Generation of temperature-sensitive human metapneumovirus strains that provide protective immunity in hamsters

Human metapneumovirus (HMPV) causes acute respiratory tract illness primarily in young children, immunocompromised individuals and the elderly. Vaccines would be desirable to prevent severe illnesses in these risk groups. Here, we describe the generation and evaluation of cold-passage (cp) temperature-sensitive (ts) HMPV strains as vaccine candidates. Repeated passage of HMPV at low temperatures in Vero cells resulted in the accumulation of mutations in the viral genome. Introduction of these mutations in a recombinant HMPV by reverse genetics resulted in a ts-phenotype, judged on the decreased shut-off temperature for virus replication in vitro. As an alternative approach, three previously described cp-respiratory syncytial virus (cp-HRSV) mutations were introduced in a recombinant HMPV, which also resulted in a low shut-off temperature in vitro. Replication of these ts-viruses containing either the cp-HMPV or cp-HRSV mutations was reduced in the upper respiratory tract (URT) and undetectable in the lower respiratory tract (LRT) of hamsters. Nevertheless, high titres of HMPV-specific antibodies were induced by both ts-viruses. Upon immunization with the ts-viruses, the LRT of hamsters were completely protected against challenge infection with a heterologous HMPV strain, and URT viral titres were reduced by 10 000-fold. In conclusion, we provide proof-of-principle for two candidate live-attenuated HMPV vaccines that induce cross-protective immunity to prevent infection of the LRT in Syrian golden hamsters. Further mapping of the molecular determinants of attenuation of HMPV should be the subject of future studies.

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Recombinant Respiratory Syncytial Viruses Lacking the C-Terminal Third of the Attachment (G) Protein Are Immunogenic and Attenuated In Vivo and In Vitro

Journal of Virology ◽

10.1128/jvi.78.11.5773-5783.2004 ◽

2004 ◽

Vol 78 (11) ◽

pp. 5773-5783 ◽

Cited By ~ 11

Author(s):

Matthew B. Elliott ◽

Karin S. Pryharski ◽

Qingzhong Yu ◽

Christopher L. Parks ◽

Todd S. Laughlin ◽

...

Keyword(s):

G Protein ◽

Reverse Genetics ◽

Point Mutations ◽

A549 Cells ◽

Temperature Sensitive ◽

Human Infants ◽

Attenuated Viruses ◽

Syncytial Virus

ABSTRACT The design of attenuated vaccines for respiratory syncytial virus (RSV) historically focused on viruses made sensitive to physiologic temperature through point mutations in the genome. These prototype vaccines were not suitable for human infants primarily because of insufficient attenuation, genetic instability, and reversion to a less-attenuated phenotype. We therefore sought to construct novel attenuated viruses with less potential for reversion through genetic alteration of the attachment G protein. Complete deletion of G protein was previously shown to result in RSV strains overly attenuated for replication in mice. Using reverse genetics, recombinant RSV (rRSV) strains were engineered with truncations at amino acid 118, 174, 193, or 213 and respectively designated rA2cpΔG118, rA2cpΔG174, rA2cpΔG193, and rA2cpΔG213. All rA2cpΔG strains were attenuated for growth in vitro and in the respiratory tracts of BALB/c mice but not restricted for growth at 37°C. The mutations did not significantly affect nascent genome synthesis in human lung epithelial (A549) cells, but infectious rA2cpΔG virus shed into the culture medium was dramatically diminished. Hence, the data suggested that a site within the C-terminal 85 amino acids of G protein is important for efficient genome packaging or budding of RSV from the infected cell. Vaccination with the rA2cpΔG strains also generated efficacious immune responses in mice that were similar to those elicited by the temperature-sensitive cpts248/404 strain previously tested in human infants. Collectively, the data indicate that the rA2cpΔG strains are immunogenic, not likely to revert to the less-attenuated phenotype, and thus candidates for further development as vaccines against RSV.

