. The formalin-inactivated-whole virus alum-precipitated vaccine, which recent evidence indicating that it was directed against post-F, was associated in naive infants, but not older children, with enhanced RSV disease (ERD) and 2 deaths upon subsequent exposure to natural RSV. The mechanisms of ERD are not well understood, but it appears that an excess of non-neutralizing antibodies coupled with a skewed T-helper 2 (Th2) immune response, and complement deposition in the lungs contributed to its development. This is a critical aspect that is being considered for the development of inactivated vaccines, and strategies to assess safety risks according to the different vaccine platforms in the infant population are required. Target Populations There are different age groups that will benefit from RSV vaccines, and these might require different approaches: young RSV-naive infants (<4–6 months), children >6 months and the elderly. Vaccination of older children (2–5 years of age) may also limit transmission, as older siblings frequently introduce RSV into the household. Infants <4–6 Months This age group has an immature/developing immune system characterized by low expression of interferon, abundance of regulatory T cells with tolerogenic reactivity and a limited B-cell repertoire because of inefficient generation of somatic hypermutations. All these factors are associated with a poor response to foreign antigens and the generation of high-affinity matured antibodies. In addition, the presence of maternal antibodies may interfere with vaccine immunogenicity. Young infants represent the main target population because the peak of severe RSV disease occurs in the first 2–3 months of life. This age group would likely benefit from maternal vaccination or neutralizing mAbs administered at birth. The main goal of maternal vaccination is to boost neutralizing RSV titers and thereby transplacental antibody transfer. However, the optimal timing for vaccination (2nd or 3rd trimester) and the durability of protection in the infant need to be defined. This coupled with the high prevalence of hypergammaglobulinemia in low- and middle-income countries, associated with HIV or malaria, which impairs transplacental antibody transfer, suggest the need for high maternal antibody titers to compete for transfer. Nevertheless, RSV antibody transfer through breast-feeding (IgG > IgA) may complement the maternal vaccination strategy.6 Vaccinating pregnant women could be questioned if it exclusively benefits the infant and not the mother. The limited data available in pregnant women are mostly derived from influenza surveillance studies with rates of RSV infection varying from 0.2% to 13%, which likely underestimates the real incidence of RSV during pregnancy. The concerns regarding adverse fetal outcomes are relatively low, because this would not be the first time the mother's immune system encounters RSV antigens and the safety profile of other vaccines used in pregnancy, such as tetanus, diptheria and acellular pertussis (Tdap) or influenza, is excellent. A number of RSV maternal vaccines are currently in clinical development (Table 1).TABLE 1.: Landscape of RSV Vaccines Undergoing Clinical TrialsOlder Infants and Children Based on the experience of the formalin-inactivated-RSV vaccine, within this age group those who are naive at the time of vaccination might be at risk of ERD with protein vaccines. This target population would likely benefit most from live-attenuated or vectored vaccines. The Elderly On the other side of the spectrum, the immunosenescence of adults >65 years of age and the presence of additional comorbidities may compromise vaccine responses and the ability to assess efficacy. This population might benefit most from adjuvanted vaccines. Clinical Endpoints The ideal vaccine should be able to prevent severe disease and limit transmission, but the lack of a standard definition of severe disease or precise markers to assess severity in infants has been a barrier for vaccine development. Clinical endpoints that define a successful vaccine might be different depending on the target population. Hospitalization and other endpoints that capture the inpatient/outpatient burden of the disease, such as a reduction in medically significant visits for RSV infection, should be considered.7 Developing composite endpoints that include a combination of viral (and possibly bacterial) factors, clinical parameters, and fast turn-around point of care biomarkers could help with patient classification and to standardize definitions.8 Also, long-term follow-up is recommended, as studies suggest that interventions reducing the acute burden of RSV disease may also impact the development of recurrent wheezing/asthma.9 Immune Correlates of Protection Serum neutralizing antibodies (IgG against pre-F > post-F and G) represent the main surrogate of protection, as shown by the effectiveness of immunoprophylaxis with anti-F mAb (palivizumab) in high risk infants. However, a standardized protective threshold has not been defined yet. Newer systems biology approaches are helping to define the optimal correlates of protection, which are complex and depend on multiple factors, rather than a single cutoff value in antibody assays, and will need to be adjusted to each target population. Other cocorrelates of protection may include, F-specific epitope antibodies, mucosal IgA, interferon responses, antibody-dependent cell-mediated cytotoxicity