Intranasal vaccination using influenza vectors is a promising approach to developing vaccines against respiratory pathogens due to the activation of the mucosa-associated immune response. However, there is no clear evidence of a vector design that could be considered preferable. To find the optimal structure of an influenza vector with a modified NS genomic segment, we constructed four vector expressing identical transgene sequences inherited from the F protein of the respiratory syncytial virus (RSV). Two vectors were designed aiming at transgene accumulation in the cytosol. Another two were supplemented with an IgGκ signal peptide prior to the transgene for its extracellular delivery. Surprisingly, adding the IgGκ substantially enhanced the T-cell immune response to the CD8 epitope of the transgene. Moreover, this strategy allowed us to obtain a better protection of mice from the RSV challenge after a single intranasal immunization. Protection was achieved without antibodies, mediated by a balanced T-cell immune response including the formation of the RSV specific effector CD8+ IFNγ+/IL10+-producing cells and the accumulation of Treg cells preventing immunopathology in the lungs of infected mice. In addition to the presented method for optimizing the influenza vector, our results highlight the possibility of achieving protection against RSV through a respiratory-associated T-cell immune response alone.

MxA is a cytoplasmic protein induced in human cells exposed to type I and III interferons. It can inhibit various viruses, including influenza A, by blocking the early steps of the viral replication cycle. The rapid advancement of mRNA-based technology has allowed us to evaluate the antiviral activity of MxA-mRNA, encoding intracellular MxA protein, and explore its potential as a therapeutic agent. In this study, we used in vitro transfection methods to obtain functional, mature MxA-mRNA and evaluate its activity within cells. We also observed an undesirable cellular response to transfection with exogenous mRNAs, which involved interferon III induction and reduced cell viability. Nevertheless, preventive administration of MxA-mRNA led to a specific 10–80-fold decrease in influenza A and B levels in cell supernatants—an effect not observed with the control GFP-Luc-mRNA. Additionally, we investigated the antiviral activity of MxA-mRNA against RNA viruses, such as SARS-CoV-2 and both serotypes of RSV, but we could not demonstrate a significant virus-specific effect of exogenous mRNA on their replication. We believe that mRNAs that encode native antiviral proteins have great therapeutic potential.

We constructed a reporter influenza A/Puerto Rico/8/1934 virus expressing truncated 124aa N-terminal NS1 protein fused to a luciferase reporter sequence (NanoLuc) without signal peptide. The reproduction activity of the vector correlated well with the luminescent activity in the lysates of infected cell cultures or mouse respiratory organ suspensions. Surprisingly, we found that luciferase enzymatic activity was present not only in the intracellular compartments but also in cell culture supernatants as well as in the sera or bronchiolar lavages of infected mice. This fact allowed us to formulate a working hypothesis about the extracellular delivery mechanism of the NS1 protein. To test this idea, we conducted co-transfection experiments in Vero cells with different combinations of plasmids encoding influenza genomic segments and chimeric NS1-NanoLuc encoding plasmid. We found that the emergence of the luciferase reporter in the extracellular compartment was promoted by the formation of the ribonucleoprotein complex (RNP) from the co-transfection of plasmids expressing PB1, PB2, PA, and NP proteins. Therefore, influenza NS1 protein may be delivered to the extracellular compartment together with the nascent RNP complexes during the maturation of virus particles.

Introduction. Respiratory syncytial virus (RSV) is the most common pathogen causing lower respiratory tract infections in children. RSV also poses a serious threat to the elderly and immunocompromised patients. Developing a therapy based on recombinant human antibodies to block the RSV fusion (F) glycoprotein is urgent to reduce the incidence of RSV infections and prevent associated complications. Aim. To design plasmid vectors for efficient production of the recombinant monoclonal antibody FM1 in a eukaryotic expression system targeting the RSV fusion (F) glycoprotein and to evaluate its activity against RSV subtypes A and B in vitro. Materials and methods. Constructs encoding the recombinant antibody FM1 were designed using genetic engineering. Recombinant antibodies were produced in the CHO-K1 cell line through transient expression. Antibody specimens were purified from the culture supernatant using affinity chromatography, with a modified protein A as the ligand. The virus-neutralizing activity of the antibody was evaluated in a microneutralization assay using several RSV strains on a Vero cell monolayer culture. Results. We developed a two-plasmid vector system to produce the recombinant FM1 antibody targeting the RSV F glycoprotein, using CHO cells as transient producers. The antibody was successfully produced, purified, and characterized, with its biological activity confirmed. The FM1 antibody demonstrated enhanced virus-neutralizing activity against reference and seasonal RSV strains of subtypes A and B compared to the control drug palivizumab. Conclusion. A recombinant FM1 antibody-based drug could address the import substitution challenge for protective measures against RSV infection. The authors are currently developing a stable FM1 producer clone with high productivity and viability and investigating the therapeutic efficacy of this antibody in a sublethal RSV infection mouse model.

