Search

In the early COVID-19 pandemic with urgent need for countermeasures, we aimed at developing a replicating viral vaccine using the highly efficacious measles vaccine as vector, a promising technology with prior clinical proof of concept. Building on our successful pre-clinical development of a measles virus (MV)-based vaccine candidate against the related SARS-CoV, we evaluated several recombinant MV expressing codon-optimized SARS-CoV-2 spike glycoprotein. Candidate V591 expressing a prefusion-stabilized spike through introduction of two proline residues in HR1 hinge loop, together with deleted S1/S2 furin cleavage site and additional inactivation of the endoplasmic reticulum retrieval signal, was the most potent in eliciting neutralizing antibodies in mice. After single immunization, V591 induced similar neutralization titers as observed in sera of convalescent patients. The cellular immune response was confirmed to be Th1 skewed. V591 conferred long-lasting protection against SARS-CoV-2 challenge in a murine model with marked decrease in viral RNA load, absence of detectable infectious virus loads, and reduced lesions in the lungs. V591 was furthermore efficacious in an established non-human primate model of disease (see companion article [S. Nambulli, N. Escriou, L. J. Rennick, M. J. Demers, N. L. Tilston-Lunel et al., J Virol 98:e01762-23, 2024, https://doi.org/10.1128/jvi.01762-23]). Thus, V591 was taken forward into phase I/II clinical trials in August 2020. Unexpected low immunogenicity in humans (O. Launay, C. Artaud, M. Lachâtre, M. Ait-Ahmed, J. Klein et al., eBioMedicine 75:103810, 2022, https://doi.org/10.1016/j.ebiom.2021.103810) revealed that the underlying mechanisms for resistance or sensitivity to pre-existing anti-measles immunity are not yet understood. Different hypotheses are discussed here, which will be important to investigate for further development of the measles-vectored vaccine platform.IMPORTANCESARS-CoV-2 emerged at the end of 2019 and rapidly spread worldwide causing the COVID-19 pandemic that urgently called for vaccines. We developed a vaccine candidate using the highly efficacious measles vaccine as vector, a technology which has proved highly promising in clinical trials for other pathogens. We report here and in the companion article by Nambulli et al. (J Virol 98:e01762-23, 2024, https://doi.org/10.1128/jvi.01762-23) the design, selection, and preclinical efficacy of the V591 vaccine candidate that was moved into clinical development in August 2020, 7 months after the identification of SARS-CoV-2 in Wuhan. These unique in-human trials of a measles vector-based COVID-19 vaccine revealed insufficient immunogenicity, which may be the consequence of previous exposure to the pediatric measles vaccine. The three studies together in mice, primates, and humans provide a unique insight into the measles-vectored vaccine platform, raising potential limitations of surrogate preclinical models and calling for further refinement of the platform., Competing Interests: J. Brunet, Z.C., M.G., H.S.-M., M.M., P.C., L. Majlessi, C.G., A.M., and N.E. are inventors of a patent application describing measles-vectored vaccine candidates against SARS-CoV-2. All other authors declare no financial or non-financial competing interests.

Measles is a highly contagious airborne viral disease. It can lead to serious complications and death and is preventable by vaccination. The live-attenuated measles vaccine (LAMV) derived from a measles virus (MV) isolated in 1954 has been in use globally for six decades and protects effectively by providing a durable humoral and cell-mediated immunity. Our study addresses the temporal stability of epitopes on the viral surface glycoprotein hemagglutinin (H) which is the major target of MV-neutralizing antibodies. We investigated the binding of seven vaccine-induced MV-H-specific monoclonal antibodies (mAbs) to cell-free synthesized MV-H proteins derived from the H gene sequences obtained from a lung specimen of a fatal case of measles pneumonia in 1912 and an isolate from a current case. The binding of four out of seven mAbs to the H protein of both MV strains provides evidence of epitopes that are stable for more than 100 years. The binding of the universally neutralizing mAbs RKI-MV-12b and RKI-MV-34c to the H protein of the 1912 MV suggests the long-term stability of highly conserved epitopes on the MV surface., (Copyright © 2024. Published by Elsevier GmbH.)

Serum titers of SARS-CoV-2-neutralizing antibodies (nAbs) correlate well with protection from symptomatic COVID-19 but decay rapidly in the months following vaccination or infection. In contrast, measles-protective nAb titers are lifelong after measles vaccination, possibly due to persistence of the live-attenuated virus in lymphoid tissues. We, therefore, sought to generate a live recombinant measles vaccine capable of driving high SARS-CoV-2 nAb responses. Since previous clinical testing of a live measles vaccine encoding a SARS-CoV-2 spike glycoprotein resulted in suboptimal anti-spike antibody titers, our new vectors were designed to encode prefusion-stabilized SARS-CoV-2 spike glycoproteins, trimerized via an inserted peptide domain, and displayed on a dodecahedral miniferritin scaffold. Additionally, to circumvent the blunting of vaccine efficacy by preformed anti-measles antibodies, we extensively modified the measles surface glycoproteins. Comprehensive in vivo mouse testing demonstrated the potent induction of high titer nAbs in measles-immune mice and confirmed the significant contributions to overall potency afforded by prefusion stabilization, trimerization, and miniferritin display of the SARS-CoV-2 spike glycoprotein. In animals primed and boosted with a measles virus (MeV) vaccine encoding the ancestral SARS-CoV-2 spike, high-titer nAb responses against ancestral virus strains were only weakly cross-reactive with the Omicron variant. However, in primed animals that were boosted with a MeV vaccine encoding the Omicron BA.1 spike, antibody titers to both ancestral and Omicron strains were robustly elevated, and the passive transfer of serum from these animals protected K18-ACE2 mice from infection and morbidity after exposure to BA.1 and WA1/2020 strains. Our results demonstrate that by engineering the antigen, we can develop potent measles-based vaccine candidates against SARS-CoV-2.IMPORTANCEAlthough the live-attenuated measles virus (MeV) is one of the safest and most efficacious human vaccines, a measles-vectored COVID-19 vaccine candidate expressing the SARS-CoV-2 spike failed to elicit neutralizing antibody (nAb) responses in a phase-1 clinical trial, especially in measles-immune individuals. Here, we constructed a comprehensive panel of MeV-based COVID-19 vaccine candidates using a MeV with extensive modifications on the envelope glycoproteins (MeV-MR). We show that artificial trimerization of the spike is critical for the induction of nAbs and that their magnitude can be significantly augmented when the spike protein is synchronously fused to a dodecahedral scaffold. Furthermore, preexisting measles immunity did not abolish heterologous immunity elicited by our vector. Our results highlight the importance of antigen optimization in the development of spike-based COVID-19 vaccines and therapies., Competing Interests: As of July 2021, M.Á.M.-A. was appointed as a scientific director at Vyriad Inc., a clinical-stage biotechnology company developing oncolytic viruses for the treatment of cancers. M.Á.M.-A. and S.J.R. are inventors on a patent application (US Patent App. 17/518,268) filed by the Mayo Clinic relating to the MeV-MR vector that has been licensed to Vyriad Inc. The Mayo Clinic has filed an invention report for the spike protein miniferritin nanoparticle described in this manuscript. The Mayo Clinic may stand to gain financially from the successful outcome of this research. This research has been reviewed by the Mayo Clinic Conflict of Interest Review Board and is being conducted in compliance with Mayo Clinic Conflict of Interest Policies. The laboratory of S.J.R. has received research support from Vyriad Inc. M.S. has received research support from ArgenX N.V. and Moderna. The laboratory of A.G.-S. has received research support from Pfizer, Senhwa Biosciences, Kenall Manufacturing, Avimex, Johnson & Johnson, Dynavax, 7Hills Pharma, Pharmamar, ImmunityBio, Accurius, Hexamer, N-fold LLC, Model Medicines, Atea Pharma, Merck, and Nanocomposix, and A.G.-S. has consulting agreements involving cash and/or stock for the following companies: Vivaldi Biosciences, Contrafect, 7Hills Pharma, Avimex, Vaxalto, Pagoda, Accurius, Esperovax, Farmak, Applied Biological Laboratories, Pharmamar, Paratus, CureLab Oncology, CureLab Veterinary, Synairgen, and Pfizer.

