HIV-1 Reverse Transcriptase Promotes Tumor Growth and Metastasis Formation via ROS-Dependent Upregulation of Twist

Funding Information: https://orcid.org/0000-0002-6160-2203 Bayurova Ekaterina [email protected] 1 2 Jansons Juris [email protected] 3 4 Skrastina Dace [email protected] 3 4 https://orcid.org/0000-0002-4980-9754 Smirnova Olga [email protected] 5 Mezale Dzeina [email protected] 3 Kostyusheva Anastasia [email protected] 6 Kostyushev Dmitry [email protected] 6 Petkov Stefan [email protected] 7 Podschwadt Philip [email protected] 7 https://orcid.org/0000-0003-0365-570X Valuev-Elliston Vladimir [email protected] 5 Sasinovich Sviataslau [email protected] 7 https://orcid.org/0000-0003-2278-4451 Korolev Sergey [email protected] 8 Warholm Per [email protected] 9 https://orcid.org/0000-0002-2260-6551 Latanova Anastasia [email protected] 1 5 https://orcid.org/0000-0003-2183-0858 Starodubova Elizaveta [email protected] 1 5 https://orcid.org/0000-0001-8506-2339 Tukhvatulin Amir [email protected] 1 Latyshev Oleg [email protected] 1 Selimov Renat [email protected] 10 Metalnikov Pavel [email protected] 10 Komarov Alexander [email protected] 10 https://orcid.org/0000-0002-3673-4714 Ivanova Olga [email protected] 5 Gorodnicheva Tatiana [email protected] 11 https://orcid.org/0000-0002-7443-6961 Kochetkov Sergey [email protected] 5 Gottikh Marina [email protected] 8 Strumfa Ilze [email protected] 3 https://orcid.org/0000-0002-5659-9679 Ivanov Alexander [email protected] 5 Gordeychuk Ilya [email protected] 1 2 12 https://orcid.org/0000-0001-9382-2254 Isaguliants Maria [email protected] 1 2 3 7 García-Rivas Gerardo 1 NF Gamaleya Research Center of Epidemiology and Microbiology Moscow Russia 2 Chumakov Federal Scientific Center for Research and Development of Immune-and-Biological Products of Russian Academy of Sciences Moscow Russia chumakovs.ru 3 Department of Pathology Riga Stradins University Riga Latvia rsu.lv 4 Latvian Biomedical Research and Study Centre Riga Latvia lu.lv 5 Engelhardt Institute of Molecular Biology Russian Academy of Sciences Moscow Russia ras.ru 6 National Medical Research Center for Tuberculosis and Infectious Diseases Moscow Russia 7 Department of Microbiology Tumor and Cell Biology Karolinska Institutet Stockholm Sweden ki.se 8 Chemistry Department and Belozersky Institute of Physico-Chemical Biology Lomonosov Moscow State University Moscow Russia msu.ru 9 Science for Life Laboratory Stockholm University Stockholm Sweden su.se 10 Russian State Center for Quality and Standardization of Veterinary Drugs and Feed (VGNKI) Moscow Russia 11 Evrogen Moscow Russia 12 Sechenov First Moscow State Medical University Moscow Russia mma.ru 2019 2 12 2019 2019 08 05 2019 01 11 2019 05 11 2019 2 12 2019 2019 Copyright © 2019 Ekaterina Bayurova et al. This is an open access article distributed under the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. HIV-induced immune suppression results in the high prevalence of HIV/AIDS-associated malignancies including Kaposi sarcoma, non-Hodgkin lymphoma, and cervical cancer. HIV-infected people are also at an increased risk of “non-AIDS-defining” malignancies not directly linked to immune suppression but associated with viral infections. Their incidence is increasing despite successful antiretroviral therapy. The mechanism behind this phenomenon remains unclear. Here, we obtained daughter clones of murine mammary gland adenocarcinoma 4T1luc2 cells expressing consensus reverse transcriptase of HIV-1 subtype A FSU_A strain (RT_A) with and without primary mutations of drug resistance. In in vitro tests, mutations of resistance to nucleoside inhibitors K65R/M184V reduced the polymerase, and to nonnucleoside inhibitors K103N/G190S, the RNase H activities of RT_A. Expression of these RT_A variants in 4T1luc2 cells led to increased production of the reactive oxygen species (ROS), lipid peroxidation, enhanced cell motility in the wound healing assay, and upregulation of expression of Vimentin and Twist . These properties, particularly, the expression of Twist , correlated with the levels of expression RT_A and/or the production of ROS. When implanted into syngeneic BALB/C mice, 4T1luc2 cells expressing nonmutated RT_A demonstrated enhanced rate of tumor growth and increased metastatic activity, dependent on the level of expression of RT_A and Twist . No enhancement was observed for the clones expressing mutated RT_A variants. Plausible mechanisms are discussed involving differential interactions of mutated and nonmutated RTs with its cellular partners involved in the regulation of ROS. This study establishes links between the expression of HIV-1 RT, production of ROS, induction of EMT, and enhanced propagation of RT-expressing tumor cells. Such scenario can be proposed as one of the mechanisms of HIV-induced/enhanced carcinogenesis not associated with immune suppression. Ministry of Science and Higher Education of the Russian Federation 075-15-2019-1660 Latvian Science Council LZP-2018/2-0308 EU VACTRAIN Russian Foundation for Basic Research 17-00-00085 17_04_00583 17_54_30002 Publisher Copyright: © 2019 Ekaterina Bayurova et al.HIV-induced immune suppression results in the high prevalence of HIV/AIDS-associated malignancies including Kaposi sarcoma, non-Hodgkin lymphoma, and cervical cancer. HIV-infected people are also at an increased risk of "non-AIDS-defining" malignancies not directly linked to immune suppression but associated with viral infections. Their incidence is increasing despite successful antiretroviral therapy. The mechanism behind this phenomenon remains unclear. Here, we obtained daughter clones of murine mammary gland adenocarcinoma 4T1luc2 cells expressing consensus reverse transcriptase of HIV-1 subtype A FSU_A strain (RT_A) with and without primary mutations of drug resistance. In in vitro tests, mutations of resistance to nucleoside inhibitors K65R/M184V reduced the polymerase, and to nonnucleoside inhibitors K103N/G190S, the RNase H activities of RT_A. Expression of these RT_A variants in 4T1luc2 cells led to increased production of the reactive oxygen species (ROS), lipid peroxidation, enhanced cell motility in the wound healing assay, and upregulation of expression of Vimentin and Twist. These properties, particularly, the expression of Twist, correlated with the levels of expression RT_A and/or the production of ROS. When implanted into syngeneic BALB/C mice, 4T1luc2 cells expressing nonmutated RT_A demonstrated enhanced rate of tumor growth and increased metastatic activity, dependent on the level of expression of RT_A and Twist. No enhancement was observed for the clones expressing mutated RT_A variants. Plausible mechanisms are discussed involving differential interactions of mutated and nonmutated RTs with its cellular partners involved in the regulation of ROS. This study establishes links between the expression of HIV-1 RT, production of ROS, induction of EMT, and enhanced propagation of RT-expressing tumor cells. Such scenario can be proposed as one of the mechanisms of HIV-induced/enhanced carcinogenesis not associated with immune suppression.publishersversionPeer reviewe

