scholarly journals Influenza H3 and H1 hemagglutinins have different genetic barriers for resistance to broadly neutralizing stem antibodies

2019 ◽  
Author(s):  
Nicholas C. Wu ◽  
Andrew J. Thompson ◽  
Juhye M. Lee ◽  
Wen Su ◽  
Britni M. Arlian ◽  
...  
Keyword(s):  
Influenza Vaccine ◽  
Neutralizing Antibodies ◽  
Viral Fitness ◽  
Resistance Mutations ◽  
Genetic Barrier ◽  
Influenza Hemagglutinin ◽  
Genetic Barriers ◽  
Universal Influenza Vaccine

ABSTRACTIn the past decade, the discovery and characterization of broadly neutralizing antibodies (bnAbs) to the highly conserved stem region of influenza hemagglutinin (HA) have provided valuable insights for development of a universal influenza vaccine. However, the genetic barrier for resistance to stem bnAbs has not been thoroughly evaluated. Here, we performed a series of deep mutational scanning experiments to probe for resistance mutations. We found that the genetic barrier to resistance to stem bnAbs is generally very low for the H3 subtype but substantially higher for the H1 subtype. Several resistance mutations in H3 cannot be neutralized by stem bnAbs at the highest concentration tested, do not reduce in vitro viral fitness and in vivo pathogenicity, and are often present in circulating strains as minor variants. Thus, H3 HAs have a higher propensity than H1 HAs to escape major stem bnAbs and creates a potential challenge in the development of a bona fide universal influenza vaccine.ONE SENTENCE SUMMARYAcquisition of resistance by influenza virus to broadly neutralizing hemagglutinin stem antibodies varies tremendously depending on subtype.

scholarly journals Different genetic barriers for resistance to HA stem antibodies in influenza H3 and H1 viruses

Science ◽  
2020 ◽  
Vol 368 (6497) ◽  
pp. 1335-1340 ◽  
Author(s):  
Nicholas C. Wu ◽  
Andrew J. Thompson ◽  
Juhye M. Lee ◽  
Wen Su ◽  
Britni M. Arlian ◽  
...  
Keyword(s):  
Influenza Vaccine ◽  
Viral Fitness ◽  
Resistance Mutations ◽  
Genetic Barrier ◽  
Influenza Hemagglutinin ◽  
Genetic Barriers ◽  
Universal Influenza Vaccine

The discovery and characterization of broadly neutralizing human antibodies (bnAbs) to the highly conserved stem region of influenza hemagglutinin (HA) have contributed to considerations of a universal influenza vaccine. However, the potential for resistance to stem bnAbs also needs to be more thoroughly evaluated. Using deep mutational scanning, with a focus on epitope residues, we found that the genetic barrier to resistance to stem bnAbs is low for the H3 subtype but substantially higher for the H1 subtype owing to structural differences in the HA stem. Several strong resistance mutations in H3 can be observed in naturally circulating strains and do not reduce in vitro viral fitness and in vivo pathogenicity. This study highlights a potential challenge for development of a truly universal influenza vaccine.

scholarly journals Development and characterization of influenza M2 ectodomain and/or HA stalk-based DC-targeting vaccines for different influenza infections

2021 ◽  
Author(s):  
Xiaojian Yao ◽  
Titus Olukitibi ◽  
Zhujun Ao ◽  
Hiva Azizi ◽  
Mona Mahmoudi ◽  
...  
Keyword(s):  
Influenza Vaccine ◽  
Matrix Protein ◽  
Influenza Infection ◽  
Extracellular Matrix Protein ◽  
Influenza Hemagglutinin ◽  
Viral Particles ◽  
Vesicular Stomatitis ◽  
Universal Influenza Vaccine ◽  
Mouse Study