The COVID-19 pandemic is caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) and has spread worldwide, with millions of cases and more than 1 million deaths to date. The gravity of the situation mandates accelerated efforts to identify safe and effective vaccines. Here, we generated measles virus (MeV)-based vaccine candidates expressing the SARS-CoV-2 spike glycoprotein (S). Insertion of the full-length S protein gene in two different MeV genomic positions resulted in modulated S protein expression. The variant with lower S protein expression levels was genetically stable and induced high levels of effective Th1-biased antibody and T cell responses in mice after two immunizations. In addition to neutralizing IgG antibody responses in a protective range, multifunctional CD8 + and CD4 + T cell responses with S protein-specific killing activity were detected. Upon challenge using a mouse-adapted SARS-CoV-2, virus loads in vaccinated mice were significantly lower, while vaccinated Syrian hamsters revealed protection in a harsh challenge setup using an early-passage human patient isolate. These results are highly encouraging and support further development of MeV-based COVID-19 vaccines., Competing Interests: The authors declare no competing interest., (Copyright © 2020 the Author(s). Published by PNAS.)

Priming and activation of CD8 + T cell responses is crucial to achieve anti-viral and anti-tumor immunity. Live attenuated measles vaccine strains have been used successfully for immunization for decades and are currently investigated in trials of oncolytic virotherapy. The available reverse genetics systems allow for insertion of additional genes, including heterologous antigens. Here, we designed recombinant measles vaccine vectors for priming and activation of antigen-specific CD8 + T cells. For proof-of-concept, we used cytotoxic T lymphocyte (CTL) lines specific for the melanoma-associated differentiation antigen tyrosinase-related protein-2 (TRP-2), or the model antigen chicken ovalbumin (OVA), respectively. We generated recombinant measles vaccine vectors with TRP-2 and OVA epitope cassette variants for expression of the full-length antigen or the respective immunodominant CD8 + epitope, with additional variants mediating secretion or proteasomal degradation of the epitope. We show that these recombinant measles virus vectors mediate varying levels of MHC class I (MHC-I)-restricted epitope presentation, leading to activation of cognate CTLs, as indicated by secretion of interferon-gamma (IFNγ) in vitro. Importantly, the recombinant OVA vaccines also mediate priming of naïve OT-I CD8 + T cells by dendritic cells. While all vaccine variants can prime and activate cognate T cells, IFNγ release was enhanced using a secreted epitope variant and a variant with epitope strings targeted to the proteasome. The principles presented in this study will facilitate the design of recombinant vaccines to elicit CD8 + responses against pathogens and tumor antigens.

Vaccine-preventable diseases (VPD) including measles and mumps have been re-emerging in countries with sustained high vaccine coverage. For mumps, waning immunity has been recognized as a major contributor to recent outbreaks. Although unvaccinated individuals account for most cases in recent measles outbreaks, the role of immune waning remains unclear. Accumulating serological and epidemiological evidence suggests that natural immunity induced by infection may be more durable compared to vaccine-induced immunity. As the proportion of population immunity via vaccination gradually increases and boosting through natural exposures becomes rare, risk of outbreaks may increase. Mechanistic insights into the coupled immuno-epidemiological dynamics of waning and boosting will be important to understand optimal vaccination strategies to combat VPD re-emergence and achieve eradication., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Objective: Analyze the genetic characteristic of Hemagglutinin(H) gene of measles viruses isolated in Henan Province in 2017. Methods: Swab samples collected from 7 lab confirmed measles cases, and we got the measles virus by Vero/Slam inoculation. Fragment of H genes were amplified by reverse transcription polymerase chain reaction(RT-PCR), then the PCR products were sequenced and analyzed. Results: The age of the 7 measles confirmed cases were between 1 and 50 years old, and all of them were males. All the 7 measles viruses were identified as H1a genotype, and the average distance of the nucleotides and the amino acids was 0.005, respectively. Compared with the Shanghai-191/China-vaccine, there were some changes in isolated virus, such as 240(th), 397(th) and 381(st) sites in the amino acid sequence. Conclusion: The measles genotype which isolated in Henan Province in 2017 was H1a. There were some difference from Shanghai-191/China-vaccine in the nucleotides sequence of H gene, which suggested that it's necessary to strengthen the monitor the variation of measles virus.

Measles is one of the most contagious viral respiratory infections and was declared to be eliminated from Canada in 1998; however, measles cases and outbreaks still occur every year through reintroduction from other parts of the world. Laboratory confirmation of measles virus (MV) RNA by real-time PCR provides a definitive diagnosis, and molecular analysis to determine the genotype is the only way to distinguish between wild-type and vaccine strains. This distinction is important since live attenuated vaccine strains are able to replicate in the patient and can be associated with rash and fever but are poorly transmissible, if at all. Prompt reporting of measles cases to local authorities, including differentiation between wild-type and vaccine strains, allows for optimal management and contact tracing. The development and validation of a multiplex real-time reverse transcription-PCR (rtRT-PCR) assay for the simultaneous detection and differentiation of the Moraten and Schwarz vaccine strains from presumptive wild-type MV in a format that can be easily implemented for high-throughput testing of patient samples are reported here. This assay is sensitive, specific, reproducible, and 100% accurate in comparison with the gold standard comparator assay., (Copyright © 2019 American Society for Microbiology.)

Infectious disease epidemics match wars and natural disasters in their capacity to threaten lives and damage economies. Like SARS previously and Zika recently, the Ebola crisis in 2015 showed how vulnerable the world is to these epidemics, with over 11,000 people dying in the outbreak. In addition to causing immense human suffering, these epidemics particularly affect low- and middle-income countries. Many of these deadly infectious diseases that have epidemic potential can become global health emergencies in the absence of effective vaccines. But very few vaccines against these threats have been developed to create proven medical products. The measles vaccine is an efficient, live attenuated, replicating virus that has been safely administered to 2 billion children over the last 40 years, affording life-long protection after a single dose. Taking advantage of these characteristics, this attenuated virus was transformed into a versatile chimeric or recombinant vaccine vector with demonstrated proof-of-principle in humans and a preclinical track record of rapid adaptability and effectiveness for a variety of pathogens. Clinical trials have shown the safety and immunogenicity of this vaccine platform in individuals with preexisting immunity to measles. This review describes the potential of this platform to develop new vaccines against emerging viral diseases., (Copyright © 2018. Published by Elsevier Masson SAS.)

Rapid differentiation of wild-type measles virus from measles vaccine strains is crucial during a measles outbreak and in a measles elimination setting. A real-time reverse transcription-PCR (rRT-PCR) for the rapid detection of measles vaccine strains was developed with high specificity and sensitivity equivalent to that of traditional measles genotyping methods. The "stressed" minor groove binder-TaqMan probe design approach achieves specificity to vaccine strains only, without compromising sensitivity. This assay, without requiring sequence genotyping, has proved to be extremely useful in outbreak settings for over 4 years at the Regional Measles Reference Laboratory for the Western Pacific Region., (Copyright © 2018 Tran et al.)

