Safety concern of recombination between self-amplifying mRNA vaccines and viruses is mitigated in vivo.

Self-amplifying mRNA (SAM) vaccines can be rapidly deployed in the event of disease outbreaks. A legitimate safety concern is the potential for recombination between alphavirus-based SAM vaccines and circulating viruses. This theoretical risk needs to be assessed in the regulatory process for SAM vaccine approval. Herein, we undertake extensive in vitro and in vivo assessments to explore recombination between SAM vaccine and a wide selection of alphaviruses and a coronavirus. SAM vaccines were found to effectively limit alphavirus co-infection through superinfection exclusion, although some co-replication was still possible. Using sensitive cell-based assays, replication-competent alphavirus chimeras were generated in vitro as a result of rare, but reproducible, RNA recombination events. The chimeras displayed no increased fitness in cell culture. Viable alphavirus chimeras were not detected in vivo in C57BL/6J, Rag1-/- and Ifnar-/- mice, in which high levels of SAM vaccine and alphavirus co-replicated in the same tissue. Furthermore, recombination between a SAM-spike vaccine and a swine coronavirus was not observed. In conclusion we state that although the ability of SAM vaccines to recombine with alphaviruses might be viewed as an environmental safety concern, several key factors substantially mitigate against in vivo emergence of chimeric viruses from SAM vaccine recipients.

Development and application of a colloidal-gold immunochromatographic strip for detecting Getah virus antibodies.

Getah virus (GETV) is a re-emerging mosquito-borne alphavirus that is highly pathogenic, mainly to pigs and horses. There are no vaccines or treatments available for GETV in swine in China. Therefore, the development of a simple, rapid, specific, and sensitive serological assay for GETV antibodies is essential for the prevention and control of GETV. Current antibody monitoring methods are time-consuming, expensive, and dependent on specialized instrumentation, and these features are not conducive to rapid detection in clinical samples. To address these problem, we developed immunochromatographic test strips (ICTS) using eukaryotically expressed soluble recombinant p62-E1 protein of GETV as a labelled antigen, which has good detection sensitivity and no cross-reactivity with other common porcine virus-positive sera. The ICTS is highly compatible with IFA and ELISA and can be stored for 1 month at 37 °C and for at least 3 months at room temperature. Hence, p62-E1-based ICTS is a rapid, accurate, and convenient method for rapid on-site detection of GETV antibodies. KEY POINTS: • We established a rapid antibody detection method that can monitor GETV infection • We developed colloidal gold test strips with high sensitivity and specificity • The development of colloidal gold test strips will aid in the field serologic detection of GETV.

Trans-Amplifying RNA: A Journey from Alphavirus Research to Future Vaccines.

Replicating RNA, including self-amplifying RNA (saRNA) and trans-amplifying RNA (taRNA), holds great potential for advancing the next generation of RNA-based vaccines. Unlike in vitro transcribed mRNA found in most current RNA vaccines, saRNA or taRNA can be massively replicated within cells in the presence of RNA-amplifying enzymes known as replicases. We recently demonstrated that this property could enhance immune responses with minimal injected RNA amounts. In saRNA-based vaccines, replicase and antigens are encoded on the same mRNA molecule, resulting in very long RNA sequences, which poses significant challenges in production, delivery, and stability. In taRNA-based vaccines, these challenges can be overcome by splitting the replication system into two parts: one that encodes replicase and the other that encodes a short antigen-encoding RNA called transreplicon. Here, we review the identification and use of transreplicon RNA in alphavirus research, with a focus on the development of novel taRNA technology as a state-of-the art vaccine platform. Additionally, we discuss remaining challenges essential to the clinical application and highlight the potential benefits related to the unique properties of this future vaccine platform.

Virus-specific antibody secreting cells reside in the peritoneal cavity and systemic immune sites of Atlantic salmon (Salmo salar) challenged intraperitoneally with salmonid alphavirus.

The development and persistence of antibody secreting cells (ASC) after antigenic challenge remain inadequately understood in teleosts. In this study, intraperitoneal (ip) injection of Atlantic salmon (Salmo salar) with salmonid alphavirus (WtSAV3) increased the total ASC response, peaking 3-6 weeks post injection (wpi) locally in the peritoneal cavity (PerC) and in systemic lymphoid tissues, while at 13 wpi the response was only elevated in PerC. At the same time point a specific ASC response was induced by WtSAV3 in PerC and systemic tissues, with the highest frequency in PerC, suggesting a local role. Inactivated SAV (InSAV1) induced comparatively lower ASC responses in all sites, and specific serum antibodies were only induced by WtSAV3 and not by InSAV1. An InSAV1 boost did not increase these responses. Expression of immune marker genes implies a role for PerC adipose tissue in the PerC immune response. Overall, the study suggests the Atlantic salmon PerC as a secondary immune site and an ASC survival niche.