A universal influenza vaccine is required for broad protection against influenza infection. Here, we revealed the efficacy of novel influenza vaccine candidates based on Ebola glycoprotein (EboGP) DC-targeting domain (EΔM) fusion protein technology. We fused influenza hemagglutinin stalk (HAcs) and extracellular matrix protein (M2e) or four copies of M2e (referred to as tetra M2e (tM2e)) with EΔM to generate EΔM-HM2e or EΔM-tM2e, respectively, and revealed that EΔM facilitates DC/macrophage targeting in vitro. In a mouse study, EΔM-HM2e- or EΔM-tM2e-pseudotyped viral particles (PVPs) induced significantly higher titers of anti-HA and/or anti-M2e antibodies. We also developed recombinant vesicular stomatitis virus (rVSV)-EΔM-HM2e and rVSV-EΔM-tM2e vaccines that resulted in rapid and potent induction of HA and/or M2 antibodies in mouse sera and mucosa. Importantly, vaccination protects mice from influenza H1N1 and H3N2 challenges. Taken together, our study suggests that recombinant rVSV-EΔM-HM2e and rVSV-EΔM-tM2e are efficacious and protective universal vaccines against influenza.

scholarly journals A bifluorescent-based assay for the identification of neutralizing antibodies against SARS-CoV-2 variants of concern in vitro and in vivo

2021 ◽  
Author(s):  
Kevin Chiem ◽  
Desarey Morales Vasquez ◽  
Jesus A. Silvas ◽  
Jun-Gyu Park ◽  
Michael S. Piepenbrink ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Vaccine Efficacy ◽  
Neutralizing Antibodies ◽  
Viral Infections ◽  
Rapid Assessment ◽  
The United States ◽  
Viral Fitness ◽  
Human Monoclonal Antibodies

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged at the end of 2019 and has been responsible for the still ongoing coronavirus disease 2019 (COVID-19) pandemic. Prophylactic vaccines have been authorized by the United States (US) Food and Drug Administration (FDA) for the prevention of COVID-19. Identification of SARS-CoV-2 neutralizing antibodies (NAbs) is important to assess vaccine protection efficacy, including their ability to protect against emerging SARS-CoV-2 variants of concern (VoC). Here we report the generation and use of a recombinant (r)SARS-CoV-2 USA/WA1/2020 (WA-1) strain expressing Venus and a rSARS-CoV-2 expressing mCherry and containing mutations K417N, E484K, and N501Y found in the receptor binding domain (RBD) of the spike (S) glycoprotein of the South African (SA) B.1.351 (beta, β) VoC, in bifluorescent-based assays to rapidly and accurately identify human monoclonal antibodies (hMAbs) able to neutralize both viral infections in vitro and in vivo. Importantly, our bifluorescent-based system accurately recapitulated findings observed using individual viruses. Moreover, fluorescent-expressing rSARS-CoV-2 and the parental wild-type (WT) rSARS-CoV-2 WA-1 had similar viral fitness in vitro , as well as similar virulence and pathogenicity in vivo in the K18 human angiotensin converting enzyme 2 (hACE2) transgenic mouse model of SARS-CoV-2 infection. We demonstrate that these new fluorescent-expressing rSARS-CoV-2 can be used in vitro and in vivo to easily identify hMAbs that simultaneously neutralize different SARS-CoV-2 strains, including VoC, for the rapid assessment of vaccine efficacy or the identification of prophylactic and/or therapeutic broadly NAbs for the treatment of SARS-CoV-2 infection. IMPORTANCE SARS-CoV-2 is responsible of the COVID-19 pandemic that has warped daily routines and socioeconomics. There is still an urgent need for prophylactics and therapeutics to treat SARS-CoV-2 infections. In this study, we demonstrate the feasibility of using bifluorescent-based assays for the rapid identification of human monoclonal antibodies (hMAbs) with neutralizing activity against SARS-CoV-2, including variants of concern (VoC) in vitro and in vivo. Importantly, results obtained with these bifluorescent-based assays recapitulate those observed with individual viruses demonstrating their feasibility to rapidly advance our understanding of vaccine efficacy and to identify broadly protective human NAbs for the therapeutic treatment of SARS-CoV-2.