The live-attenuated measles virus (MV) vaccine based on the Hu191 strain has played a significant role in controlling measles in China. However, it has considerable adverse effects that may cause public health burden. We hypothesize that the safety and efficacy of MV vaccine can be improved by altering the S-adenosylmethionine (SAM) binding site in the conserved region VI of the large polymerase protein. To test this hypothesis, we established an efficient reverse genetics system for the rMV-Hu191 strain and generated two recombinant MV-Hu191 carrying mutations in the SAM binding site. These two mutants grew to high titer in Vero cells, were genetically stable, and were significantly more attenuated in vitro and in vivo compared to the parental rMV-Hu191 vaccine strain. Importantly, both MV-Hu191 mutants triggered a higher neutralizing antibody than rMV-Hu191 vaccine and provided complete protection against MV challenge. These results demonstrate its potential for an improved MV vaccine candidate., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Background/purpose: Along with the improving vaccine coverage, suspected vaccine-associated measles has been reported in Zhejiang Province, China. In order to maintain the accuracy of the measles surveillance system, it is critical to discriminate between measles vaccine and wild-type virus., Methods: Eight suspected cases of vaccine-associated measles were reported in Zhejiang Province during 2011 and 2014. Sera collected within 4 days and throat swabs collected within 6 days after rash onset were tested with immunoglobulin M and measles virus (MeV) RNA to confirm MeV infection. In order to further identify the vaccine-associated cases, throat swabs with positive MeV RNA were tested using an allelic discrimination real-time reverse transcriptase polymerase chain reaction (rRT-PCR) assay developed in this study, RT-PCR-restriction fragment length polymorphism (RFLP) recommended by the National Measles Laboratory, and RT-PCR followed by sequencing and genotyping., Results: Combining anti-measles immunoglobulin M and RNA testing, eight cases were confirmed as MeV infection. Of the eight, two were identified as vaccine-associated cases by the allelic discrimination rRT-PCR assay, and one was identified by RT-PCR-RFLP. Subsequent sequencing and genotyping confirmed that the sequences of the two cases were identical to that of the Chinese vaccine strain. The developed allelic discrimination rRT-PCR was 10 times more sensitive than the RT-PCR-RFLP assay when RNA standards generated from three genotypes of MeV were tested., Conclusion: Vaccine-associated measles has been identified in Zhejiang. The developed allelic discrimination rRT-PCR assay is rapid and sensitive, which will facilitate the surveillance for vaccine-associated measles., (Copyright © 2015. Published by Elsevier B.V.)

Attenuated measles virus (MV) is one of the most effective and safe vaccines available, making it an attractive candidate vector for preventing other infectious diseases. Yet the great capacity of this vaccine still needs to be understood at the molecular level. MV vaccine strains have different type I interferon (IFN)-inducing abilities that partially depend on the presence of 5' copy-back defective interfering genomes (DI-RNAs). DI-RNAs are pathogen-associated molecular patterns recognized by RIG-I-like receptors (RLRs) (RIG-I, MDA5, and LGP2) that activate innate immune signaling and shape the adaptive immune response. In this study, we characterized the DI-RNAs produced by various modified recombinant MVs (rMVs), including vaccine candidates, as well as wild-type MV. All tested rMVs produced 5' copy-back DI-RNAs that were different in length and nucleotide sequence but still respected the so-called "rule of six." We correlated the presence of DI-RNAs with a larger stimulation of the IFN-β pathway and compared their immunostimulatory potentials. Importantly, we revealed that encapsidation of DI-RNA molecules within the MV nucleocapsid abolished their immunoactive properties. Furthermore, we identified specific interactions of DI-RNAs with both RIG-I and LGP2 but not MDA5. Our results suggest that DI-RNAs produced by rMV vaccine candidates may indeed strengthen their efficiency by triggering RLR signaling. IMPORTANCE Having been administered to hundreds of millions of children, the live attenuated measles virus (MV) vaccine is the safest and most widely used human vaccine, providing high protection with long-term memory. Additionally, recombinant MVs carrying heterologous antigens are promising vectors for new vaccines. The great capacity of this vaccine still needs to be elucidated at the molecular level. Here we document that recombinant MVs produce defective interfering genomes that have high immunostimulatory properties via their binding to RIG-I and LGP2 proteins, both of which are cytosolic nonself RNA sensors of innate immunity. Defective interfering genome production during viral replication should be considered of great importance due to the immunostimulatory properties of these genomes as intrinsic adjuvants produced by the vector that increase recognition by the innate immune system., (Copyright © 2017 American Society for Microbiology.)

Current measles vaccines suffer from poor effectiveness in young infants due primarily to the inhibitory effect of residual maternal immunity on vaccine responses. The development of a measles vaccine that resists such passive immunity would strongly contribute to the stalled effort toward measles eradication. In this concise communication, we show that a measles virus (MV) with enhanced hemagglutinin (H) expression and incorporation, termed MVvac2-H2, retained its enhanced immunogenicity, previously established in older mice, when administered to very young, genetically modified, MV-susceptible mice in the presence of passive anti-measles immunity. This immunity level mimics the sub-neutralizing immunity prevalent in infants too young to be vaccinated. Additionally, toward a more physiological small animal model of maternal anti-measles immunity interference, we document vertical transfer of passive anti-MV immunity in genetically-modified, MV susceptible mice and show in this physiological model a better MVvac2-H2 immunogenic profile than that of the parental vaccine strain. In sum, these data support the notion that enhancing MV hemagglutinin incorporation can circumvent in vivo neutralization. This strategy merits additional exploration as an alternative pediatric measles vaccine., Competing Interests: The authors declare no conflict of interest.

During measles outbreaks, it is important to be able to rapidly distinguish between measles cases and vaccine reactions to avoid unnecessary outbreak response measures such as case isolation and contact investigations. We have developed a real-time reverse transcription-PCR (RT-PCR) method specific for genotype A measles virus (MeV) (MeVA RT-quantitative PCR [RT-qPCR]) that can identify measles vaccine strains rapidly, with high throughput, and without the need for sequencing to determine the genotype. We have evaluated the method independently in three measles reference laboratories using two platforms, the Roche LightCycler 480 system and the Applied Biosystems (ABI) 7500 real-time PCR system. In comparison to the standard real-time RT-PCR method, the MeVA RT-qPCR showed 99.5% specificity for genotype A and 94% sensitivity for both platforms. The new assay was able to detect RNA from five currently used vaccine strains, AIK-C, CAM-70, Edmonston-Zagreb, Moraten, and Shanghai-191. The MeVA RT-qPCR assay has been used successfully for measles surveillance in reference laboratories, and it could be readily deployed to national and subnational laboratories on a wide scale., (Copyright © 2017 American Society for Microbiology.)

Rapid differentiation of vaccine from wild-type strains in suspect measles cases is a valuable epidemiological tool that informs the public health response to this highly infectious disease. Few public health laboratories sequence measles virus-positive specimens to determine genotype, and the vaccine-specific real-time reverse transcriptase PCR (rRT-PCR) assay described by F. Roy et al. (J. Clin. Microbiol. 55:735-743, 2017, https://doi.org/10.1128/JCM.01879-16) offers a rapid, easily adoptable method to identify measles vaccine strains in suspect cases., (Copyright © 2017 American Society for Microbiology.)

This chapter describes the development of recombinant measles virus (MV)-based vaccines starting from plasmid DNA. Live-attenuated measles vaccines are very efficient and safe. Since the availability of a reverse genetic system to manipulate MV genomes and to generate respective recombinant viruses, a considerable number of recombinant viruses has been generated that present antigens of foreign pathogens during MV replication. Thereby, robust humoral and cellular immune responses can be induced, which have shown protective capacity in a substantial number of experiments.For this purpose, the foreign antigen-encoding genes are cloned into additional transcription units of plasmid based full-length MV vaccine strain genomes, which in turn are used to rescue recombinant MV by providing both full-length viral RNA genomes respective anti-genomes together with all protein components of the viral ribonucleoprotein complex after transient transfection of the so-called rescue cells. Infectious centers form among these transfected cells, which allow clonal isolation of single recombinant viruses that are subsequently amplified, characterized in vitro, and then evaluated for their immunogenicity in appropriate preclinical animal models.

