Broadly Reactive Nanobody Targeting the H3 Hemagglutinin of the Influenza A Virus.

Monoclonal antibodies and recombinant antibody fragments are a very promising therapeutic tool to combat infectious diseases. Due to their unique paratope structure, nanobodies (VHHs) hold several advantages over conventional monoclonal antibodies, especially in relation to viral infections. Influenza A viruses (IAVs) remain a major threat to public health. The hemagglutinin (HA) protein is the main protective and immunodominant antigen of IAVs. In this study, three broadly reactive nanobodies (D9.2, E12.2, and D4.2) to H3N2 influenza strains were isolated and Fc-fusion proteins (VHH-Fcs) were obtained and characterized in vitro. This modification improved the nanobodies' binding activity and allowed for their interaction with a wider range of strains. The D9.2-Fc antibody showed a 100% protection rate against mortality in vivo in a mouse lethal model. Furthermore, we demonstrated that the observed protection has to do with Fc-FcγR interactions. These results indicate that D9.2-Fc can serve as an effective antiviral agent against the H3N2 influenza infection.

[Review of candidate vaccines for the prevention of Lassa fever].

The Lassa virus one of the main etiological agent of hemorrhagic fevers in the world: according to WHO estimates, it affects 100,000 to 300,000 people annually, which results in up to 10,000 deaths [1]. Although expansion of Lassa fever caused by this pathogen is mostly limited to the West African countries: Sierra Leone, Liberia, Guinea and Nigeria, imported cases have been historically documented in Europe, the United States of America (USA), Canada, Japan, and Israel [2]. In 2017, WHO included the Lassa virus in the list of priority pathogens in need of accelerated research, development of vaccines, therapeutic agents and diagnostic tools regarding infections they cause [3]. This review describes main technological platforms used for the development of vaccines for the prevention of Lassa fever.

Development of Neutralizing Nanobodies to the Hemagglutinin Stem Domain of Influenza A Viruses.

The influenza virus infection claims ~650,000 lives annually. Taking into account the evolving resistance of the pathogen to antiviral drugs and the waning effectiveness of vaccination among certain populations, new approaches to the treatment of influenza are needed. The current study is aimed at obtaining single-domain antibodies (Nanobodies®) to the highly conserved stem domain of influenza A virus hemagglutinin by phage display. Two high-affinity neutralizing clones of Nanobodies® with a particular specificity were selected; they ensured 100% neutralization of the H1N1 and H5N2 influenza viruses in vivo. The obtained data demonstrate that it is possible to develop highly effective VHH-based drugs for the treatment of influenza.

Nanobodies Are Potential Therapeutic Agents for the Ebola Virus Infection.

Ebola fever is an acute, highly contagious viral disease with a mortality rate that can reach 90%. There are currently no licensed therapeutic agents specific to Ebola in the world. Monoclonal antibodies (MAbs) with viral-neutralizing activity and high specificity to the Ebola virus glycoprotein (EBOV GP) are considered as highly effective potential antiviral drugs. Over the past decade, nanobodies (single-domain antibodies, non-canonical camelid antibodies) have found wide use in the diagnosis and treatment of various infectious and non-infectious diseases. In this study, a panel of nanobodies specifically binding to EBOV GP was obtained using recombinant human adenovirus 5, expressing GP (Ad5-GP) for alpaca (Vicugna pacos) immunization, for the first time. Based on specific activity assay results, affinity constants, and the virus-neutralizing activity against the recombinant vesicular stomatitis virus pseudotyped with EBOV GP (rVSV-GP), the most promising clone (aEv6) was selected. The aEv6 clone was then modified with the human IgG1 Fc fragment to improve its pharmacokinetic and immunologic properties. To assess the protective activity of the chimeric molecule aEv6-Fc, a lethal model of murine rVSV-GP infection was developed by using immunosuppression. The results obtained in lethal model mice have demonstrated the protective effect of aEv6-Fc. Thus, the nanobody and its modified derivative obtained in this study have shown potential protective value against Ebola virus.

Preclinical Studies of Immunogenity, Protectivity, and Safety of the Combined Vector Vaccine for Prevention of the Middle East Respiratory Syndrome.