Safety and immunogenicity of rAd26 and rAd5 vector-based heterologous prime-boost COVID-19 vaccine against SARS-CoV-2 in healthy adolescents: an open-label, non-randomized, multicenter, phase 1/2, dose-escalation study

To protect young individuals against SARS-CoV-2 infection, we conducted an open-label, prospective, non-randomised dose-escalation Phase 1/2 clinical trial to evaluate the immunogenicity and safety of the prime-boost “Sputnik V” vaccine administered at 1/10 and 1/5 doses to adolescents aged 12–17 years. The study began with the vaccination of the older cohort (15-to-17-year-old participants) with the lower (1/10) dose of vaccine and then expanded to the whole group (12-to-17-year-old participants). Next, 1/5 dose was used according to the same scheme. Both doses were well tolerated by all age groups. No serious or severe adverse events were detected. Most of the solicited adverse reactions were mild. No significant differences in total frequencies of adverse events were registered between low and high doses in age-pooled groups (69.6% versus 66.7%). In contrast, the 1/5 dose induced significantly higher humoral and T cell-mediated immune responses than the 1/10 dose. The 1/5 vaccine dose elicited higher antigen-binding (both S and RBD-specific) as well as virus-neutralising antibody titres at the maximum of response (day 42), also resulting in a statistically significant difference at a distanced timepoint (day 180) compared to the 1/10 vaccine dose. Higher dose resulted in increased cross-neutralization of Delta and Omicron variants.;Clinical Trial RegistrationClinicalTrials.gov, NCT04954092, LP-007632