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Replacement of the F and G Proteins of Respiratory Syncytial Virus (RSV) Subgroup A with Those of Subgroup B Generates Chimeric Live Attenuated RSV Subgroup B Vaccine Candidates

Journal of Virology ◽

10.1128/jvi.73.12.9773-9780.1999 ◽

1999 ◽

Vol 73 (12) ◽

pp. 9773-9780 ◽

Cited By ~ 32

Author(s):

S. S. Whitehead ◽

M. G. Hill ◽

C. Y. Firestone ◽

M. St. Claire ◽

W. R. Elkins ◽

...

Keyword(s):

Respiratory Syncytial Virus ◽

Reverse Genetics ◽

Gene Replacement ◽

Chimeric Virus ◽

Antigenic Determinants ◽

Temperature Sensitive ◽

Protective Antigens ◽

Syncytial Virus ◽

High Level

ABSTRACT Human respiratory syncytial virus (RSV) exists as two antigenic subgroups, A and B, both of which should be represented in a vaccine. The F and G glycoproteins are the major neutralization and protective antigens, and the G protein in particular is highly divergent between the subgroups. The existing system for reverse genetics is based on the A2 strain of RSV subgroup A, and most efforts to develop a live attenuated RSV vaccine have focused on strain A2 or other subgroup A viruses. In the present study, the development of a live attenuated subgroup B component was expedited by the replacement of the F and G glycoproteins of recombinant A2 virus with their counterparts from the RSV subgroup B strain B1. This gene replacement was initially done for wild-type (wt) recombinant A2 virus to create awt AB chimeric virus and then for a series of A2 derivatives which contain various combinations of A2-derived attenuating mutations located in genes other than F and G. Thewt AB virus replicated in cell culture with an efficiency which was comparable to that of the wt A2 and B1 parents. AB viruses containing temperature-sensitive mutations in the A2 background exhibited levels of temperature sensitivity in vitro which were similar to those of A2 viruses bearing the same mutations. In chimpanzees, the replication of the wt AB chimera was intermediate between that of the A2 and B1 wt viruses and was accompanied by moderate rhinorrhea, as previously seen in this species. An AB chimeric virus, rABcp248/404/1030, which was constructed to contain a mixture of attenuating mutations derived from two different biologically attenuated A2 viruses, was highly attenuated in both the upper and lower respiratory tracts of chimpanzees. This attenuated AB chimeric virus was immunogenic and conferred a high level of resistance on chimpanzees to challenge with wt AB virus. The rABcp248/404/1030 chimeric virus is a promising vaccine candidate for RSV subgroup B and will be evaluated next in humans. Furthermore, these results suggest that additional attenuating mutations derived from strain A2 can be inserted into the A2 background of the recombinant chimeric AB virus as necessary to modify the attenuation phenotype in a reasonably predictable manner to achieve an optimal balance between attenuation and immunogenicity in a virus bearing the subgroup B antigenic determinants.

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Infection of Nonhuman Primates with Recombinant Human Metapneumovirus Lacking the SH, G, or M2-2 Protein Categorizes Each as a Nonessential Accessory Protein and Identifies Vaccine Candidates

Journal of Virology ◽

10.1128/jvi.79.19.12608-12613.2005 ◽

2005 ◽

Vol 79 (19) ◽

pp. 12608-12613 ◽

Cited By ~ 112

Author(s):

Stéphane Biacchesi ◽

Quynh N. Pham ◽

Mario H. Skiadopoulos ◽

Brian R. Murphy ◽

Peter L. Collins ◽

...