The Middle East Respiratory Syndrome (MERS) is an acute inflammatory disease of the respiratory system caused by the MERS-CoV coronavirus. The mortality rate for MERS is about 34.5%. Due to its high mortality rate, the lack of therapeutic and prophylactic agents, and the continuing threat of the spread of MERS beyond its current confines, developing a vaccine is a pressing task, because vaccination would help limit the spread of MERS and reduce its death toll. We have developed a combined vector vaccine for the prevention of MERS based on recombinant human adenovirus serotypes 26 and 5. Studies of its immunogenicity have shown that vaccination of animals (mice and primates) induces a robust humoral immune response that lasts for at least six months. Studies of the cellular immune response in mice after vaccination showed the emergence of a specific CD4+ and CD8+ T cell response. A study of the vaccine protectivity conducted in a model of transgenic mice carrying the human DPP4 receptor gene showed that our vaccination protected 100% of the animals from the lethal infection caused by the MERS-CoV virus (MERS-CoV EMC/2012, 100LD50 per mouse). Studies of the safety and tolerability of the developed vaccine in rodents, rabbits, and primates showed a good safety profile and tolerance in animals; they revealed no contraindications for clinical testing.

Immunogenicity of Different Forms of Middle East Respiratory Syndrome S Glycoprotein.

The Middle East respiratory syndrome coronavirus (MERS-CoV) was identified in 2012 during the first Middle East respiratory syndrome (MERS) outbreaks. MERS-CoV causes an acute lower-respiratory infection in humans, with a fatality rate of ~35.5%. Currently, there are no registered vaccines or means of therapeutic protection against MERS in the world. The MERS-CoV S glycoprotein plays the most important role in the viral life cycle (virus internalization). The S protein is an immunodominant antigen and the main target for neutralizing antibodies. In the present study, the immunogenicities of five different forms of the MERS-CoV S glycoprotein were compared: the full-length S glycoprotein, the full-length S glycoprotein with the transmembrane domain of the G glycoprotein of VSV (S-G), the receptor-binding domain (RBD) of the S glycoprotein, the membrane-fused RBD (the RBD fused with the transmembrane domain of the VSV G glycoprotein (RBD-G)), and the RBD fused with Fc of human IgG1 (RBD-Fc). Recombinant vectors based on human adenoviruses type 5 (rAd5) were used as delivery vehicles. Vaccination with all of the developed rAd5 vectors elicited a balanced Th1/Th2 response in mice. The most robust humoral immune response was induced after the animal had been vaccinated with the membrane-fused RBD (rAd5-RBD-G). Only immunization with membrane forms of the glycoprotein (rAd5-S, rAd5-S-G, and rAd5-RBD-G) elicited neutralizing antibodies among all vaccinated animals. The most significant cellular immune response was induced after vaccination of the animals with the full-length S (rAd5-S). These investigations suggest that the full-length S and the membrane form of the RBD (RBD-G) are the most promising vaccine candidates among all the studied forms of S glycoprotein.

[The use of monoclonal antibodies for the treatment of Ebola virus disease.]

Some drugs candidates for treatment of Ebola virus disease (EVD), have been studied, monoclonal antibody (mAb) cocktails have shown great potential as EVD therapeutics. The advantages of mAb therapy include low toxicity, high specifcity and versatility, with the range of biological effects being dependent upon the Fc region. Functions of mAbs include pathogen opsonisation, complement activation, antibody-dependent cell cytotoxicity and virus neutralization characteristics. The most known mAb cocktail, used as therapeutic, is ZMapр, manufactured by «Leaf Biopharmaceutical¼ from 2004. The elaborated mAb cocktails, structures and properties s of mAbs, the protective characteristics of mAbs and development of new pan-ebolavirus mAbs are reviewed in this article.

Virus-Vectored Ebola Vaccines.

The Ebola virus disease (EVD) is one of the most dangerous infections affecting humans and animals. The first EVD outbreaks occurred in 1976 in Sudan and Zaire. Since then, more than 20 outbreaks have occurred; the largest of which (2014-2016) evolved into an epidemic in West Africa and claimed the lives of more than 11,000 people. Although vaccination is the most effective way to prevent epidemics, there was no licensed vaccine for EVD at the beginning of the latest outbreak. The development of the first vaccines for EVD started in 1980 and has come a long technological way, from inactivated to genetically engineered vaccines based on recombinant viral vectors. This review focuses on virus-vectored Ebola vaccines that have demonstrated the greatest efficacy in preclinical trials and are currently under different phases of clinical trial. Particular attention is paid to the mechanisms of immune response development, which are important for protection from EVD, and the key vaccine parameters necessary for inducing long-term protective immunity against EVD.