In 2013, a major chikungunya virus (CHIKV) epidemic reached the Americas. In the past 2 years, >1.7 million people have been infected. In light of the current epidemic, with millions of people in North and South America at risk, efforts to rapidly develop effective vaccines have increased. Here, we focus on CHIKV vaccines that use viral-vector technologies. This group of vaccine candidates shares an ability to potently induce humoral and cellular immune responses by use of highly attenuated and safe vaccine backbones. So far, well-described vectors such as modified vaccinia virus Ankara, complex adenovirus, vesicular stomatitis virus, alphavirus-based chimeras, and measles vaccine Schwarz strain (MV/Schw) have been described as potential vaccines. We summarize here the recent data on these experimental vaccines, with a focus on the preclinical and clinical activities on the MV/Schw-based candidate, which is the first CHIKV-vectored vaccine that has completed a clinical trial., (© The Author 2016. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail journals.permissions@oup.com.)

Unlabelled: Imported measles virus (MV) outbreaks are maintained by poor vaccine responders and unvaccinated people. A convenient but more immunogenic vaccination strategy would enhance vaccine performance, contributing to measles eradication efforts. We report here the generation of alternative pediatric vaccines against MV with increased expression of the H protein in the background of the current MV vaccine strain. We generated two recombinants: MVvac2-H2, with increased full-length H expression resulting in a 3-fold increase in H incorporation into virions, and MVvac2-Hsol, vectoring a truncated, soluble form of the H protein that is secreted into the supernatants of infected cells. Replication fitness was conserved despite the duplication of the H cistron for both vectors. The modification to the envelope of MVvac2-H2 conferred upon this virus a measurable level of resistance to in vitro neutralization by MV polyclonal immune sera without altering its thermostability. Most interestingly, both recombinant MVs with enhanced H expression were significantly more immunogenic than their parental strain in outbred mice, while MVvac2-H2 additionally proved more immunogenic after a single, human-range dose in genetically modified MV-susceptible mice., Importance: Measles incidence was reduced drastically following the introduction of attenuated vaccines, but progress toward the eradication of this virus has stalled, and MV still threatens unvaccinated populations. Due to the contributions of primary vaccine failures and too-young-to-be-vaccinated infants to this problem, more immunogenic measles vaccines are highly desirable. We generated two experimental MV vaccines based on a current vaccine's genome but with enriched production of the H protein, the main MV antigen in provoking immunity. One vaccine incorporated H at higher rates in the viral envelope, and the other secreted a soluble H protein from infected cells. The increased expression of H by these vectors improved neutralizing responses induced in two small-animal models of MV immunogenicity. The enhanced immunogenicity of these vectors, mainly from the MV that incorporates additional H, suggests their value as potential alternative pediatric MV vaccines., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Attenuated measles virus (MV) is one of the most effective and safe vaccines available, making it attractive candidate vector to prevent infectious diseases. Attenuated MV have acquired the ability to use the complement regulator CD46 as a major receptor to mediate virus entry and intercellular fusion. Therefore, attenuated MV strains preferentially infect and destroy a wide variety of cancer cells making them also attractive oncolytic vectors. The use of recombinant MV vector has to comply with various regulatory requirements, particularly relating to the assessment of potential risks for human health and the environment. The present article highlights the main characteristics of MV and recombinant MV vectors used for vaccination and virotherapy and discusses these features from a biosafety point of view.

Unlabelled: The live attenuated measles virus vaccine is highly immunostimulatory. Identification and characterization of its components that activate the innate immune response might provide new strategies and agents for the rational design and development of chemically defined adjuvants. In this study, we report on the activation of type I interferon (IFN) production by a defective interfering (DI) RNA isolated from the Hu-191 vaccine strain of measles virus. We found that the Hu-191 virus induced IFN-β much more potently than the Edmonston strain. In the search for IFN-inducing species in Hu-191, we identified a DI RNA specifically expressed by this strain. This DI RNA, which was of the copy-back type, was predicted to fold into a hairpin structure with a long double-stranded stem region of 206 bp, and it potently induced the expression of IFN-β. Its IFN-β-inducing activity was further enhanced when both cytoplasmic RNA sensor RIG-I and its partner, PACT, were overexpressed. On the contrary, this activity was abrogated in cells deficient in PACT or RIG-I. The DI RNA was found to be associated with PACT in infected cells. In addition, both the 5'-di/triphosphate end and the double-stranded stem region on the DI RNA were essential for its activation of PACT and RIG-I. Taken together, our findings support a model in which a viral DI RNA is sensed by PACT and RIG-I to initiate an innate antiviral response. Our work might also provide a foundation for identifying physiological PACT ligands and developing novel adjuvants or antivirals., Importance: The live attenuated measles virus vaccine is one of the most successful human vaccines and has largely contained the devastating impact of a highly contagious virus. Identifying the components in this vaccine that stimulate the host immune response and understanding their mechanism of action might help to design and develop better adjuvants, vaccines, antivirals, and immunotherapeutic agents. We identified and characterized a defective interfering RNA from the Hu-191 vaccine strain of measles virus which has safely been used in millions of people for many years. We further demonstrated that this RNA potently induces an antiviral immune response through cellular sensors of viral RNA known as PACT and RIG-I. Similar types of viral RNA that bind with and activate PACT and RIG-I might retain the immunostimulatory property of measles virus vaccines but would not induce adaptive immunity. They are potentially useful as chemically defined vaccine adjuvants, antivirals, and immunostimulatory agents., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Unlabelled: Although live-attenuated measles virus (MV) vaccines have been used successfully for over 50 years, the target cells that sustain virus replication in vivo are still unknown. We generated a reverse genetics system for the live-attenuated MV vaccine strain Edmonston-Zagreb (EZ), allowing recovery of recombinant (r)MV(EZ). Three recombinant viruses were generated that contained the open reading frame encoding enhanced green fluorescent protein (EGFP) within an additional transcriptional unit (ATU) at various positions within the genome. rMV(EZ)EGFP(1), rMV(EZ)EGFP(3), and rMV(EZ)EGFP(6) contained the ATU upstream of the N gene, following the P gene, and following the H gene, respectively. The viruses were compared in vitro by growth curves, which indicated that rMV(EZ)EGFP(1) was overattenuated. Intratracheal infection of cynomolgus macaques with these recombinant viruses revealed differences in immunogenicity. rMV(EZ)EGFP(1) and rMV(EZ)EGFP(6) did not induce satisfactory serum antibody responses, whereas both in vitro and in vivo rMV(EZ)EGFP(3) was functionally equivalent to the commercial MV(EZ)-containing vaccine. Intramuscular vaccination of macaques with rMV(EZ)EGFP(3) resulted in the identification of EGFP(+) cells in the muscle at days 3, 5, and 7 postvaccination. Phenotypic characterization of these cells demonstrated that muscle cells were not infected and that dendritic cells and macrophages were the predominant target cells of live-attenuated MV., Importance: Even though MV strain Edmonston-Zagreb has long been used as a live-attenuated vaccine (LAV) to protect against measles, nothing is known about the primary cells in which the virus replicates in vivo. This is vital information given the push to move toward needle-free routes of vaccination, since vaccine virus replication is essential for vaccination efficacy. We have generated a number of recombinant MV strains expressing enhanced green fluorescent protein. The virus that best mimicked the nonrecombinant vaccine virus was formulated according to protocols for production of commercial vaccine virus batches, and was subsequently used to assess viral tropism in nonhuman primates. The virus primarily replicated in professional antigen-presenting cells, which may explain why this LAV is so immunogenic and efficacious., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Measles was an inevitable infection during the human development with substantial degree of morbidity and mortality. The severity of measles virus (MV) infection was largely contained by the development of a live attenuated vaccine that was introduced into the vaccination programs. However, all efforts to eradicate the disease failed and continued to annually result in significant deaths. The development of molecular biology techniques allowed the rescue of MV from cDNA that enabled important insights into a variety of aspects of the biology of the virus and its pathogenesis. Subsequently these technologies facilitated the development of novel vaccine candidates that induce immunity against measles and other pathogens. Based on the promising prospective, the use of MV as a recombinant vaccine and a therapeutic vector is addressed.