Chlamydial Type III Secretion System Needle Protein Induces Protective Immunity against Chlamydia muridarum Intravaginal Infection

Chlamydia trachomatis imposes serious health problems and causes infertility. Because of asymptomatic onset, it often escapes antibiotic treatment. Therefore, vaccines offer a better option for the prevention of unwanted inflammatory sequelae. The existence of serologically distinct serovars of C. trachomatis suggests that a vaccine will need to provide protection against multiple serovars. Chlamydia spp. use a highly conserved type III secretion system (T3SS) composed of structural and effector proteins which is an essential virulence factor. In this study, we expressed the T3SS needle protein of Chlamydia muridarum, TC_0037, an ortholog of C. trachomatis CdsF, in a replication-defective adenoviral vector (AdTC_0037) and evaluated its protective efficacy in an intravaginal Chlamydia muridarum model. For better immune responses, we employed a heterologous prime-boost immunization protocol in which mice were intranasally primed with AdTC_0037 and subcutaneously boosted with recombinant TC_0037 and Toll-like receptor 4 agonist monophosphoryl lipid A mixed in a squalene nanoscale emulsion. We found that immunization with TC_0037 antigen induced specific humoral and T cell responses, decreased Chlamydia loads in the genital tract, and abrogated pathology of upper genital organs. Together, our results suggest that TC_0037, a highly conserved chlamydial T3SS protein, is a good candidate for inclusion in a Chlamydia vaccine

Chlamydial Type III Secretion System Needle Protein Induces Protective Immunity against Chlamydia muridarum Intravaginal Infection

Chlamydia trachomatis imposes serious health problems and causes infertility. Because of asymptomatic onset, it often escapes antibiotic treatment. Therefore, vaccines offer a better option for the prevention of unwanted inflammatory sequelae. The existence of serologically distinct serovars of C. trachomatis suggests that a vaccine will need to provide protection against multiple serovars. Chlamydia spp. use a highly conserved type III secretion system (T3SS) composed of structural and effector proteins which is an essential virulence factor. In this study, we expressed the T3SS needle protein of Chlamydia muridarum, TC_0037, an ortholog of C. trachomatis CdsF, in a replication-defective adenoviral vector (AdTC_0037) and evaluated its protective efficacy in an intravaginal Chlamydia muridarum model. For better immune responses, we employed a heterologous prime-boost immunization protocol in which mice were intranasally primed with AdTC_0037 and subcutaneously boosted with recombinant TC_0037 and Toll-like receptor 4 agonist monophosphoryl lipid A mixed in a squalene nanoscale emulsion. We found that immunization with TC_0037 antigen induced specific humoral and T cell responses, decreased Chlamydia loads in the genital tract, and abrogated pathology of upper genital organs. Together, our results suggest that TC_0037, a highly conserved chlamydial T3SS protein, is a good candidate for inclusion in a Chlamydia vaccine

Stimulation of Dectin-1 and Dectin-2 during Parenteral Immunization, but Not Mincle, Induces Secretory IgA in Intestinal Mucosa

Induction of a robust and long-lived mucosal immune response during vaccination is critical to achieve protection against numerous pathogens. However, traditional injected vaccines are generally poor inducers of mucosal immunity. One of the effective strategies to improve vaccine efficacy is incorporation of adjuvant molecules that enhance and polarize adaptive immune reactions. Effects of Syk-coupled lectin receptor agonists as adjuvants to induce mucosal immune reactions during parenteral immunization are not fully studied. We now report that the agonists trehalose-6,6-dibehenate (TDB), curdlan, and furfurman, which stimulate Dectin-1, Dectin-2, and Mincle, respectively, activate transcription factors (NF-κB, NFAT, and AP-1) to various extents in murine RAW 264.7 macrophages, even though similar pathways are activated. The agonists also elicit differential expression of maturation markers in bone marrow-derived dendritic cells, as well as differential cytokine secretion from these cells and from splenic mononuclear cells. In vivo assays also show that agonists of Dectin-1 and Dectin-2, but not Mincle, induce heavy IgA secretion in intestinal mucosa even when delivered parenterally. Strikingly, this effect appears to be formulation-independent. Collectively, the data suggest that adjuvants based on Dectin-1 and Dectin-2 agonists may significantly improve the efficacy of parenteral vaccines by inducing robust local immune reactions in intestinal mucosa

