Universal paramyxovirus vaccine design by stabilizing regions involved in structural transformation of the fusion protein.

The Paramyxoviridae family encompasses medically significant RNA viruses, including human respiroviruses 1 and 3 (RV1, RV3), and zoonotic pathogens like Nipah virus (NiV). RV3, previously known as parainfluenza type 3, for which no vaccines or antivirals have been approved, causes respiratory tract infections in vulnerable populations. The RV3 fusion (F) protein is inherently metastable and will likely require prefusion (preF) stabilization for vaccine effectiveness. Here we used structure-based design to stabilize regions involved in structural transformation to generate a preF protein vaccine antigen with high expression and stability, and which, by stabilizing the coiled-coil stem region, does not require a heterologous trimerization domain. The preF candidate induces strong neutralizing antibody responses in both female naïve and pre-exposed mice and provides protection in a cotton rat challenge model (female). Despite the evolutionary distance of paramyxovirus F proteins, their structural transformation and local regions of instability are conserved, which allows successful transfer of stabilizing substitutions to the distant preF proteins of RV1 and NiV. This work presents a successful vaccine antigen design for RV3 and provides a toolbox for future paramyxovirus vaccine design and pandemic preparedness.

Safety and Immunogenicity of an mRNA-Based hMPV/PIV3 Combination Vaccine in Seropositive Children.

OBJECTIVES: Human metapneumovirus (hMPV) and parainfluenza virus type 3 (PIV3) are common respiratory illnesses in children. The safety and immunogenicity of an investigational mRNA-based vaccine, mRNA-1653, encoding membrane-anchored fusion proteins of hMPV and PIV3, was evaluated in hMPV/PIV3-seropositive children. METHODS: In this phase 1b randomized, observer-blind, placebo-controlled, dose-ranging study, hMPV/PIV3-seropositive children were enrolled sequentially into 2 dose levels of mRNA-1653 administered 2 months apart; children aged 12 to 36 months were randomized (1:1) to receive 10-µg of mRNA-1653 or placebo and children aged 12 to 59 months were randomized (3:1) to receive 30-µg of mRNA-1653 or placebo. RESULTS: Overall, 27 participants aged 18 to 55 months were randomized; 15 participants received 10-µg of mRNA-1653 (n = 8) or placebo (n = 7), whereas 12 participants received 30-µg of mRNA-1653 (n = 9) or placebo (n = 3). mRNA-1653 was well-tolerated at both dose levels. The only reported solicited local adverse reaction was tenderness at injection site; solicited systemic adverse reactions included grade 1 or 2 chills, irritability, loss of appetite, and sleepiness. A single 10-µg or 30-µg mRNA-1653 injection increased hMPV and PIV3 neutralizing antibody titers (geometric mean fold-rise ratio over baseline: hMPV-A = 2.9-6.1; hMPV-B = 6.2-13.2; PIV3 = 2.8-3.0) and preF and postF binding antibody concentrations (geometric mean fold-rise ratio: hMPV preF = 5.3-6.1; postF = 4.6-6.5 and PIV3 preF = 13.9-14.2; postF = 11.0-12.1); a second injection did not further increase antibody levels in these seropositive children. Binding antibody responses were generally preF biased. CONCLUSIONS: mRNA-1653 was well-tolerated and boosted hMPV and PIV3 antibody levels in seropositive children aged 12 to 59 months, supporting the continued development of mRNA-1653 or its components for the prevention of hMPV and PIV3.

Prevention of respiratory virus transmission by resident memory CD8+ T cells.