Keyword(s):

Respiratory Tract ◽

Reverse Genetics ◽

Topical Administration ◽

Human Metapneumovirus ◽

Anatomical Site ◽

Upper Respiratory Tract ◽

Wild Type ◽

Reading Frame ◽

Vaccine Candidates ◽

Upper Respiratory

ABSTRACT Recombinant human metapneumovirus (HMPV) in which the SH, G, or M2 gene or open reading frame was deleted by reverse genetics was evaluated for replication and vaccine efficacy following topical administration to the respiratory tract of African green monkeys, a permissive primate host. Replication of the ΔSH virus was only marginally less efficient than that of wild-type HMPV, whereas the ΔG and ΔM2-2 viruses were reduced sixfold and 160-fold in the upper respiratory tract and 3,200-fold and 4,000-fold in the lower respiratory tract, respectively. Even with the highly attenuated mutants, there was unequivocal HMPV replication at each anatomical site in each animal. Thus, none of these three proteins is essential for HMPV replication in a primate host, although G and M2-2 increased the efficiency of replication. Each gene-deletion virus was highly immunogenic and protective against wild-type HMPV challenge. The ΔG and ΔM2-2 viruses are promising vaccine candidates that are based on independent mechanisms of attenuation and are appropriate for clinical evaluation.

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Isolation and Characterization of Monoclonal Antibodies Which Neutralize Human Metapneumovirus In Vitro and In Vivo

Journal of Virology ◽

10.1128/jvi.00318-06 ◽

2006 ◽

Vol 80 (16) ◽

pp. 7799-7806 ◽

Cited By ~ 64

Author(s):

Nancy D. Ulbrandt ◽

Hong Ji ◽

Nita K. Patel ◽

Jeffrey M. Riggs ◽

Yambasu A. Brewah ◽

...

Keyword(s):

Monoclonal Antibody ◽

Monoclonal Antibodies ◽

Respiratory Tract ◽

Neutralizing Antibodies ◽

Lower Respiratory Tract ◽

Human Metapneumovirus ◽

F Protein ◽

Isolation And Characterization ◽

Syncytial Virus

ABSTRACT Human metapneumovirus (hMPV) is a recently described member of the Paramyxoviridae family/Pneumovirinae subfamily and shares many common features with respiratory syncytial virus (RSV), another member of the same subfamily. hMPV causes respiratory tract illnesses that, similar to human RSV, occur predominantly during the winter months and have symptoms that range from mild to severe cough, bronchiolitis, and pneumonia. Like RSV, the hMPV virus can be subdivided into two genetic subgroups, A and B. With RSV, a single monoclonal antibody directed at the fusion (F) protein can prevent severe lower respiratory tract RSV infection. Because of the high level of sequence conservation of the F protein across all the hMPV subgroups, this protein is likely to be the preferred antigenic target for the generation of cross-subgroup neutralizing antibodies. Here we describe the generation of a panel of neutralizing monoclonal antibodies that bind to the hMPV F protein. A subset of these antibodies has the ability to neutralize prototypic strains of both the A and B hMPV subgroups in vitro. Two of these antibodies exhibited high-affinity binding to the F protein and were shown to protect hamsters against infection with hMPV. The data suggest that a monoclonal antibody could be used prophylactically to prevent lower respiratory tract disease caused by hMPV.

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Vinegar and Its Active Component Acetic Acid Inhibit SARS-CoV-2 Infection In Vitro and Ex Vivo

10.1101/2020.07.08.193193 ◽

2020 ◽

Author(s):

Isabel Pagani ◽

Silvia Ghezzi ◽

Massimo Clementi ◽

Guido Poli ◽

Mario Bussi ◽

...