Today, immune compromised interferon-α-receptor deficient mice expressing hCD46 (IFNARCD46tg) are usually used for measles virus (MV) based vaccine characterization. However, for the development of MV-based recombinant vaccine candidates (rMV), an immune competent mouse model is desirable in order to induce and evaluate meaningful immune response. In this study, humoral and cellular immune response induced by rMV in immune competent mice expressing human MV receptor CD46 (hCD46tg) were compared with those induced in wild-type black/6, and IFNARCD46tg mice.   All three strains developed humoral and cellular response against MV, whereas only hCD46tg and IFNARCD46tg mice developed a humoral response against the transgene. Differences were observed in the magnitude of the response, where the IFNARCD46tg mice displayed the strongest immune responses, followed by the hCD46tg mice and the black/6 mice. Interestingly, hCD46tg and wt black/6 mice showed a predominant CD4(+) T-cell response against MV-N, whereas IFNARCD46tg mice developed both, CD4(+) and CD8(+) T-cell response against MV-N. Analysis of the cytokine profile of MV-N specific CD4(+) T-cells and transgene (SIVgag) specific CD8(+) T-cells revealed qualitative differences of the T-cell responses; noticeably a significant reduction of the frequency of CD4(+)IL-2(+) expressing cells in IFNARCD46tg mice as compared with hCD46tg or wt black/6 mice. We show in this study significant quantitative and qualitative differences in immune responses between immune competent and immune-compromised mice. Our results therefore highlight the importance of the animal model and support the use of hCD46tg mice as mouse model for the characterization of the immunological profile induced by recombinant measles virus vaccine candidates.

Measles, mumps and rubella are vaccine-preventable diseases; however limited epidemiological data are available from low-income or developing countries. Thus, it is important to investigate the transmission of these viruses in different geographical regions. In this context, a cell culture-based rapid and reliable immuno-colorimetric assay (ICA) was established and its utility studied. Twenty-three measles, six mumps and six rubella virus isolates and three vaccine strains were studied. Detection by ICA was compared with plaque and RT-PCR assays. In addition, ICA was used to detect viruses in throat swabs (n = 24) collected from patients with suspected measles or mumps. Similarly, ICA was used in a focus reduction neutralization test (FRNT) and the results compared with those obtained by a commercial IgG enzyme immuno assay. Measles and mumps virus were detected 2 days post-infection in Vero or Vero-human signaling lymphocytic activation molecule cells, whereas rubella virus was detected 3 days post-infection in Vero cells. The blue stained viral foci were visible by the naked eye or through a magnifying glass. In conclusion, ICA was successfully used on 35 virus isolates, three vaccine strains and clinical specimens collected from suspected cases of measles and mumps. Furthermore, an application of ICA in a neutralization test (i.e., FRNT) was documented; this may be useful for sero-epidemiological, cross-neutralization and pre/post-vaccine studies., (© 2014 The Societies and Wiley Publishing Asia Pty Ltd.)

The recent identification of a novel human coronavirus responsible of a SARS-like illness in the Middle-East a decade after the SARS pandemic, demonstrates that reemergence of a SARS-like coronavirus from an animal reservoir remains a credible threat. Because SARS is contracted by aerosolized contamination of the respiratory tract, a vaccine inducing mucosal long-term protection would be an asset to control new epidemics. To this aim, we generated live attenuated recombinant measles vaccine (MV) candidates expressing either the membrane-anchored SARS-CoV spike (S) protein or its secreted soluble ectodomain (Ssol). In mice susceptible to measles virus, recombinant MV expressing the anchored full-length S induced the highest titers of neutralizing antibodies and fully protected immunized animals from intranasal infectious challenge with SARS-CoV. As compared to immunization with adjuvanted recombinant Ssol protein, recombinant MV induced stronger and Th1-biased responses, a hallmark of live attenuated viruses and a highly desirable feature for an antiviral vaccine., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Nipah virus (NiV), a paramyxovirus, was first discovered in Malaysia in 1998 in an outbreak of infection in pigs and humans, and incurred a high fatality rate in humans. We established a system that enabled the rescue of replicating NiVs from a cloned DNA. Using the system, we analyzed the functions of accessory proteins in infected cells and the implications in in vivo pathogenicity. Further, we have developed a recombinant measles virus (rMV) vaccine expressing NiV envelope glycoproteins, which appeared to be an appropriate to NiV vaccine candidate for use in humans.

DNA vaccines formulated with the cationic lipid-based adjuvant Vaxfectin induce protective immunity in macaques after intradermal (i.d.) or intramuscular (i.m.) delivery of 0.5 to 1 mg of codon-optimized DNA encoding the hemagglutinin (H) and fusion (F) proteins of measles virus (MeV). To characterize the effect of Vaxfectin at lower doses of H+F DNA, rhesus macaques were vaccinated twice with 20 μg of DNA plus Vaxfectin i.d., 100 μg of DNA plus Vaxfectin i.d., 100 μg of DNA plus Vaxfectin i.m. or 100 μg of DNA plus phosphate-buffered saline (PBS) i.m. using a needleless Biojector device. The levels of neutralizing (P = 0.036) and binding (P = 0.0001) antibodies were higher after 20 or 100 μg of DNA plus Vaxfectin than after 100 μg of DNA plus PBS. Gamma interferon (IFN-γ)-producing T cells were induced more rapidly than antibody, but were not improved with Vaxfectin. At 18 months after vaccination, monkeys were challenged with wild-type MeV. None developed rash or viremia, but all showed evidence of infection. Antibody levels increased, and IFN-γ- and interleukin-17-producing T cells, including cells specific for the nucleoprotein absent from the vaccine, were induced. At 3 months after challenge, MeV RNA was detected in the leukocytes of two monkeys. The levels of antibody peaked 2 to 4 weeks after challenge and then declined in vaccinated animals reflecting low numbers of bone marrow-resident plasma cells. Therefore, Vaxfectin was dose sparing and substantially improved the antibody response to the H+F DNA vaccine. This immune response led to protection from disease (rash/viremia) but not from infection. Antibody responses after challenge were more transient in vaccinated animals than in an unvaccinated animal.

Because of increasing measles vaccine coverage, the proportion of patients with modified measles has been increasing. Such patients have low-grade fever with very mild eruptions similar to vaccine-related adverse events. Differentiation between these two pathogenic conditions is required to improve the quality of laboratory-based measles surveillance. In this study, vaccine-specific and wild-type specific primer sets were designed for loop-mediated isothermal amplification in the N gene, and vaccine strains, C1, D3, D4, D5, D8, D9, G3 and H1 wild strains were examined. Three vaccine strains were efficiently amplified using a vaccine-specific primer set with an approximately 10-times higher sensitivity than wild-type primer. Modified measles was differentiated from vaccine-associated cases by this system, but limitations were encountered with the other genotypes., (© 2013 The Societies and Wiley Publishing Asia Pty Ltd.)