Camelid VHHs Fused to Human Fc Fragments Provide Long Term Protection Against Botulinum Neurotoxin A in Mice

The bacterium Clostridium botulinum is the causative agent of botulism—a severe intoxication caused by botulinum neurotoxin (BoNT) and characterized by damage to the nervous system. In an effort to develop novel C. botulinum immunotherapeutics, camelid single-domain antibodies (sdAbs, VHHs, or nanobodies) could be used due to their unique structure and characteristics. In this study, VHHs were produced using phage display technology. A total of 15 different monoclonal VHHs were selected based on their comlementarity-determining region 3 (CDR3) sequences. Different toxin lethal dose (LD50) challenges with each selected phage clone were conducted in vivo to check their neutralizing potency. We demonstrated that modification of neutralizing VHHs with a human immunoglobulin G (IgG)1 Fc (fragment crystallizable) fragment (fusionbody, VHH-Fc) significantly increased the circulation time in the blood (up to 14 days). At the same time, VHH-Fc showed the protective activity 1000 times higher than monomeric form when challenged with 5 LD50. Moreover, VHH-Fcs remained protective even 14 days after antibody administration. These results indicate that this VHH-Fc could be used as an effective long term antitoxin protection against botulinum type A

Targeting TLR-4 with a novel pharmaceutical grade plant derived agonist, Immunomax®, as a therapeutic strategy for metastatic breast cancer.

BackgroundPreviously we demonstrated that the resection of primary 4T1 tumors only slightly prolongs mouse survival, but importantly, creates a "window of opportunity" with attenuated suppressor cell and increased activated T cell populations. This suggests that additional activation of the immune system by immunostimulatory agents during this period may enhance anti-tumor immunity and potentially eradicate micro-metastatic disease in this stringent model. We hypothesized that the immunostimulator Immunomax®, which is comprised of a plant-derived polysaccharide, is non-toxic in humans and stimulates immune defense during the infectious diseases treatment, may have also anti-tumor activity and be beneficial in the adjuvant setting when endogenous anti-tumor responses are present and during the "window of opportunity" in post-resection metastatic breast cancer model. Here we provide the initial report that Immunomax® demonstrates the capacity to eliminate micro-metastatic disease in the post-resection, 4T1 mouse model of breast cancer.MethodsThe efficacy of Immunomax® was evaluated by analyzing survival rate and the number of spontaneous clonogenic tumor cells in the lung homogenates of mice. The frequencies of activated NK, CD4(+) and CD8(+) cells as well as myeloid-derived suppressor cells and Treg cells were evaluated using flow cytometry. Highly purified mouse and human dendritic and NK cells were sorted and the effect of Immunomax® on activation status of these cells was assessed by flow cytometry. The property of Immunomax® as TLR-4 agonist was determined by NF-κB/SEAP reporter gene assay, WB, RT-PCR.ResultsImmunomax® injections significantly prolonged overall survival and cured 31% of mice. This immunostimulator activates DCs via the TLR-4, which in turn stimulates tumoricidal NK cells and in vitro, completely inhibits growth of 4T1 cells. Incubation of PBMC from healthy donors with Immunomax® activates NK cells via activation of plasmacytoid DC leading significantly higher efficacy in killing of human NK-target cells K562 compared with non-treated cells.ConclusionThis is the first demonstration that Immunomax® is a TLR-4 agonist and the first report of a documented role for this pharmaceutical grade immunostimulator in augmenting anti-tumor activity, suggesting that incorporation of Immunomax® into developing breast cancer therapeutic strategies may be beneficial and with less potential toxicity than checkpoint inhibitors

Targeting TLR-4 with a novel pharmaceutical grade plant derived agonist, Immunomax®, as a therapeutic strategy for metastatic breast cancer.