Keyword(s):

Acetic Acid ◽

Antiviral Activity ◽

Respiratory Tract ◽

Active Component ◽

Ex Vivo ◽

Vero Cells ◽

Nasopharyngeal Swab ◽

Upper Respiratory Tract ◽

Upper Respiratory

AbstractEffective and economical measures are needed to either prevent or inhibit the replication of SARS-CoV-2, the causative agent of COVID-19, in the upper respiratory tract. As fumigation of vinegar at low concentration (0.34%) ameliorated the symptoms of mild SARS-CoV-2 infection, we tested in vitro the potential antiviral activity of vinegar and of its active component, acetic acid. We here demonstrate that both vinegar and acetic acid indeed strongly inactivate SARS-CoV-2 infectivity in Vero cells. Furthermore, vinegar treatment caused a 90% inhibition of the infectious titer when directly applied to a nasopharyngeal swab transfer medium of a COVID-19 patient. These effects were potentiated if conduced at a temperature of 45 °C vs. 37 °C, a condition that is transiently generated in the upper respiratory tract during fumigation. Our findings are consistent and extend the results of studies performed in the early and mid-20th century on the disinfectant capacity of organic acids and can provide an affordable home-made aid to prevent or contain SARS-CoV-2 infection of the upper respiratory tract.

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Probiotics in the comprehensive prevention of respiratory infections in children

Лечащий врач ◽

10.51793/os.2021.24.9.004 ◽

2021 ◽

Author(s):

С.В. Николаева ◽

Д.В. Усенко ◽

Ю.Н. Хлыповка ◽

А.В. Горелов

Keyword(s):

Helicobacter Pylori ◽

Respiratory Tract ◽

Viral Infections ◽

Respiratory Infections ◽

Influenza Viruses ◽

Acute Respiratory Infections ◽

Upper Respiratory Tract ◽

Potential Clinical Benefit ◽

Syncytial Virus

Вирусные инфекции дыхательных путей являются наиболее частой причиной инфекционных заболеваний, особенно у детей. В большинстве случаев, особенно при поражении верхних дыхательных путей, острые респираторные вирусные инфекции протекают в легкой или среднетяжелой форме и часто купируются самостоятельно. Фармакологические средства для лечения или профилактики данных инфекций у детей в настоящее время ограничены. Многочисленные исследования доказали эффективность пробиотиков в лечении и профилактике заболеваний желудочно-кишечного тракта, таких как инфекционные и антибиотик-ассоциированные диареи, диареи путешественников, некротизирующий энтероколит, инфекция Helicobacter pylori, а также атопических заболеваний. Становится актуальным изучение эффективности пробиотиков в качестве средств профилактики острых респираторных инфекций среди детей и взрослых. Данные in vitro демонстрируют, что пробиотики обладают штаммоспецифическим иммуномодулирующим действием на иммунные клетки. Показано, что пробиотики эффективны в подавлении репликации различных респираторных вирусов, включая вирусы гриппа и респираторно-синцитиальный вирус. Подобные эффекты были продемонстрированы на мышах, было показано, что пробиотики способны снижать титры вируса в тканях легких и модулировать экспрессию противовирусных и провоспалительных генов до и после вирусной инфекции. Доклинические исследования также показывают уменьшение симптомов заболевания у мышей, что указывает на потенциальную клиническую пользу. Данные литературы по изучению применения пробиотиков и синбиотиков при вирусных инфекциях респираторного тракта показывают, что их использование связано с более низкой частотой и меньшей продолжительностью легких форм респираторной инфекции как у детей, так и у взрослых. Целесообразно дальнейшее проведение исследований для получения адекватных выводов об эффективности пробиотиков и синбиотиков при острых респираторных инфекциях. Viral infections of the respiratory tract are the most common cause of infectious diseases, especially in children. In most cases, especially when the upper respiratory tract is affected, acute respiratory viral infections are mild to moderate and often stop spontaneously. Pharmacological agents for the treatment or prevention of these infections in children are currently limited. Numerous studies have proven the effectiveness of probiotics in the treatment and prevention of diseases of the gastrointestinal tract, such as infectious and antibiotic-associated diarrhea, traveler's diarrhea, necrotizing enterocolitis, Helicobacter pylori infection, as well as atopic diseases. It is becoming urgent to study the effectiveness of probiotics as prophylactic agents for acute respiratory infections. among children and adults. In vitro data demonstrate that probiotics have strain-specific immunomodulatory effects on immune cells. Probiotics have been shown to be effective in suppressing the replication of various respiratory viruses, including influenza viruses and respiratory syncytial virus. Similar effects have been demonstrated in mice with the ability of probiotics to reduce viral titers in lung tissues and modulate the expression of antiviral and pro-inflammatory genes before and after viral infection. Preclinical studies also show improvement in symptoms in mice, indicating potential clinical benefit. Literature data on the use of probiotics and synbiotics for viral infections of the respiratory tract show that their use is associated with a lower frequency and duration of mild forms of respiratory infection in both children and adults. It is advisable to further conduct research necessary to obtain adequate conclusions about the effectiveness of probiotics and synbiotics in acute respiratory infections.