Displaying alphavirus physicochemical consensus antigens on immunogenic liposomes enhances antibody elicitation in mice.

Cobalt-porphyrin phospholipid displays recombinant protein antigens on liposome surfaces via antigen polyhistidine-tag (His-tag), and when combined with monophosphorylated lipid A and QS-21 yields the "CPQ" vaccine adjuvant system. In this proof of principle study, CPQ was used to generate vaccine prototypes that elicited antibodies for two different alphaviruses (AV). Mice were immunized with computationally designed, His-tagged, physicochemical property consensus (PCPcon) protein antigens representing the variable B-domain of the envelope protein 2 (E2) from the serotype specific Venezuelan Equine Encephalitis Virus (VEEVcon) or a broad-spectrum AV-antigen termed EVCcon. The CPQ adjuvant enhanced the antigenicity of both proteins without eliciting detectable anti-His-tag antibodies. Antibodies elicited from mice immunized with antigens admixed with CPQ showed orders-of-magnitude higher levels of antigen-specific IgG compared to alternative control adjuvants. The ELISA results correlated with antiviral activity against VEEV strain TC83 and more weakly to Chikungunya virus 118/25. Thus, display of E.coli-produced His-tagged E2 protein segments on the surface of immunogenic liposomes elicits high levels of antigen-specific and AV neutralizing antibodies in mice with vaccination, while facilitating vaccine preparation and providing dose-sparing potential.

MAVS disruption impairs downstream signaling and results in higher virus replication levels of salmonid alphavirus subtype 3 but not infectious pancreatic necrosis virus in vitro.

The mitochondrial anti-viral signaling (MAVS) protein is an intermediary adaptor protein of retinoic acid-inducible gene-1 (RIG-I) like receptor (RLR) signaling, which activates the transcription factor interferon (IFN) regulatory factor 3 (IRF3) and NF-kB to produce type I IFNs. MAVS expression has been reported in different fish species, but few studies have shown its functional role in anti-viral responses to fish viruses. In this study, we used the transcription activator-like effector nuclease (TALEN) as a gene editing tool to disrupt the function of MAVS in Chinook salmon (Oncorhynchus tshawytscha) embryonic cells (CHSE) to understand its role in induction of interferon I responses to infections with the (+) RNA virus salmonid alphavirus subtype 3 (SAV-3), and the dsRNA virus infectious pancreatic necrosis virus (IPNV) infection. A MAVS-disrupted CHSE clone with a 7-aa polypeptide (GVFVSRV) deletion mutation at the N-terminal of the CARD domain infected with SAV-3 resulted in significantly lower expression of IRF3, IFNa, and ISGs and increased viral titer (1.5 log10) compared to wild-type. In contrast, the IPNV titer in MAVS-disrupted cells was not different from the wild-type. Furthermore, overexpression of salmon MAVS in MAVS-disrupted CHSE cells rescued the impaired type I IFN-mediated anti-viral effect against SAV-3.

Mayaro virus: imported cases of human infection in São Paulo State, Brazil

Mayaro virus (MAYV) is an arbovirus (Togaviridae: Alphavirus) enzootic in tropical South America and maintained in a sylvan cycle involving wild vertebrates and Haemagogus mosquitoes. MAYV cases occur sporadically in persons with a history of recent activities inside or around forests. This paper reports three cases of MAYV fever detected in men infected in Camapuã, MS, Brazil. Serum samples collected at four days and two months after the onset of the symptoms and examined by hemagglutination inhibition test, revealed monotypic seroconversion to MAYV. Isolation of the virus was obtained from one of the samples by inoculation of the first blood samples into newborn mice. A suspension of the infected mouse brain was inoculated into C6/36 cells culture and the virus was identified by indirect immunofluorescent assay with alphavirus polyclonal antibodies. RT-PCR, performed with RNA extracted from the supernatant of C6/36 infected cells in the presence of alphavirus generic primers as well as specific MAYV primers, confirmed these results. The reported cases illustrate the importance of laboratory confirmation in establishing a correct diagnosis. Clinical symptoms are not always indicative of a disease caused by an arbovirus. Also MAYV causes febrile illness, which may be mistaken for dengue.