An ideal vaccine both attenuates virus growth and disease in infected individuals and reduces the spread of infections in the population, thereby generating herd immunity. Although this strategy has proved successful by generating humoral immunity to measles, yellow fever and polio, many respiratory viruses evolve to evade pre-existing antibodies1. One approach for improving the breadth of antiviral immunity against escape variants is through the generation of memory T cells in the respiratory tract, which are positioned to respond rapidly to respiratory virus infections2-6. However, it is unknown whether memory T cells alone can effectively surveil the respiratory tract to the extent that they eliminate or greatly reduce viral transmission following exposure of an individual to infection. Here we use a mouse model of natural parainfluenza virus transmission to quantify the extent to which memory CD8+ T cells resident in the respiratory tract can provide herd immunity by reducing both the susceptibility of acquiring infection and the extent of transmission, even in the absence of virus-specific antibodies. We demonstrate that protection by resident memory CD8+ T cells requires the antiviral cytokine interferon-γ (IFNγ) and leads to altered transcriptional programming of epithelial cells within the respiratory tract. These results suggest that tissue-resident CD8+ T cells in the respiratory tract can have important roles in protecting the host against viral disease and limiting viral spread throughout the population.

Cross-protective antibodies against common endemic respiratory viruses.

Respiratory syncytial virus (RSV), human metapneumovirus (HMPV), and human parainfluenza virus types one (HPIV1) and three (HPIV3) can cause severe disease and death in immunocompromised patients, the elderly, and those with underlying lung disease. A protective monoclonal antibody exists for RSV, but clinical use is limited to high-risk infant populations. Hence, therapeutic options for these viruses in vulnerable patient populations are currently limited. Here, we present the discovery, in vitro characterization, and in vivo efficacy testing of two cross-neutralizing monoclonal antibodies, one targeting both HPIV3 and HPIV1 and the other targeting both RSV and HMPV. The 3 × 1 antibody is capable of targeting multiple parainfluenza viruses; the MxR antibody shares features with other previously reported monoclonal antibodies that are capable of neutralizing both RSV and HMPV. We obtained structures using cryo-electron microscopy of these antibodies in complex with their antigens at 3.62 Å resolution for 3 × 1 bound to HPIV3 and at 2.24 Å for MxR bound to RSV, providing a structural basis for in vitro binding and neutralization. Together, a cocktail of 3 × 1 and MxR could have clinical utility in providing broad protection against four of the respiratory viruses that cause significant morbidity and mortality in at-risk individuals.

An Unusual Resurgence of Human Metapneumovirus in Western Australia Following the Reduction of Non-Pharmaceutical Interventions to Prevent SARS-CoV-2 Transmission.

Non-pharmaceutical interventions (NPIs) to reduce SARS-CoV-2 transmission disrupted respiratory virus seasonality. We examined the unusual return of human metapneumovirus (hMPV) in Western Australia following a period of absence in 2020. We analysed hMPV laboratory testing data from 1 January 2017 to 31 December 2021. Whole-genome sequencing of selected hMPV-positive samples was performed using a tiled-amplicon approach. Following an absence in spring 2020, an unusual hMPV surge was observed during the wet summer season in the tropical Northern region in late 2020. Following a six-month delay, an intense winter season occurred in the subtropical/temperate Southern and Metropolitan regions. Compared to 2017-2019, hMPV incidence in 2021 increased by 3-fold, with a greater than 4-fold increase in children aged 1-4 years. There was a collapse in hMPV diversity in 2020, with the emergence of a single subtype. NPIs contributed to an absent 2020 season and a clonal hMPV resurgence. The summer surge and delayed winter season suggest that prevailing temperature and humidity are keys determinant of hMPV transmission. The increased incidence in 2021 was linked to an expanded cohort of hMPV-naïve 1-4-year-old children and waning population immunity. Further intense and unusual respiratory virus seasons are expected as COVID-19 associated NPIs are removed.

Structural basis for ultrapotent antibody-mediated neutralization of human metapneumovirus.

Human metapneumovirus (hMPV) is a leading cause of morbidity and hospitalization among children worldwide, however, no vaccines or therapeutics are currently available for hMPV disease prevention and treatment. The hMPV fusion (F) protein is the sole target of neutralizing antibodies. To map the immunodominant epitopes on the hMPV F protein, we isolated a panel of human monoclonal antibodies (mAbs), and the mAbs were assessed for binding avidity, neutralization potency, and epitope specificity. We found the majority of the mAbs target diverse epitopes on the hMPV F protein, and we discovered multiple mAb binding approaches for antigenic site III. The most potent mAb, MPV467, which had picomolar potency, was examined in prophylactic and therapeutic mouse challenge studies, and MPV467 limited virus replication in mouse lungs when administered 24 h before or 72 h after viral infection. We determined the structure of MPV467 in complex with the hMPV F protein using cryo-electron microscopy to a resolution of 3.3 Å, which revealed a complex novel prefusion-specific epitope overlapping antigenic sites II and V on a single protomer. Overall, our data reveal insights into the immunodominant antigenic epitopes on the hMPV F protein, identify a mAb therapy for hMPV F disease prevention and treatment, and provide the discovery of a prefusion-specific epitope on the hMPV F protein.