scholarly journals In Vivo and In Vitro Escape from Neutralizing Antibodies 2G12, 2F5, and 4E10

2007 ◽  
Vol 81 (16) ◽  
pp. 8793-8808 ◽  
Author(s):  
Amapola Manrique ◽  
Peter Rusert ◽  
Beda Joos ◽  
Marek Fischer ◽  
Herbert Kuster ◽  
...  
Keyword(s):  
Neutralizing Antibodies ◽  
In Vitro Selection ◽  
Passive Immunization ◽  
Resistance Mutations ◽  
Antibody Treatment ◽  
Glycosylation Sites ◽  
Depth Analysis ◽  
Immunodeficiency Virus

ABSTRACT Recently, passive immunization of human immunodeficiency virus (HIV)-infected individuals with monoclonal antibodies (MAbs) 2G12, 2F5, and 4E10 provided evidence of the in vivo activity of 2G12 but raised concerns about the function of the two membrane-proximal external region (MPER)-specific MAbs (A. Trkola, H. Kuster, P. Rusert, B. Joos, M. Fischer, C. Leemann, A. Manrique, M. Huber, M. Rehr, A. Oxenius, R. Weber, G. Stiegler, B. Vcelar, H. Katinger, L. Aceto, and H. F. Gunthard, Nat. Med. 11:615-622, 2005). In the light of MPER-targeting vaccines under development, we performed an in-depth analysis of the emergence of mutations conferring resistance to these three MAbs to further elucidate their activity. Clonal analysis of the MPER of plasma virus samples derived during antibody treatment confirmed that no changes in this region had occurred in vivo. Sequence analysis of the 2G12 epitope relevant N-glycosylation sites of viruses derived from 13 patients during the trial supported the phenotypic evaluation, demonstrating that mutations in these sites are associated with resistance. In vitro selection experiments with isolates of four of these individuals corroborated the in vivo finding that virus strains rapidly escape 2G12 pressure. Notably, in vitro resistance mutations differed, in most cases, from those found in vivo. Importantly, in vitro selection with 2F5 and 4E10 demonstrated that resistance to these MAbs can be difficult to achieve and can lead to selection of variants with impaired infectivity. This remarkable vulnerability of the virus to interference within the MPER calls for a further evaluation of the safety and efficacy of MPER-targeting therapeutic and vaccination strategies.

scholarly journals A bifluorescent-based assay for the identification of neutralizing antibodies against SARS-CoV-2 variants of concern in vitro and in vivo

2021 ◽  
Author(s):  
Kevin Chiem ◽  
Desarey Morales Vasquez ◽  
Jesus Silvas ◽  
Jun-Gyu Park ◽  
Michael Piepenbrink ◽  
...  
Keyword(s):  
South African ◽  
Neutralizing Antibodies ◽  
Viral Infections ◽  
Rapid Assessment ◽  
The United States ◽  
Viral Fitness ◽  
Angiotensin Converting Enzyme 2 ◽  
Protection Efficacy

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged at the end of 2019 and has been responsible for the still ongoing coronavirus disease 2019 (COVID-19) pandemic. Prophylactic vaccines have been authorized by the United States (US) Food and Drug Administration (FDA) for the prevention of COVID-19. Identification of SARS-CoV-2 neutralizing antibodies (NAbs) is important to assess vaccine protection efficacy, including their ability to protect against emerging SARS-CoV-2 variants of concern (VoC). Here we report the generation and use of a recombinant (r)SARS-CoV-2 USA/WA1/2020 (WA-1) strain expressing Venus and a rSARS-CoV-2 expressing mCherry and containing mutations K417N, E484K, and N501Y found in the receptor binding domain (RBD) of the spike (S) glycoprotein of the South African (SA) B.1.351 (beta, β) VoC, in bifluorescent-based assays to rapidly and accurately identify human monoclonal antibodies (hMAbs) able to neutralize both viral infections in vitro and in vivo. Importantly, our bifluorescent-based system accurately recapitulated findings observed using individual viruses. Moreover, fluorescent-expressing rSARS-CoV-2 and the parental wild-type (WT) rSARS-CoV-2 WA-1 had similar viral fitness in vitro, as well as similar virulence and pathogenicity in vivo in the K18 human angiotensin converting enzyme 2 (hACE2) transgenic mouse model of SARS-CoV-2 infection. We demonstrate that these new fluorescent-expressing rSARS-CoV-2 can be used in vitro and in vivo to easily identify hMAbs that simultaneously neutralize different SARS-CoV-2 strains, including VoC, for the rapid assessment of vaccine efficacy or the identification of prophylactic and/or therapeutic broadly NAbs for the treatment of SARS-CoV-2 infection.