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Identification of antibody neutralization epitopes on the fusion protein of human metapneumovirus

Journal of General Virology ◽

10.1099/vir.0.2008/005199-0 ◽

2008 ◽

Vol 89 (12) ◽

pp. 3113-3118 ◽

Cited By ~ 30

Author(s):

Nancy D. Ulbrandt ◽

Hong Ji ◽

Nita K. Patel ◽

Arnita S. Barnes ◽

Susan Wilson ◽

...

Keyword(s):

Respiratory Tract ◽

Neutralizing Antibodies ◽

Human Metapneumovirus ◽

F Protein ◽

Respiratory Tract Illnesses ◽

Rsv Infection ◽

Resistant Mutants ◽

Syncytial Virus

Human metapneumovirus (hMPV) is genetically related to respiratory syncytial virus (RSV); both cause respiratory tract illnesses ranging from a mild cough to bronchiolitis and pneumonia. The F protein-directed monoclonal antibody (mAb) palivizumab has been shown to prevent severe lower respiratory tract RSV infection in animals and humans. We have previously reported on a panel of mAbs against the hMPV F protein that neutralize hMPV in vitro and, in two cases, in vivo. Here we describe the generation of hMPV mAb-resistant mutants (MARMs) to these neutralizing antibodies. Sequencing the F proteins of the hMPV MARMs identified several neutralizing epitopes. Interestingly, some of the epitopes mapped on the hMPV F protein coincide with homologous regions mapped previously on the RSV F protein, including the site against which the broadly protective mAb palivizumab is directed. This suggests that these homologous regions play important, conserved functions in both viruses.

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Evaluation of the Safety and Immune Efficacy of Recombinant Human Respiratory Syncytial Virus Strain Long Live Attenuated Vaccine Candidates

Virologica Sinica ◽

10.1007/s12250-021-00345-3 ◽

2021 ◽

Author(s):

Li-Nan Wang ◽

Xiang-Lei Peng ◽

Min Xu ◽

Yuan-Bo Zheng ◽

Yue-Ying Jiao ◽

...

Keyword(s):