and cell-mediated immunity. In addition, a balanced Th1/Th2 immune response, indicated by a high IgG2a/IgG1 ratio, is desirable. Other Factors The lack of an ideal animal model has also slowed down RSV vaccine development. Human challenge models mostly reproduce upper but not LRTI, limiting the generalizability of the results or the ability to assess the impact of vaccines on disease severity. There are also gaps in RSV epidemiology with lack of accurate information defining the temporal and geographic patterns of RSV circulation in inpatients/outpatients, across different age groups or RSV-associated mortality. Implementing robust multiplex polymerase chain reaction-based surveillance platforms could help to assess the impact of interventions on the burden of RSV disease, to identify possible escape mutants, or the contribution of other respiratory viruses causing RSV-like illnesses. VACCINE STRATEGIES The most effective approach to protect young infants and children from severe RSV infection may be a combined strategy using passive and active immunization: either maternal vaccination with stabilized pre-F or virus-like particles containing the F protein or mAb against pre-F at birth; followed by pediatric active immunization with a live vaccine, either attenuated RSV or the pre-F protein expressed from a virus vector. There are 39 vaccines candidates under development (http://www.path.org); of those 19 are undergoing clinical trials (Table 1).10 Protein Vaccines Particle Based The recombinant adjuvanted RSV post-F nanoparticle vaccine is the most advanced vaccine in clinical development. Results from a phase-3 clinical trial that enrolled 4636 pregnant women on the third trimester demonstrated a decrease in RSV hospitalizations in the offspring; however, the study did not meet the primary endpoint defined as prevention of medically significant RSV LRTI. The potential approval of this vaccine is being evaluated. It also aims to target elderly individuals and children >6 months to 5 years of age. Subunit Vaccines These vaccines consist of purified, adjuvanted proteins and use stabilized pre-F as the main antigen with promising results. They are mainly directed at pregnant women or the elderly because of the risk of ERD in RSV-naive infants. Other subunit vaccines in clinical or preclinical stages are using SH or G as main vaccine antigens. Live Vaccines Vector Based There are 5 vector-based vaccines in clinical development. The first 4 use adenovirus as a vector, while the other uses a modified vaccinia Ankara virus. Two of them are intended for use in pediatric seronegative patients. All of these vaccines express RSV F (pre-F > post-F depending on the vaccine) and 2 of them also express other viral antigens (N, M2 or G proteins). Live-attenuated vaccines (LAVs) represent an attractive alternative for older infants and young children. LAVs are administered intranasally and are able to elicit broad innate, humoral and cellular responses and replicate in the respiratory tract despite the presence of maternal antibodies. Importantly, these vaccines have not been associated with ERD and are considered safer in infants. The use of reverse genetics has made possible to incorporate different mutations in the viral genome, making LAV sufficiently immunogenic and, except for rhinorrhea, not associated with adverse events. There are 6 intranasal LAVs undergoing phase-1 clinical trials; 4 are using attenuated RSV, one Sendai virus as a backbone expressing RSV F and the last one is a chimeric vaccine using bacille Calmette-Guerin (BCG). The BCG/RSV vaccine is the only LAV intended to be administered systemically (subdermal) and in newborns. MONOCLONAL ANTIBODIES mAbs are also being evaluated for the prevention of RSV LRTI in young infants. Of those, suptavumab (REGN-2222), that targeted the pre-F-specific site V, has been discontinued from the market after it failed to prevent serious RSV LRTI in premature infants (primary endpoint). During the study, RSV type B was the predominant circulating strain and developed escape mutations that conferred resistance to this mAb. MK-1654 is an extended half-life mAb currently undergoing phase-I clinical trials and it is directed against antigenic site-IV (present the pre-F and post-F forms). Nirsevimab (MEDI8897) is a highly potent human neutralizing IgG1Κ targeting the pre-F-specific antigenic site ϕ. It also has an extended half-life because of modifications in the FC region using YTE technology. MEDI8897 is entering phase-3 clinical trials with the intent to provide passive immunization for prevention of severe RSV LRTI to all infants (preterm and full term), using a fixed, once per season intramuscular dose. SUMMARY Over the past decade, there have been significant advances in our knowledge of RSV molecular and structural biology and in the understanding of the human immune response to RSV. Despite the barriers, there are several opportunities for RSV vaccine development to protect the most vulnerable populations. The increasing interest of academic, industry and international bodies, such as the World Health Organization or Bill & Melinda Gates Foundation, is helping to move the field forward, promoting the implementation of surveillance platforms and standardization of clinical definitions, assays and surrogate markers of protection.

Differential Immune Responses and Pulmonary Pathophysiology Are Induced by Two Different Strains of Respiratory Syncytial Virus