Avian metapneumovirus subtype B vaccination in commercial broiler chicks: heterologous protection and selected host transcription responses to subtype A or B challenge.

Avian metapneumovirus (aMPV) causes respiratory disease and drops in egg production in chickens, and is routinely controlled by vaccination. However, the host's immune response to virulent challenge in vaccinated or unvaccinated broiler chickens is poorly characterized. We show that subtype B vaccination offers heterologous (subtype A challenge) and homologous (subtype B challenge) protection. Subtype B challenge caused significantly greater humoral antibody titres in vaccinated and unvaccinated chickens. In turbinate and lung tissues of unvaccinated-challenged chickens, IgA and IgY mRNA transcription was significantly up-regulated after subtype B challenge compared to subtype A. Cellular immunity (CD8-α and CD8-ß) gene transcripts were significantly up-regulated during early and later stages of infection from subtype B or subtype A, respectively. Immune gene transcriptional responses (IL-1ß, IL-6 and IL-18) were significantly up-regulated after challenge. Gene transcription results showed that mRNA expression levels of CD8-α, CD8-ß, TLR3 and IL-6, particularly in turbinate and trachea tissues, are useful parameters to include in future aMPV vaccination-challenge studies.

Novel HLA-B7-restricted human metapneumovirus epitopes enhance viral clearance in mice and are recognized by human CD8+ T cells.

Human metapneumovirus (HMPV) is a leading cause of acute lower respiratory tract illness in children and adults. Repeated infections are common and can be severe in young, elderly, and immunocompromised persons due to short-lived protective humoral immunity. In turn, few protective T cell epitopes have been identified in humans. Thus, we infected transgenic mice expressing the common human HLA MHC-I allele B*07:02 (HLA-B7) with HMPV and screened a robust library of overlapping and computationally predicted HLA-B7 binding peptides. Six HLA-B7-restricted CD8+ T cell epitopes were identified using ELISPOT screening in the F, M, and N proteins, with M195-203 (M195) eliciting the strongest responses. MHC-tetramer flow cytometric staining confirmed HLA-B7 epitope-specific CD8+ T cells migrated to lungs and spleen of HMPV-immune mice. Immunization with pooled HLA-B7-restricted peptides reduced viral titer and protected mice from virulent infection. Finally, we confirmed that CD8+ T cells from HLA-B7 positive humans also recognize the identified epitopes. These results enable identification of HMPV-specific CD8+ T cells in humans and help to inform future HMPV vaccine design.

Efficacy of a novel avian metapneumovirus live vaccine candidate based on vaccination route and age.

This article describes a series of animal studies for the development of an avian metapneumovirus (aMPV) live vaccine. Although aMPV causes continual economic loss in the poultry industry, there are no live aMPV vaccines available in Korea. Furthermore, information is limited with respect to standard field practices for vaccinations at an early age. Here, the development of an aMPV live vaccine was attempted, and its efficacy was investigated with respect to the vaccination route and age to develop a method for controlling aMPV. Before vaccine development, an animal challenge model was established using the aMPV field isolate to identify the most effective time and site for collecting samples for evaluation. After attenuation of the virulent aMPV in Vero cells, a safety and efficacy test was conducted for the vaccine candidate. As a novel aMPV live vaccine candidate, aMPV K655/07HP displayed sufficient safety in day-old chicks with 10 vaccine doses. The efficacy test using 1-week-old chicks showed weaker humoral immune response than that in 4-week-old chicks. However, the candidate vaccine provided complete protection against infection caused by the challenge virus for all ages of vaccinated chicks. In conclusion, an effective aMPV challenge model was established for studying aMPV in chickens, which offered important, insightful information. The safety and efficacy study suggested that the new aMPV candidate vaccine could be used to effectively reduce the economic losses incurred because of aMPV infection.