Respiratory Syncytial Virus ◽

Gene Deletion ◽

Human Respiratory Syncytial Virus ◽

Temperature Sensitive ◽

T7 Promoter ◽

Vaccine Candidates ◽

Rsv Infection ◽

Syncytial Virus

AbstractHuman respiratory syncytial virus (RSV) infection is the leading cause of lower respiratory tract illness (LRTI), and no vaccine against LRTI has proven to be safe and effective in infants. Our study assessed attenuated recombinant RSVs as vaccine candidates to prevent RSV infection in mice. The constructed recombinant plasmids harbored (5′ to 3′) a T7 promoter, hammerhead ribozyme, RSV Long strain antigenomic cDNA with cold-passaged (cp) mutations or cp combined with temperature-sensitive attenuated mutations from the A2 strain (A2cpts) or further combined with SH gene deletion (A2cptsΔSH), HDV ribozyme (δ), and a T7 terminator. These vectors were subsequently co-transfected with four helper plasmids encoding N, P, L, and M2-1 viral proteins into BHK/T7-9 cells, and the recovered viruses were then passaged in Vero cells. The rescued recombinant RSVs (rRSVs) were named rRSV-Long/A2cp, rRSV-Long/A2cpts, and rRSV-Long/A2cptsΔSH, respectively, and stably passaged in vitro, without reversion to wild type (wt) at sites containing introduced mutations or deletion. Although rRSV-Long/A2cpts and rRSV-Long/A2cptsΔSH displayed temperature-sensitive (ts) phenotype in vitro and in vivo, all rRSVs were significantly attenuated in vivo. Furthermore, BALB/c mice immunized with rRSVs produced Th1-biased immune response, resisted wtRSV infection, and were free from enhanced respiratory disease. We showed that the combination of ΔSH with attenuation (att) mutations of cpts contributed to improving att phenotype, efficacy, and gene stability of rRSV. By successfully introducing att mutations and SH gene deletion into the RSV Long parent and producing three rRSV strains, we have laid an important foundation for the development of RSV live attenuated vaccines.

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Viral etiology and epidemiology of pediatric patients hospitalized for acute respiratory tract infections in Macao: a retrospective study from 2014 to 2017

BMC Infectious Diseases ◽

10.1186/s12879-021-05996-x ◽

2021 ◽

Vol 21 (1) ◽

Author(s):

Cheng Lei ◽

Lisong Yang ◽

Cheong Tat Lou ◽

Fan Yang ◽

Kin Ian SiTou ◽

...

Keyword(s):

Respiratory Tract ◽

Lower Respiratory Tract ◽

Respiratory Tract Infections ◽

Pediatric Patients ◽

Respiratory Infections ◽

Upper Respiratory Tract ◽

Viral Etiology ◽

Upper Respiratory ◽

Syncytial Virus ◽

Tract Infections

Abstract Background Acute respiratory infections (ARIs) are among the leading causes of hospitalization in children. Understanding the local dominant viral etiologies is important to inform infection control practices and clinical management. This study aimed to investigate the viral etiology and epidemiology of respiratory infections among pediatric inpatients in Macao. Methods A retrospective study using electronic health records between 2014 and 2017 at Kiang Wu Hospital was performed. Nasopharyngeal swab specimens were obtained from hospitalized children aged 13 years or younger with respiratory tract diseases. xMAP multiplex assays were employed to detect respiratory agents including 10 respiratory viruses. Data were analyzed to describe the frequency and seasonality. Results Of the 4880 children enrolled in the study, 3767 (77.1%) were positive for at least one of the 13 viral pathogens tested, of which 2707 (55.5%) being male and 2635 (70.0%) under 2 years old. Among the positive results, there were 3091 (82.0%) single infections and 676 (18.0%) multiple infections. The predominant viruses included human rhinovirus/enterovirus (HRV/EV 27.4%), adenovirus (ADV, 15.8%), respiratory syncytial virus B (RSVB, 7.8%) and respiratory syncytial virus A (RSVA, 7.8%). The detection of viral infection was the most prevalent in autumn (960/1176, 81.6%), followed by spring (1095/1406, 77.9%), winter (768/992, 77.4%), and summer (944/1306, 72.3%), with HRV/EV and ADV being most commonly detected throughout the 4 years of study period. The detection rate of viral infection was highest among ARI patients presented with croup (123/141, 87.2%), followed by lower respiratory tract infection (1924/2356, 81.7%) and upper respiratory tract infection (1720/2383, 72.2%). FluA, FluB and ADV were positive factors for upper respiratory tract infections. On the other hand, infection with RSVA, RSVB, PIV3, PIV4, HMPV, and EV/RHV were positively associated with lower respiratory tract infections; and PIV1, PIV2, and PIV3 were positively associated with croup. Conclusions This is the first study in Macao to determine the viral etiology and epidemiology of pediatric patients hospitalized for ARIs. The study findings can contribute to the awareness of pathogen, appropriate preventative measure, accurate diagnosis, and proper clinical management of respiratory viral infections among children in Macao.

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