BackgroundPreviously we demonstrated that the resection of primary 4T1 tumors only slightly prolongs mouse survival, but importantly, creates a "window of opportunity" with attenuated suppressor cell and increased activated T cell populations. This suggests that additional activation of the immune system by immunostimulatory agents during this period may enhance anti-tumor immunity and potentially eradicate micro-metastatic disease in this stringent model. We hypothesized that the immunostimulator Immunomax®, which is comprised of a plant-derived polysaccharide, is non-toxic in humans and stimulates immune defense during the infectious diseases treatment, may have also anti-tumor activity and be beneficial in the adjuvant setting when endogenous anti-tumor responses are present and during the "window of opportunity" in post-resection metastatic breast cancer model. Here we provide the initial report that Immunomax® demonstrates the capacity to eliminate micro-metastatic disease in the post-resection, 4T1 mouse model of breast cancer.MethodsThe efficacy of Immunomax® was evaluated by analyzing survival rate and the number of spontaneous clonogenic tumor cells in the lung homogenates of mice. The frequencies of activated NK, CD4(+) and CD8(+) cells as well as myeloid-derived suppressor cells and Treg cells were evaluated using flow cytometry. Highly purified mouse and human dendritic and NK cells were sorted and the effect of Immunomax® on activation status of these cells was assessed by flow cytometry. The property of Immunomax® as TLR-4 agonist was determined by NF-κB/SEAP reporter gene assay, WB, RT-PCR.ResultsImmunomax® injections significantly prolonged overall survival and cured 31% of mice. This immunostimulator activates DCs via the TLR-4, which in turn stimulates tumoricidal NK cells and in vitro, completely inhibits growth of 4T1 cells. Incubation of PBMC from healthy donors with Immunomax® activates NK cells via activation of plasmacytoid DC leading significantly higher efficacy in killing of human NK-target cells K562 compared with non-treated cells.ConclusionThis is the first demonstration that Immunomax® is a TLR-4 agonist and the first report of a documented role for this pharmaceutical grade immunostimulator in augmenting anti-tumor activity, suggesting that incorporation of Immunomax® into developing breast cancer therapeutic strategies may be beneficial and with less potential toxicity than checkpoint inhibitors

Genetic Passive Immunization with Adenoviral Vector Expressing Chimeric Nanobody-Fc Molecules as Therapy for Genital Infection Caused by Mycoplasma hominis.

Developing pathogen-specific recombinant antibody fragments (especially nanobodies) is a very promising strategy for the treatment of infectious disease. Nanobodies have great potential for gene therapy application due to their single-gene nature. Historically, Mycoplasma hominis has not been considered pathogenic bacteria due to the lack of acute infection and partially due to multiple studies demonstrating high frequency of isolation of M. hominis samples from asymptomatic patients. However, recent studies on the role of latent M. hominis infection in oncologic transformation, especially prostate cancer, and reports that M. hominis infects Trichomonas and confers antibiotic resistance to Trichomonas, have generated new interest in this field. In the present study we have generated specific nanobody against M. hominis (aMh), for which the identified target is the ABC-transporter substrate-binding protein. aMh exhibits specific antibacterial action against M. hominis. In an attempt to improve the therapeutic properties, we have developed the adenoviral vector-based gene therapy approach for passive immunization with nanobodies against M. hominis. For better penetration into the mucous layer of the genital tract, we fused aMh with the Fc-fragment of IgG. Application of this comprehensive approach with a single systemic administration of recombinant adenovirus expressing aMh-Fc demonstrated both prophylactic and therapeutic effects in a mouse model of genital M. hominis infection

Cross-Reactive Fc-Fused Single-Domain Antibodies to Hemagglutinin Stem Region Protect Mice from Group 1 Influenza a Virus Infection

The continued evolution of influenza viruses reduces the effectiveness of vaccination and antiviral drugs. The identification of novel and universal agents for influenza prophylaxis and treatment is an urgent need. We have previously described two potent single-domain antibodies (VHH), G2.3 and H1.2, which bind to the stem domain of hemagglutinin and efficiently neutralize H1N1 and H5N2 influenza viruses in vivo. In this study, we modified these VHHs with Fc-fragment to enhance their antiviral activity. Reformatting of G2.3 into bivalent Fc-fusion molecule increased its in vitro neutralizing activity against H1N1 and H2N3 viruses up to 80-fold and, moreover, resulted in obtaining the ability to neutralize H5N2 and H9N2 subtypes. We demonstrated that a dose as low as 0.6 mg/kg of G2.3-Fc or H1.2-Fc administered systemically or locally before infection could protect mice from lethal challenges with both H1N1 and H5N2 viruses. Furthermore, G2.3-Fc reduced the lung viral load to an undetectable level. Both VHH-Fc antibodies showed in vivo therapeutic efficacy when delivered via systemic or local route. The findings support G2.3-Fc as a potential therapeutic agent for both prophylaxis and therapy of Group 1 influenza A infection