O vírus Mayaro (MAYV) é um arbovírus do gênero Alphavirus, família Togaviridae, enzoótico na América do Sul, sendo mantido em ciclo silvestre envolvendo vertebrados e mosquitos Haemagogus. Casos de MAYV são esporádicos e ocorrem em pessoas com história de recentes atividades dentro ou próximo a florestas. Este artigo relata infecção por MAYV detectada em três pacientes, infectados em Camapuã, MS, Brasil. Amostras de sangue, coletadas no 4° dia e no 2° mês após o início dos sintomas, foram usadas para teste de inibição da hemaglutinação, que revelou soroconversão monotípica para MAYV. O isolamento do vírus foi obtido somente de uma das amostras, por inoculação em camundongos lactentes. Suspensão de cérebro de camundongo infectado foi inoculada em cultura de células C6/36 e o vírus foi identificado por imunofluorescência indireta com anticorpos policlonais para alphavirus. RT-PCR realizado com RNA extraído do sobrenadante de células C6/36 infectadas, na presença de "primers" genéricos para alphavirus assim como "primers" para MAYV, confirmou os resultados. Os casos relatados ilustram a importância da confirmação laboratorial em estabelecer um diagnóstico correto. Os sintomas clínicos não são sempre indicativos de uma doença causada por arbovírus. MAYV causa doença febril, que pode ser confundida com dengue.

Recombinant viruses as vaccines against viral diseases

Vaccine approaches to infectious diseases are widely applied and appreciated. Amongst them, vectors based on recombinant viruses have shown great promise and play an important role in the development of new vaccines. Many viruses have been investigated for their ability to express proteins from foreign pathogens and induce specific immunological responses against these antigens in vivo. Generally, gene-based vaccines can stimulate potent humoral and cellular immune responses and viral vectors might be an effective strategy for both the delivery of antigen-encoding genes and the facilitation and enhancement of antigen presentation. In order to be utilized as a vaccine carrier, the ideal viral vector should be safe and enable efficient presentation of required pathogen-specific antigens to the immune system. It should also exhibit low intrinsic immunogenicity to allow for its re-administration in order to boost relevant specific immune responses. Furthermore, the vector system must meet criteria that enable its production on a large-scale basis. Several viral vaccine vectors have thus emerged to date, all of them having relative advantages and limits depending on the proposed application, and thus far none of them have proven to be ideal vaccine carriers. In this review we describe the potential, as well as some of the foreseeable obstacles associated with viral vaccine vectors and their use in preventive medicine.

Study of two different enzyme immunoassays for the detection of Mayaro virus antibodies

This paper presents the evaluation of an enzyme immunoassay in which Mayaro virus-infected cultured cells ara used as antigen (EIA-ICC) and an IgM antibody capture ELISA (MAC-ELISA) for Mayaro serologic diagnosis using 114 human sera obtained during a Mayaro outbreak occurred in Bolivia, in 1987. Results were compared with those obtained by haemagglutination-inhibition test (HAI). MAC-ELISA was the most sensitive technique for anti-Mayaro IgM detection. MAC-ELISA was twice sensitive as IgM EIA-ICC. The data shows that MAC-ELISA is a practical and valid technique for diagnosis of recent mayaro infection. IgG-ICC showed hight sensitivity and high specificity compared to HAI. The combination of anti-Mayaro IgG and IgM EIA-ICC results presented the highest sensitivity of the study. Anti-Mayaro IgG and IgM simultaneous detection by ELISA-ICC can be used for recent infection diagnosis (in spite of a less sensitive IgM detection than by MAC-ELISA), for surveillance and sero-epidemiologic studies, and for studies of IgG and IgM responses to Mayaro infection

Estudos sorológicos para pesquisa de anticorpos de arbovirus em populacao humana da regiao do Vale do Ribeira. II Inquerito em pacientes do Hospital Regional de Pariquera-Acu, 1980. / Serological studies for the research of arbovirus antibodies in human population of the Ribeira Valley region. II - Survey in patients of Pariquera-Acu Regional Hospital, 1980

Foi realizado inquerito sorologico para pesquisa de anticorpos inibidores de hemaglutinacao de arbovirus em 516 moradores das zonas urbana e rural da regiao do Vale do Ribeira, Brasil, area extensamente coberta de florestas onde ocorreu recentemente uma epidemia de encefalite atribuida ao Flavivirus Rocio. Verificou-se que 24,2% destas pessoas tinham anticorpos IH para um ou mais arbovirus (11,2% para Alphavirus; 13,2% para Flavivirus 4,6% para Bunyavirus Caraparu e 0,8% para outros arbovirus). Alguns dos investigados, sem antecedente de vacinacao contra febre amarela apresentaram anticorpos neutralizantes para o virus da encefalite equina do Leste, St.Louis e da febre amarela, os dois ultimos ainda nao isolados na regiao. A analise das caracteristicas dos individuos com sorologia positiva sugeria que a transmissao de arboviroses nao era fato recente e estava se fazendo em pelo menos 9 municipios da area, nao so no ambiente silvestre como fora do mesmo. Os individuos do sexo masculino e entre estes os que trabalham em pesca, em geral no periodo vespertino e noturno, apresentaram maior risco a infeccoes arboviricas