scholarly journals In Vivo Validation of a Bioinformatics Based Tool to Identify Reduced Replication Capacity in HIV-1

2010 ◽  
Vol 4 (1) ◽  
pp. 225-232
Author(s):  
Christina M.R Kitchen ◽  
Paul Krogstad ◽  
Scott G Kitchen
Keyword(s):  
Drug Resistance ◽  
In Silico ◽  
Viral Fitness ◽  
Biological Assessment ◽  
Relative Fitness ◽  
Replication Capacity ◽  
Resistance Mutations ◽  
Hiv 1

Although antiretroviral drug resistance is common in treated HIV infected individuals, it is not a consistent indicator of HIV morbidity and mortality. To the contrary, HIV resistance-associated mutations may lead to changes in viral fitness that are beneficial to infected individuals. Using a bioinformatics-based model to assess the effects of numerous drug resistance mutations, we determined that the D30N mutation in HIV-1 protease had the largest decrease in replication capacity among known protease resistance mutations. To test thisin silicoresult in anin vivoenvironment, we constructed several drug-resistant mutant HIV-1 strains and compared their relative fitness utilizing the SCID-hu mouse model. We found HIV-1 containing the D30N mutation had a significant defectin vivo, showing impaired replication kinetics and a decreased ability to deplete CD4+ thymocytes, compared to the wild-type or virus without the D30N mutation. In comparison, virus containing the M184V mutation in reverse transcriptase, which shows decreased replication capacityin vitro, did not have an effect on viral fitnessin vivo. Thus, in this study we have verified anin silicobioinformatics result with a biological assessment to identify a unique mutation in HIV-1 that has a significant fitness defectin vivo.

scholarly journals Blocking the GITR-GITRL pathway to overcome resistance to therapy in sarcomatoid malignant pleural mesothelioma

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Meilin Chan ◽  
Licun Wu ◽  
Zhihong Yun ◽  
Trevor D. McKee ◽  
Michael Cabanero ◽  
...  
Keyword(s):  
Tumor Growth ◽  
Malignant Pleural Mesothelioma ◽  
Neutralizing Antibodies ◽  
Pleural Mesothelioma ◽  
Spheroid Formation ◽  
Resistance To Therapy ◽  
Sarcomatoid Mesothelioma

AbstractMalignant pleural mesothelioma (MPM) is an aggressive neoplasm originating from the pleura. Non-epithelioid (biphasic and sarcomatoid) MPM are particularly resistant to therapy. We investigated the role of the GITR-GITRL pathway in mediating the resistance to therapy. We found that GITR and GITRL expressions were higher in the sarcomatoid cell line (CRL5946) than in non-sarcomatoid cell lines (CRL5915 and CRL5820), and that cisplatin and Cs-137 irradiation increased GITR and GITRL expressions on tumor cells. Transcriptome analysis demonstrated that the GITR-GITRL pathway was promoting tumor growth and inhibiting cell apoptosis. Furthermore, GITR+ and GITRL+ cells demonstrated increased spheroid formation in vitro and in vivo. Using patient derived xenografts (PDXs), we demonstrated that anti-GITR neutralizing antibodies attenuated tumor growth in sarcomatoid PDX mice. Tumor immunostaining demonstrated higher levels of GITR and GITRL expressions in non-epithelioid compared to epithelioid tumors. Among 73 patients uniformly treated with accelerated radiation therapy followed by surgery, the intensity of GITR expression after radiation negatively correlated with survival in non-epithelioid MPM patients. In conclusion, the GITR-GITRL pathway is an important mechanism of autocrine proliferation in sarcomatoid mesothelioma, associated with tumor stemness and resistance to therapy. Blocking the GITR-GITRL pathway could be a new therapeutic target for non-epithelioid mesothelioma.