Licensed live attenuated virus vaccines capable of expressing transgenes from other pathogens have the potential to reduce the number of childhood immunizations by eliciting robust immunity to multiple pathogens simultaneously. Recombinant attenuated measles virus (rMV) derived from the Edmonston Zagreb vaccine strain was engineered to express simian immunodeficiency virus (SIV) Gag protein for the purpose of evaluating the immunogenicity of rMV as a vaccine vector in rhesus macaques. rMV-Gag immunization alone elicited robust measles-specific humoral and cellular responses, but failed to elicit transgene (Gag)-specific immune responses, following aerosol or intratracheal/intramuscular delivery. However, when administered as a priming vaccine to a heterologous boost with recombinant adenovirus serotype 5 expressing the same transgene, rMV-Gag significantly enhanced Gag-specific T lymphocyte responses following rAd5 immunization. Gag-specific humoral responses were not enhanced, however, which may be due to either the transgene or the vector. Cellular response priming by rMV against the transgene was highly effective even when using a suboptimal dose of rAd5 for the boost. These data demonstrate feasibility of using rMV as a priming component of heterologous prime-boost vaccine regimens for pathogens requiring strong cellular responses., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

A major difference between vaccine and wild-type strains of measles virus (MV) in vitro is the wider cell specificity of vaccine strains, resulting from the receptor usage of the hemagglutinin (H) protein. Wild-type H proteins recognize the signaling lymphocyte activation molecule (SLAM) (CD150), which is expressed on certain cells of the immune system, whereas vaccine H proteins recognize CD46, which is ubiquitously expressed on all nucleated human and monkey cells, in addition to SLAM. To examine the effect of the H protein on the tropism and attenuation of MV, we generated enhanced green fluorescent protein (EGFP)-expressing recombinant wild-type MV strains bearing the Edmonston vaccine H protein (MV-EdH) and compared them to EGFP-expressing wild-type MV strains. In vitro, MV-EdH replicated in SLAM(+) as well as CD46(+) cells, including primary cell cultures from cynomolgus monkey tissues, whereas the wild-type MV replicated only in SLAM(+) cells. However, in macaques, both wild-type MV and MV-EdH strains infected lymphoid and respiratory organs, and widespread infection of MV-EdH was not observed. Flow cytometric analysis indicated that SLAM(+) lymphocyte cells were infected preferentially with both strains. Interestingly, EGFP expression of MV-EdH in tissues and lymphocytes was significantly weaker than that of the wild-type MV. Taken together, these results indicate that the CD46-binding activity of the vaccine H protein is important for determining the cell specificity of MV in vitro but not the tropism in vivo. They also suggest that the vaccine H protein attenuates MV growth in vivo.

Live attenuated measles virus is one of the most efficient and safest vaccines available, making it an attractive candidate vector for a HIV/AIDS vaccine aimed at eliciting cell-mediated immune responses (CMI). Here we have characterized the potency of CMI responses generated in mice and non-human primates after intramuscular immunisation with a candidate recombinant measles vaccine carrying an HIV-1 insert encoding Clade B Gag, RT and Nef (MV1-F4). Eight Mauritian derived, MHC-typed cynomolgus macaques were immunised with 10(5) TCID(50) of MV1-F4, four of which were boosted 28 days later with the same vaccine. F4 and measles virus (MV)-specific cytokine producing T cell responses were detected in 6 and 7 out of 8 vaccinees, respectively. Vaccinees with either M6 or recombinant MHC haplotypes demonstrated the strongest cytokine responses to F4 peptides. Polyfunctional analysis revealed a pattern of TNFα and IL-2 responses by CD4+ T cells and TNFα and IFNγ responses by CD8+ T cells to F4 peptides. HIV-specific CD4+ and CD8+ T cells expressing cytokines waned in peripheral blood lymphocytes by day 84, but CD8+ T cell responses to F4 peptides could still be detected in lymphoid tissues more than 3 months after vaccination. Anti-F4 and anti-MV antibody responses were detected in 6 and 8 out of 8 vaccinees, respectively. Titres of anti-F4 and MV antibodies were boosted in vaccinees that received a second immunisation. MV1-F4 carrying HIV-1 Clade B inserts induces robust boostable immunity in non-human primates. These results support further exploration of the MV1-F4 vector modality in vaccination strategies that may limit HIV-1 infectivity.

Objective: Vitamins A and D, and their receptors, are important regulators of the immune system, including vaccine immune response. We assessed the association between polymorphisms in the vitamin A receptors [retinoic acid receptor α, retinoic acid receptor β (RARB), and retinoic acid receptor γ] and vitamin D receptor (VDR)/retinoid X receptor α (RXRA) genes and interindividual variations in immune responses after two doses of measles vaccine in 745 children., Methods: Using a tag single nucleotide polymorphism (SNP) approach, we genotyped 745 healthy children for the 391 polymorphisms in vitamin A receptor and VDR genes., Results: The RARB haplotype (rs6800566/rs6550976/rs9834818) was significantly associated with variations in both measles antibody (global, P=0.013) and cytokine secretion levels, such as interleukin (IL)-10 (global, P=0.006), interferon (IFN)-α (global, P=0.008), and tumor necrosis factor-α (global, P=0.039) in the Caucasian subgroup. Specifically, the RARB haplotype, AAC, was associated with higher (t-statistic: 3.27, P=0.001) measles antibody levels. At the other end of the spectrum, haplotype GG for rs6550978/rs6777544 was associated with lower antibody levels (t-statistic: -2.32, P=0.020) in the Caucasian subgroup. In a sensitivity analysis, the RARB haplotype, CTGGGCAA, remained marginally significant (P<0.02) when the single SNP rs12630816 was included in the model for IL-10 secretion levels. A significant association was found between lower measles-specific IFN-γ Enzyme-linked immunosorbent spot responses and haplotypes rs11102986/rs11103473/rs11103482/rs10776909/rs12004589/rs35780541/rs2266677/rs875444 (global, P=0.004) and rs6537944/rs3118571 (global, P<0.001) in the RXRA gene for Caucasians. We also found associations between multiple RARB, VDR, and RXRA SNPs/haplotypes and measles-specific IL-2, IL-6, IL-10, IFN-α, IFN-γ, IFNλ-1, and TNF-α cytokine secretions., Conclusion: Our results suggest that specific allelic variations and haplotypes in the vitamin A receptor and VDR genes may influence adaptive immune responses to measles vaccine.

The incidence of measles in China has increased over the last decade. To evaluate the genetic variation of measles strains, 16 measles wild-type virus strains were isolated from 14 vaccinated cases and 2 nonvaccinated cases in Jilin Province during 2005-2006, and their nucleoprotein (N) and hemagglutinin (H) genes were amplified by RT-PCR. The amplified products were sequenced and compared with the Edmonston virus and the existing vaccine strains (Changchun-47 and Shanghai-191). The results showed that the variation rate between the vaccine and wild-type strains was 9.8-12.0% in the N gene and 5.9-6.9% in the H gene, respectively. In addition, cross-neutralization assays revealed that although sera obtained from infants following primary vaccination effectively neutralized vaccine strains, the capacity in neutralizing H1 wild-type measles virus isolates was decreased fourfold. Antigenic ratios testing revealed that the antigenic relatedness between wild-type measles viruses and existing vaccine strains was notably low. These data suggest that the increased incidence of measles in Jilin Province may be attributed to the antigenic drift between wild-type and vaccine strains. Our findings strengthen the recommendation of supplemental immunization with existing vaccines and also strongly suggest a need for developing new vaccines to better control measles virus outbreaks.