Background/Objectives: Although influenza A viruses with partially truncated NS1 proteins are substantially attenuated and immunogenic due to enhanced innate immune activation; residual NS1-mediated antagonism of antiviral innate responses may support viral replication in the lower respiratory tract and constrain optimal immune responses. Strategies to further improve their immunogenicity and protective efficacy by incorporating immunomodulatory cytokines, such as IL-2, have been successfully explored. Methods: Here, we extended this approach to chemokine expression by engineering an NS1-truncated PR8-based virus (PR8/NS124) to express the immunomodulatory chemokine CXCL10 from the NS segment and compared it with the parental vector. Results: The recombinant NS124_SS_CXCL10 virus replicated to high titers in embryonated chicken eggs and MDCK cells. In vivo, however, CXCL10 expression reduced viral replication in mouse lungs by ~10⁴-fold, resulting in a near-non-replicating phenotype. In contrast to the parental virus, the vector did not induce weight loss and exhibited a strongly attenuated phenotype. This effect was associated with altered innate immune signaling, including increased IRF7 expression and early induction of IFN-α responses in the lungs, together with modulation of TLR-dependent sensing pathways in the upper respiratory tract. Despite severely impaired replication, intranasal immunization induced antigen-specific T-cell responses comparable to those elicited by the parental vector. Following intraperitoneal immunization, when replication of both vectors was minimal, the CXCL10-expressing vector induced significantly higher frequencies of antigen-specific CD8⁺ and CD4⁺ effector-memory T cells. This was accompanied by enhanced antigen-specific T-cell recall responses in the lungs following intranasal challenge. Importantly, the CXCL10-expressing vector demonstrated protective efficacy comparable to that of the parental NS124 vector against heterologous H3N2 challenge while exhibiting an improved safety profile. Conclusions: These findings support the incorporation of CXCL10 as a strategy to improve the safety and T-cell immunogenicity of NS1-truncated influenza vectors.

Хлопковые крысы широко используются в мире в качестве экспериментальной животной модели в исследованиях вакцин и химиопрепаратов против респираторно-синцитиальной вирусной (РСВ) инфекции. Востребованность данной модели обусловлена тем, что хлопковые крысы более восприимчивы к заражению РСВ, чем большинство лабораторных линий мышей, а также несут функциональный набор генов, кодирующих белки Mx1 и Mx2, которые являются важнейшими компонентами врожденной системы противовирусной защиты человека. Несмотря на небольшой размер, хлопковые крысы сложны в содержании и при проведении манипуляций, поскольку они сохранили поведенческие реакции диких животных, избегают контакта и быстро передвигаются. В этой статье мы обсуждаем препятствия на пути внедрения РСВ-модели хлопковых крыс в лабораторные исследования и представляем собственный опыт работы с этими животными. Наиболее длительным этапом был период времени, затраченный на переговоры с питомником и подготовку документов для получения животных. По прибытии хлопковых крыс (самки, возраст 6-8 нед) размещали в отдельном помещении вивария в индивидуальных клетках, снабженных стерилизованным подстилом. Животные быстро адаптировались к новым условиям и начали стабильно набирать массу тела с первых суток карантинного периода. Для моделирования РСВ-инфекции использовали две заражающие дозы вируса - 5 или 6 lg БОЕ/животное. Животным вводили вирус интраназально без наркоза или под ингаляционным наркозом (смесь изофлурана и пропиленгликоля). При этом инфекция без использования наркоза протекала бессимптомно в независимости от заражающей дозы вируса, хотя и сопровождалась продуктивной репликацией вируса главным образом в верхних дыхательных путях. Заражение животных под наркозом приводило к активной репликации вируса в легких, значительной потере массы тела и существенным повреждениям легких при использовании высокой заражающей дозы вируса. Иммунизация хлопковых крыс инактивированным формалином респираторно-синцитиальным вирусом привела к появлению классических маркеров усиления респираторной патологии в гистологических срезах легких и к снижению массы тела животных. Полученные результаты позволяют использовать отработанную модель хлопковых крыс для изучения защитных свойств и безопасности вакцин против...