Evaluation of pre- and post-fusion Human metapneumovirus F proteins as subunit vaccine candidates in mice.

Human metapneumovirus (hMPV) is an important respiratory pathogen especially in young children and elderly subjects. Our objective was to assess the immunogenicity and protection conferred by predominant pre- and post-fusion (F) hMPV-F constructs in Balb/C mice. Immunizations without adjuvant were not immunogenic whereas alum-adjuvanted hMPV-F proteins, regardless of their conformations, generated comparable neutralizing antibody titers with undetectable pulmonary viral titers following viral challenge. In conclusion, we found no apparent advantage for mixtures of predominant pre-fusion F proteins over post-fusion conformations for hMPV vaccination in opposite to recent data obtained with the human respiratory syncytial virus.

Progress in the development of virus-like particle vaccines against respiratory viruses.

Introduction: Influenza virus, human respiratory syncytial virus (RSV), and human metapneumovirus (HMPV) are important human respiratory pathogens. Recombinant virus-like particle (VLP) vaccines are suggested to be potential promising platforms to protect against these respiratory viruses. This review updates important progress in the development of VLP vaccines against respiratory viruses.Areas Covered: This review summarizes progress in developing VLP and nanoparticle-based vaccines against influenza virus, RSV, and HMPV. The PubMed was mainly used to search for important research articles published since 2010 although earlier key articles were also referenced. The research area covered includes VLP and nanoparticle platform vaccines against seasonal, pandemic, and avian influenza viruses as well as RSV and HMPV respiratory viruses. The production methods, immunogenic properties, and vaccine efficacy of respiratory VLP vaccines in preclinical animal models and clinical studies were reviewed in this article.Expert opinion: Previous and current preclinical and clinical studies suggest that recombinant VLP and nanoparticle vaccines are expected to be developed as promising alternative platforms against respiratory viruses in future. Therefore, continued research efforts are warranted.

Induction of Trained Immunity by Recombinant Vaccines.

Vaccines represent an important strategy to protect humans against a wide variety of pathogens and have even led to eradicating some diseases. Although every vaccine is developed to induce specific protection for a particular pathogen, some vaccine formulations can also promote trained immunity, which is a non-specific memory-like feature developed by the innate immune system. It is thought that trained immunity can protect against a wide variety of pathogens other than those contained in the vaccine formulation. The non-specific memory of the trained immunity-based vaccines (TIbV) seems beneficial for the immunized individual, as it may represent a powerful strategy that contributes to the control of pathogen outbreaks, reducing morbidity and mortality. A wide variety of respiratory viruses, including respiratory syncytial virus (hRSV) and metapneumovirus (hMPV), cause serious illness in children under 5 years old and the elderly. To address this public health problem, we have developed recombinant BCG vaccines that have shown to be safe and immunogenic against hRSV or hMPV. Besides the induction of specific adaptive immunity against the viral antigens, these vaccines could generate trained immunity against other respiratory pathogens. Here, we discuss some of the features of trained immunity induced by BCG and put forward the notion that recombinant BCGs expressing hRSV or hMPV antigens have the capacity to simultaneously induce specific adaptive immunity and non-specific trained immunity. These recombinant BCG vaccines could be considered as TIbV capable of inducing simultaneously the development of specific protection against hRSV or hMPV, as well as non-specific trained-immunity-based protection against other pathogenic viruses.

Persuasive Effects of Linguistic Agency Assignment and Linguistic Markers of Argumentation in Health Messages about an Emerging Sexually Transmitted Disease.