The complete genomic sequences of 9 measles vaccine strains were compared with the sequence of the Edmonston wild-type virus. AIK-C, Moraten, Rubeovax, Schwarz, and Zagreb are vaccine strains of the Edmonston lineage, whereas CAM-70, Changchun-47, Leningrad-4 and Shanghai-191 were derived from 4 different wild-type isolates. Nucleotide substitutions were found in the noncoding regions of the genomes as well as in all coding regions, leading to deduced amino acid substitutions in all 8 viral proteins. Although the precise mechanisms involved in the attenuation of individual measles vaccines remain to be elucidated, in vitro assays of viral protein functions and recombinant viruses with defined genetic modifications have been used to characterize the differences between vaccine and wild-type strains. Although almost every protein contributes to an attenuated phenotype, substitutions affecting host cell tropism, virus assembly, and the ability to inhibit cellular antiviral defense mechanisms play an especially important role in attenuation., (Published by Oxford University Press on behalf of the Infectious Diseases Society of America 2011.)

The Schwarz FF-8 (FF-8) and AIK-C measles virus vaccine strains are currently used for vaccination in Japan. Here, the complete genome nucleotide sequence of the FF-8 strain has been determined and its genome sequence found to be remarkably similar to that of the AIK-C strain. These two strains are differentiated only by two nucleotide differences in the phosphoprotein gene. Since the FF-8 strain does not possess the amino acid substitutions in the phospho- and fusion proteins which are responsible for the temperature-sensitivity and small syncytium formation phenotypes of the AIK-C strain, respectively, other unidentified common mechanisms likely attenuate both the FF-8 and AIK-C strains., (© 2011 The Societies and Blackwell Publishing Asia Pty Ltd.)

Background: Plasmid-based measles vaccines offer great promises over the conventional fertilised egg method such as ease of manufacture and mimic wild-type intracellular antigen expression. The increasing number of clinical trials on plasmid-based measles vaccines has triggered the need to make more in less time., Results: In this work, we investigated the process variables necessary to improve the volumetric and specific yields of a model plasmid-based measles vaccine (pcDNA3F) harboured in E. coli DH5α. Results from growth medium optimisation in 500 mL shake flasks by response surface methodology (RSM) generated a maximum volumetric yield of 13.65 mg/L which was 1.75 folds higher than that of the base medium. A controlled fed-batch fermentation employing strategic glycerol feeding and optimised growth conditions resulted in a remarkable pcDNA3F volumetric yield of 110 mg/L and a specific yield of 14 mg/g. In addition, growth pH modification and temperature fluctuation between 35 and 45°C were successfully employed to improve plasmid production., Conclusion: Production of a high copy number plasmid DNA containing a foreign gene of interest is often hampered by the low plasmid volumetric yield which results from the over expression of foreign proteins and metabolic repressors. In this work, a simple bioprocess framework was employed and successfully improved the production of pcDNA3F.

We sought to examine ADAR-1 editing of measles and influenza virus genomes derived from inactivated seasonal influenza and live attenuated measles virus vaccines grown on chicken cells as the culture substrate. Using highly sensitive 3DI-PCR (R. Suspène et al., Nucleic Acids Res. 36:e72, 2008), it was possible to show that ADAR-1 could hyperdeaminate adenosine residues in both measles virus and influenza virus A genomes. Detailed analysis of the dinucleotide editing context showed preferences for 5'ArA and 5'UrA, which is typical of editing in mammalian cells. The hyperedited mutant frequency, including genomes and antigenomes, was a log greater for influenza virus compared to measles virus, suggesting a greater sensitivity to restriction by ADAR-1.

Respiratory syncytial virus (RSV) is the most common cause of respiratory infection in infants, and no vaccine is available. In this report, recombinant AIK-C measles vaccines, expressing the RSV G or F protein of subgroup A (MVAIK/RSV/G or F), were investigated as a RSV vaccine candidate. MVAIK/RSV/G or F had the original ts phenotype and expressed RSV/G or F protein. Cross-reactive neutralizing antibodies against RSV subgroups A and B were detected in cotton rats immunized intramuscularly with MVAIK/RSV/F but not MVAIK/RSV/G. In cotton rats infected with RSV, RSV was recovered and lung histopathological finding was compatible with interstitial pneumonia, demonstrating thickening of alveolar walls and infiltration of mononuclear cells. When cotton rats immunized with MVAIK/RSV/F were challenged with homologous RSV subgroup A, no infectious RSV was recovered and very mild inflammation was noted without RSV antigen expression. When they were challenged with subgroup B, protective efficacy decreased. When cotton rats immunized with MVAIK/RSV/G were challenged with RSV subgroup A, low levels of infectious virus were recovered from lung. When challenged with subgroup B, no protective effects was demonstrated, demonstrating large amounts of RSV antigen in bronchial-epithelial cells. MVAIK/RSV/F is promising candidate and protective effects should be confirmed in monkey model., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Measles remains a leading cause of death worldwide among children because it suppresses immune function. The measles virus (MV) P gene encodes three proteins (P, V, and C) that interfere with innate immunity, controlling STAT1, STAT2, mda5, and perhaps other key regulators of immune function. We identified here three residues in the shared domain of the P and V proteins-tyrosine 110, valine 112, and histidine 115-that function to retain STAT1 in the cytoplasm and inhibit interferon transcription. This information was used to generate a recombinant measles virus unable to antagonize STAT1 function (STAT1-blind MV) differing only in these three residues from a wild-type strain of well-defined virulence. This virus was used to assess the relevance of P and V interactions with STAT1 for virulence in primates. When a group of six rhesus monkeys (Macaca mulatta) was inoculated intranasally with STAT1-blind MV, viremia was short-lived, and the skin rash and other clinical signs observed with wild-type MV were absent. The STAT1-blind virus less efficiently controlled the inflammatory response, as measured by enhanced transcription of interleukin-6 and tumor necrosis factor alpha in peripheral blood mononuclear cells from infected hosts. Importantly, neutralizing antibody titers and MV-specific T-cell responses were equivalent in hosts infected with either virus. These findings indicate that efficient MV interactions with STAT1 are required to sustain virulence in a natural host by controlling the inflammatory response against the virus. They also suggest that selectively STAT1-blind MV may have utility as vectors for targeted oncolysis and vaccination.

Several problems associated with the available anti-measles vaccine emphasize the need for a single shot anti-measles vaccine which is efficacious by mucosal route of administration and functional in the presence of anti-measles neutralizing antibodies. To achieve these goals, we constructed two recombinant human adenoviruses (collectively designated Ad-F/H) carrying genes for measles virus (MV) fusion (F) and haemagglutinin (H) proteins. Single intranasal or intramuscular vaccination of mice and cotton rats with Ad-F/H elicited high MV-specific serum neutralizing-antibody titers. Furthermore, bronchoalveolar lavage samples from mice vaccinated intranasally with Ad-F/H showed a 100-fold increase in MV-specific IgA titers compared with intramuscularly vaccinated mice. Moreover, Ad-F/H vaccine administered intranasally, but not intramuscularly, completely protected challenged cotton rats from MV replication in the lungs., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Breast cancer is the second leading cause of malignant effusions in cancer patients. Pleural effusion indicates incurable disease with limited palliative treatment options and poor outcome. Here, we demonstrate the therapeutic efficacy of measles virus (MV) vaccine strain derivative against malignant pleural effusion in an MDA-MB-231 xenograft model of advanced breast cancer. Both systemic intravenous (i.v.) and intrapleural (t.t.) administered virus caused massive infection and syncytia formation in the pleural tumor deposits. Intrapleural administration of 1.5 x 10(6) plaque-forming units (PFU) total dose of MV significantly improved median survival by approximately 80% compared to the control animal group. Furthermore, we tested human dendritic cells as carriers for delivery of oncolytic MV infection to breast cancer pleural metastases. Carrier-delivered MV infection prevented accumulation of the pleural exudate and also significantly improved the survival of the treated mice. This is the first demonstration of the therapeutic potential of oncolytic virotherapy against malignant pleural effusions in a pre-clinical model of advanced breast cancer.