Safety and immunogenicity of GamEvac-Combi, a heterologous VSV- and Ad5-vectored Ebola vaccine: An open phase I/II trial in healthy adults in Russia.

Ebola hemorrhagic fever, also known as Ebola virus disease or EVD, is one of the most dangerous viral diseases in humans and animals. In this open-label, dose-escalation clinical trial, we assessed the safety, side effects, and immunogenicity of a novel, heterologous prime-boost vaccine against Ebola, which was administered in 2 doses to 84 healthy adults of both sexes between 18 and 55 years. The vaccine consists of live-attenuated recombinant vesicular stomatitis virus (VSV) and adenovirus serotype-5 (Ad5) expressing Ebola envelope glycoprotein. The most common adverse event was pain at the injection site, although no serious adverse events were reported. The vaccine did not significantly impact blood, urine, and immune indices. Seroconversion rate was 100 %. Antigen-specific IgG geometric mean titer at day 42 was 3,277 (95 % confidence interval 2,401-4,473) in volunteers immunized at full dose. Neutralizing antibodies were detected in 93.1 % of volunteers immunized at full dose, with geometric mean titer 20. Antigen-specific response in peripheral blood mononuclear cells was also detected in 100 % of participants, as well as in CD4+ and CD8+ T cells in 82.8 % and 58.6 % of participants vaccinated at full dose, respectively. The data indicate that the vaccine is safe and induces strong humoral and cellular immune response in up to 100 % of healthy adult volunteers, and provide a rationale for testing efficacy in Phase III trials. Indeed, the strong immune response to the vaccine may elicit long-term protection. This trial was registered with grls.rosminzdrav.ru (No. 495*), and with zakupki.gov.ru (No. 0373100043215000055).

Triggering of Toll-like Receptor-2 in Mouse Myelomonocytic Leukaemia Cells WEHI-3B Leads to the Suppression of Apoptosis and Promotes Tumor Progression in Vivo.

Toll-like receptors are the essential components of innate immunity. It is shown that TLRs play an essential role in the immune resistance of an organism to bacterial and viral infections. The binding of TLR to its own ligands results in the activation of several adapter molecules and kinases, inducing the activation of the main pro-inflammatory transcriptional factors, which in turn induce the activation of the main pro-inflammatory transcriptional factors. This activation results in the development of both the innate immune response triggered by the enhanced expression of a number of pro-inflammatory cytokines and antimicrobial peptides and that of the adaptive immune response, via the activation of dendritic cells and enhancement of antigen presentation, etc. The ability of TLR agonists to bolster the immune reaction makes them promising for use in the therapy of infectious diseases and in the chemotherapy of malignant neoformations. However, different TLR ligands may have either antitumor activity (lipopolysaccharide, imiquimod, CpG) or, conversely, could beef up the resistance of tumor cells to apoptosis, stimulating their proliferation under certain conditions (lipopolysaccharide, lipopeptide). It has been shown that the TLR2-dependent signalling pathway in the myelomonocytic mouse leukaemia cell line WEHI-3B leads to the constitutive activation of the transcriptional factor NF-kB, suppression of apoptosis in tumor cells, and progression of myelomonocytic mouse leukaemiain vivo, upon the addition of TLR2 agonist (synthetic lipopeptide Pam2CSK4) or following the infection of tumor cells withMycoplasma arginini.

Toll-Like Receptors (TLRs): The Role in Tumor Progression.

Toll-like receptors (TLRs) are major components of the innate immune system that recognize the conserved molecular structures of pathogens (pathogen-associated molecular patterns; PAMPs). TLRs are found in many different cell types, ranging from epithelial to immunocompetent cells. TLR binding triggers the expression of several adapter proteins and downstream kinases, leading to the induction of key pro-inflammatory mediators. This results in the activation of both the innate immune response (elevated expression of antiapoptotic proteins, proinflammatory cytokines, and antibacterial proteins), as well as the adaptive immune response (maturation of the dendritic cells, antigen presentation, etc.). In consequence of their ability to enhance the specific and nonspecific immune reactions of an organism, TLR agonists are widely used in the therapy of infectious diseases and, as adjuvants, in the therapy of malignant neoplasia. However, to date, TLRs have had the opposite effects on tumor progression. On the one hand, TLR ligands can suppress tumor growth. On the other hand, TLR agonists can promote the survival of malignant cells and increase their resistance to chemotherapy. The purpose of this review is to summarize the available data on the effects of TLRs and their agonists on tumor progression, as well as the mechanisms underlying the differences in the effects of TLRs on tumor growth.