Answering the call by some health communication researchers to give greater attention to message strategies at the level of word choices and sentence structures, this study examined how the linguistic marking of argumentative orientation and linguistic agency assignment affects young adults' reactions to an informational message about a sexually transmitted infection presented as a new emerging health threat. Participants were randomly assigned to read one of the four versions of a fact sheet defined by a 2 × 2 (agency assignment x marking of argumentation orientation) factorial design and thereafter completed a questionnaire. Results indicated that the assignment of agency to the virus (vs. human) increased the perceived severity of the health threat, perceived susceptibility to it, persuasiveness of the message, and safer sex intentions. The same outcomes occurred when the message was phrased with a high marking of the argumentative orientation rather than a low marking. These findings suggest that a better understanding of language variable effects can boost the efficacy of promotional health messages.

Molecular epidemiology of infectious bronchitis virus and avian metapneumovirus in Greece.

Respiratory diseases like infectious bronchitis virus (IBV) and avian metapneumovirus (aMPV) have been held accountable for major losses for poultry production. Nevertheless, scarce information was present dealing with the prevalence and molecular epidemiology of these infections in Greece and the efficacy of currently applied control strategies. To fill this gap, a specific epidemiological study was designed. A total of 106 broiler and layer farms, including 10 backyard and 96 commercial flocks, were sampled between March 2016 and May 2017, and the obtained tracheal swabs were tested for IBV and aMPV using RT-PCR based techniques followed by sequencing. For each farm, data regarding production type, flock features, clinical signs, and vaccination program were also recorded. Different associations between vaccination protocol, production type, animal category, birds density, age, presence of clinical signs, and IBV and/or aMPV infection were tested. Both IBV and aMPV field strain prevalence were proven high, approximately 20 and 30%, respectively, being the GI-19 lineage (14 out of 19; 73.6%) and B subtype (30 out of 30; 100%), the most commonly detected IBV and aMPV genetic types. Infection with IBV field strains was significantly associated with clinical sign presence (odds ratio = 8.55 [95CI = 2.17-42.90]). Remarkably, only the vaccination protocol involving a double vaccination at 1 D of age was proven protective against IBV-induced symptomatology, with the odds of developing disease being 4.14 [95CI = 1.34-14.51] times lower. No association was demonstrated between aMPV infection and clinical outbreaks or between aMPV and IBV detection, suggesting the marginal role of the former pathogen in poultry farming. Globally, the present study provides the first detailed investigation of the epidemiological scenario of 2 viruses traditionally considered of pivotal relevance in poultry farming and demonstrates that remarkable benefits could be obtained with just minor adjustments in vaccination protocols.

Infant Pneumococcal Carriage During Influenza, RSV, and hMPV Respiratory Illness Within a Maternal Influenza Immunization Trial.

In this post-hoc analysis of midnasal pneumococcal carriage in a community-based, randomized prenatal influenza vaccination trial in Nepal with weekly infant respiratory illness surveillance, 457 of 605 (75.5%) infants with influenza, respiratory syncytial virus (RSV), or human metapneumovirus (hMPV) illness had pneumococcus detected. Pneumococcal carriage did not impact rates of lower respiratory tract disease for these 3 viruses. Influenza-positive infants born to mothers given influenza vaccine had lower pneumococcal carriage rates compared to influenza-positive infants born to mothers receiving placebo (58.1% versus 71.6%, P = 0.03). Maternal influenza immunization may impact infant acquisition of pneumococcus during influenza infection. Clinical Trials Registration. NCT01034254.

Two similar commercial live attenuated AMPV vaccines prepared by random passage of the identical field isolate, have unrelated sequences.

Since late '80 s Avian metapneumovirus subtype A causes sufficient disease in Europe for commercial companies to have started developing live attenuated vaccines. Here, two of those vaccines were fully consensus sequenced alongside their progenitor field strain (#8544). Sequences comparison shows that the attenuation of field strain #8544 was associated with no common substitutions between the two derived vaccines. This finding suggests that the attenuation of field viruses via serial passage on cell cultures or tissues is the result of a random process, rather than a mechanism aiming to achieve a specific sequence. Furthermore, field vaccination strategies would greatly benefit by the unambiguous vaccine markers identified in this study, enabling a prompt and confident vaccines detection.