Measles remains a major cause of child mortality, in part due to an inability to vaccinate young infants with the current live attenuated virus vaccine (LAV). To explore new approaches to infant vaccination, chimeric Venezuelan equine encephalitis/Sindbis virus (VEE/SIN) replicon particles were used to express the hemagglutinin (H) and fusion (F) proteins of measles virus (MV). Juvenile rhesus macaques vaccinated intradermally with a single dose of VEE/SIN expressing H or H and F proteins (VEE/SIN-H or VEE/SIN-H+F, respectively) developed high titers of MV-specific neutralizing antibody and gamma-interferon (IFN-gamma)-producing T cells. Infant macaques vaccinated with two doses of VEE/SIN-H+F also developed neutralizing antibody and IFN-gamma-producing T cells. Control animals were vaccinated with LAV or with a formalin-inactivated measles vaccine (FIMV). Neutralizing antibody remained above the protective level for more than 1 year after vaccination with VEE/SIN-H, VEE/SIN-H+F, or LAV. When challenged with wild-type MV 12 to 17 months after vaccination, all vaccinated juvenile and infant monkeys vaccinated with VEE/SIN-H, VEE/SIN-H+F, and LAV were protected from rash and viremia, while FIMV-vaccinated monkeys were not. Antibody was boosted by challenge in all groups. T-cell responses to challenge were biphasic, with peaks at 7 to 25 days and at 90 to 110 days in all groups, except for the LAV group. Recrudescent T-cell activity coincided with the presence of MV RNA in peripheral blood mononuclear cells. We conclude that VEE/SIN expressing H or H and F induces durable immune responses that protect from measles and offers a promising new approach for measles vaccination. The viral and immunological factors associated with long-term control of MV replication require further investigation.

Measles virus Edmonston strain (MV-Edm) is thought to have remarkable oncolytic activity that selectively destroys human tumor cells. The P/V/C protein of wild-type MV was shown to resist the antiviral effects of interferon (IFN)-alpha. Here, we engineered new MVs by arming MV-Edm tag strain (a V-defective vaccine-lineage strain, MV-Etag) with the P or N, P, and L genes of wild-type MV (MV-P and MV-NPL, respectively). The oncolytic activities of the MVs were determined in human renal cell carcinoma (RCC) cell lines and primary human RCC cells by the MTT assay. The antitumor efficacy of the MVs was evaluated in A-498 xenografts in nude mice. IFN-alpha effectively inhibited the replication of MV-Etag and MV-P, but not MV-NPL. MV-NPL more efficiently induced cytopathic effects (CPEs) in OS-RC-2 cells, even in the presence of human IFN-alpha. MV-NPL replicated more rapidly than MV-P and MV-Etag in A-498 cells. Apoptosis was induced earlier in A-498 cells by MV-NPL than MV-Etag and MV-P. MV-NPL showed more significant antitumoral effects and had prolonged replication compared to MV-Etag and MV-P. In this study, we demonstrated that the newly engineered MV-NPL has more effective oncolytic activity and may help establish an innovative cancer therapy.

Prostate cancer cells overexpress the measles virus (MV) receptor CD46. Herein, we evaluated the antitumor activity of an oncolytic derivative of the MV Edmonston (MV-Edm) vaccine strain engineered to express the human sodium iodide symporter (NIS; MV-NIS virus). MV-NIS showed significant cytopathic effect (CPE) against prostate cancer cell lines in vitro. Infected cells effectively concentrated radioiodide isotopes as measured in vitro by Iodide-125 ((125)I) uptake assays. Virus localization and spread in vivo could be effectively followed by imaging of (123)I uptake. In vivo administration of MV-NIS either locally or systemically (total dose of 9 x 10(6) TCID(50)) resulted in significant tumor regression (P < 0.05) and prolongation of survival (P < 0.01). Administration of (131)I further enhanced the antitumor effect of MV-NIS virotherapy (P < 0.05). In conclusion, MV-NIS is an oncolytic vector with significant antitumor activity against prostate cancer, which can be further enhanced by (131)I administration. The NIS transgene allows viral localization and monitoring by noninvasive imaging which can facilitate dose optimization in a clinical setting.

Replication-competent viruses are being tested as tumor therapy agents. The fundamental premise of this therapy is the selective infection of the tumor cell population with the amplification of the virus. Spread of the virus in the tumor ultimately should lead to eradication of the cancer. Tumor virotherapy is unlike any other form of cancer therapy as the outcome depends on the dynamics that emerge from the interaction between the virus and tumor cell populations both of which change in time. We explore these interactions using a model that captures the salient biological features of this system in combination with in vivo data. Our results show that various therapeutic outcomes are possible ranging from tumor eradication to oscillatory behavior. Data from in vivo studies support these conclusions and validate our modeling approach. Such realistic models can be used to understand experimental observations, explore alternative therapeutic scenarios and develop techniques to optimize therapy.

Attenuated live vaccines of measles virus (MV) have been developed from clinical isolates by serial propagation in heterologous cells, mainly chicken embryonic cells. The safety and effectiveness of these vaccines have been well established. However, the molecular mechanism of their attenuation remains a subject of investigation. The CAM-70 MV vaccine strain was developed from the Tanabe strain by serial propagation in chicken embryonic cells. In the present study, we assessed the contribution of each gene in the CAM-70 strain to efficient growth in chicken embryonic fibroblasts (CEF). We used a cloned MV IC323 based on the wild-type IC-B strain and generated a series of IC323s that possess one or more of the CAM-70 genes. Then, we examined the infection of CEF and CEF expressing human signaling lymphocyte activation molecule with the recombinant MVs. Our results demonstrated that MV needs to adapt to CEF at both the entry and postentry steps and that the CAM-70 matrix protein gene plays an important role in adaptation to CEF at the early stage of the virus replication cycle. The CAM-70 large protein gene was responsible for the efficient transcription and replication in CEF, and the CAM-70 hemagglutinin and fusion protein genes were responsible for efficient entry. Investigations focusing on these genes might elucidate unknown molecular mechanisms underlying the attenuation of MV.

The P, V, and C proteins of measles virus are encoded in overlapping reading frames of the P gene, which makes it difficult to analyze the functions of the individual proteins in the context of virus infection. We established a system to analyze the C protein independently from the P and V proteins by placing its gene in an additional transcription unit between the H and L genes. Analyses with this system indicated that a highly attenuated Edmonston lineage vaccine strain encodes a fully functional C protein, and the P and/or V protein is involved in the attenuated phenotype.

Objective: To establish a simple and quick method for identifying China vaccine strains and wild strains of Measles Virus., Methods: To search the enzyme site in Hemagglutinin gene of measles virus for different domestic vaccine strains and wild strains of measles virus, and design the RT-PCR primer within the range covering the enzyme site, and then to confirm the specificity and sensibility of the RT-PCR method, and then identify the RT-PCR product by RFLP., Results: The one-step RT-PCR method is sensitive, the measles virus of 4.64 TCID50 can be detected at least. No positive bands can be found in the non-measles virus strains, it means that the RT-PCR method has good specificity, the PCR products of Chian measles vaccine strains of Shang-191 and Chang-47 were all cut into two fragments (287 bp and 151 bp) by Afi II, but two measles wild virus strains can't be cut by Afl II., Conclusion: The RT-PCR-RFLP method which we established is a rapid and simple method for identifying China vaccine strain and wild strain.