scholarly journals Gilteritinib overcomes lorlatinib resistance in ALK-rearranged cancer

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Hayato Mizuta ◽  
Koutaroh Okada ◽  
Mitsugu Araki ◽  
Jun Adachi ◽  
Ai Takemoto ◽  
...  
Keyword(s):  
Gene Rearrangement ◽  
Kinase Inhibitors ◽  
Inhibitory Effect ◽  
In Vivo Model ◽  
Resistance Mutations ◽  
Lung Cancer Patients ◽  
Short Period ◽  
Tki Resistance

AbstractALK gene rearrangement was observed in 3%–5% of non-small cell lung cancer patients, and multiple ALK-tyrosine kinase inhibitors (TKIs) have been sequentially used. Multiple ALK-TKI resistance mutations have been identified from the patients, and several compound mutations, such as I1171N + F1174I or I1171N + L1198H are resistant to all the approved ALK-TKIs. In this study, we found that gilteritinib has an inhibitory effect on ALK-TKI–resistant single mutants and I1171N compound mutants in vitro and in vivo. Surprisingly, EML4-ALK I1171N + F1174I compound mutant-expressing tumors were not completely shrunk but regrew within a short period of time after alectinib or lorlatinib treatment. However, the relapsed tumor was markedly shrunk after switching to the gilteritinib in vivo model. In addition, gilteritinib was effective against NTRK-rearranged cancers including entrectinib-resistant NTRK1 G667C-mutant and ROS1 fusion-positive cancer.

scholarly journals Effects of Size and Surface Properties of Nanodiamonds on the Immunogenicity of Plant-Based H5 Protein of A/H5N1 Virus in Mice

Nanomaterials ◽  
2021 ◽  
Vol 11 (6) ◽  
pp. 1597
Author(s):  
Thuong Thi Ho ◽  
Van Thi Pham ◽  
Tra Thi Nguyen ◽  
Vy Thai Trinh ◽  
Tram Vi ◽  
...  
Keyword(s):  
High Pressure ◽  
Neutralizing Antibodies ◽  
H5n1 Virus ◽  
Vaccine Development ◽  
Particle Characterization ◽  
Innovative Strategy ◽  
Specific Igg ◽  
Positively Charged

Nanodiamond (ND) has recently emerged as a potential nanomaterial for nanovaccine development. Here, a plant-based haemagglutinin protein (H5.c2) of A/H5N1 virus was conjugated with detonation NDs (DND) of 3.7 nm in diameter (ND4), and high-pressure and high-temperature (HPHT) oxidative NDs of ~40–70 nm (ND40) and ~100–250 nm (ND100) in diameter. Our results revealed that the surface charge, but not the size of NDs, is crucial to the protein conjugation, as well as the in vitro and in vivo behaviors of H5.c2:ND conjugates. Positively charged ND4 does not effectively form stable conjugates with H5.c2, and has no impact on the immunogenicity of the protein both in vitro and in vivo. In contrast, the negatively oxidized NDs (ND40 and ND100) are excellent protein antigen carriers. When compared to free H5.c2, H5.c2:ND40, and H5.c2:ND100 conjugates are highly immunogenic with hemagglutination titers that are both 16 times higher than that of the free H5.c2 protein. Notably, H5.c2:ND40 and H5.c2:ND100 conjugates induce over 3-folds stronger production of both H5.c2-specific-IgG and neutralizing antibodies against A/H5N1 than free H5.c2 in mice. These findings support the innovative strategy of using negatively oxidized ND particles as novel antigen carriers for vaccine development, while also highlighting the importance of particle characterization before use.

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