Nitazoxanide inhibits paramyxovirus replication by targeting the Fusion protein folding: role of glycoprotein-specific thiol oxidoreductase ERp57.

Paramyxoviridae, a large family of enveloped viruses harboring a nonsegmented negative-sense RNA genome, include important human pathogens as measles, mumps, respiratory syncytial virus (RSV), parainfluenza viruses, and henipaviruses, which cause some of the deadliest emerging zoonoses. There is no effective antiviral chemotherapy for most of these pathogens. Paramyxoviruses evolved a sophisticated membrane-fusion machine consisting of receptor-binding proteins and the fusion F-protein, critical for virus infectivity. Herein we identify the antiprotozoal/antimicrobial nitazoxanide as a potential anti-paramyxovirus drug targeting the F-protein. We show that nitazoxanide and its circulating-metabolite tizoxanide act at post-entry level by provoking Sendai virus and RSV F-protein aggregate formation, halting F-trafficking to the host plasma membrane. F-protein folding depends on ER-resident glycoprotein-specific thiol-oxidoreductase ERp57 for correct disulfide-bond architecture. We found that tizoxanide behaves as an ERp57 non-competitive inhibitor; the putative drug binding-site was located at the ERp57-b/b' non-catalytic domains interface. ERp57-silencing mimicked thiazolide-induced F-protein alterations, suggesting an important role of this foldase in thiazolides anti-paramyxovirus activity. Nitazoxanide is used in the clinic as a safe and effective antiprotozoal/antimicrobial drug; its antiviral activity was shown in patients infected with hepatitis-C virus, rotavirus and influenza viruses. Our results now suggest that nitazoxanide may be effective also against paramyxovirus infection.

Immunization with a Mixture of Nucleoprotein from Human Metapneumovirus and AbISCO-100 Adjuvant Reduces Viral Infection in Mice Model.

The human metapneumovirus (hMPV) is the second leading cause globally of acute infection of the respiratory tract in children, infecting the upper and lower airways. The hMPV may induce an inappropriate Th2-type immune response, which causes severe pulmonary inflammation, leading to the obstruction of airways. Despite its severe epidemiological relevance, no vaccines are currently available for the prevention of hMPV-induced illness. In this investigation, we demonstrated that immunization of mice with the recombinant hMPV nucleoprotein (hMPV-N) mixed with the AbISCO-100 adjuvant reduced viral replication in lungs following challenge with the virus. We found that immunized mice had reduced weight loss, decreased granulocytes in the lung, an increased level of specific nucleoprotein antibodies of IgG1 and IgG2a-isotypes, and a local profile of Th1/Th17-type cytokines. Our results suggest that immunization with the hMPV-N and the AbISCO-100 adjuvant induces a reduction of viral infection and could be considered for the development of an hMPV vaccine.

Vaccines for the Paramyxoviruses and Pneumoviruses: Successes, Candidates, and Hurdles.

Human parainfluenza viruses (family Paramyxoviridae), human metapneumovirus, and respiratory syncytial virus (family Pneumoviridae) infect most infants and children within the first few years of life and are the etiologic agents for many serious acute respiratory illnesses. These virus infections are also associated with long-term diseases that impact quality of life, including asthma. Despite over a half-century of vaccine research, development, and clinical trials, no vaccine has been licensed to date for the paramyxoviruses or pneumoviruses for the youngest infants. In this study, we describe the recent reclassification of paramyxoviruses and pneumoviruses into distinct families by the International Committee on the Taxonomy of Viruses. We also discuss some past unsuccessful vaccine trials and some currently preferred vaccine strategies. Finally, we discuss hurdles that must be overcome to support successful respiratory virus vaccine development for the youngest children.

Evaluation of a Live Attenuated Human Metapneumovirus Vaccine in Adults and Children.

We conducted a phase I clinical trial of an experimental live attenuated recombinant human metapneumovirus (HMPV) vaccine (rHMPV-Pa) sequentially in adults, HMPV-seropositive children, and HMPV-seronegative children, the target population for vaccination. rHMPV-Pa was appropriately restricted in replication in adults and HMPV-seropositive children but was overattenuated for HMPV-seronegative children.