Broad betacoronavirus neutralization by a stem helix–specific human antibody

The spillovers of betacoronaviruses in humans and the emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants highlight the need for broad coronavirus countermeasures. We describe five monoclonal antibodies (mAbs) cross-reacting with the stem helix of multiple betacoronavirus spike glycoproteins isolated from COVID-19 convalescent individuals. Using structural and functional studies, we show that the mAb with the greatest breadth (S2P6) neutralizes pseudotyped viruses from three different subgenera through the inhibition of membrane fusion, and we delineate the molecular basis for its cross-reactivity. S2P6 reduces viral burden in hamsters challenged with SARS-CoV-2 through viral neutralization and Fc-mediated effector functions. Stem helix antibodies are rare, oftentimes of narrow specificity, and can acquire neutralization breadth through somatic mutations. These data provide a framework for structure-guided design of pan-betacoronavirus vaccines eliciting broad protection

ACE2-binding exposes the SARS-CoV-2 fusion peptide to broadly neutralizing coronavirus antibodies

The coronavirus spike glycoprotein attaches to host receptors and mediates viral fusion. Using a broad screening approach, we isolated seven monoclonal antibodies (mAbs) that bind to all human-infecting coronavirus spike proteins from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) immune donors. These mAbs recognize the fusion peptide and acquire affinity and breadth through somatic mutations. Despite targeting a conserved motif, only some mAbs show broad neutralizing activity in vitro against alpha- and betacoronaviruses, including animal coronaviruses WIV-1 and PDF-2180. Two selected mAbs also neutralize Omicron BA.1 and BA.2 authentic viruses and reduce viral burden and pathology in vivo. Structural and functional analyses showed that the fusion peptide–specific mAbs bound with different modalities to a cryptic epitope hidden in prefusion stabilized spike, which became exposed upon binding of angiotensin-converting enzyme 2 (ACE2) or ACE2-mimicking mAbs

Karakterizacija protutijela specifičnih za MCMV protein MCK-2 i gO

Cytomegalovirus (CMV) is β-herpesvirus, which establishes lifelong latent infection in host. CMV is transmitted through bodily fluids and represents the most frequent congenital infection in humans. Infection of healthy individuals with human CMV (HCMV) is usually asymptomatic, however, it can cause severe disease in immunocompromised individuals, as well as in infants infected in the prenatal period. CMV has broad tropism, infecting majority of cell types and organs. Viral glycoproteins mediate fusion with host cell membranes and entry of virus into target cells. Glycoprotein complexes gH/gL are determining virus specificity for infection of distinct cell types. Due to the species-specific nature of CMV, mouse CMV (MCMV) has been widely used as a model to study HCMV infection. Two gH/gL complexes have been identified in MCMV, gH/gL/gO and gH/gL/MCK-2. However, the biology and role of these complexes is still incompletely defined, in part due to the lack of antibodies specific for these proteins. Here I characterized new antibodies specific for gO and MCK-2 using flow cytometry, Western blot and enzyme-linked immunosorbent assay. Antibodies were generated by immunizing mice with proteins produced in eukaryotic cells and subsequent fusion of B cells with myeloma cells. Obtained hybridomas were preliminary screened by ELISA and positive antibodies were further tested in Western blot and flow cytometry. In total, seven out of fifty-one tested MCK-2 antibodies were specific for MCK-2 in flow cytometry. Five of them were positive in Western blot as well. Furthermore, five out of twelve tested gO antibodies specific for gO were identified by flow cytometry, and additional four by Western blot. Identified antibodies will be used in future studies of MCMV gH/gL complexes.Citomegalovirus (CMV) je β-herpesvirus koji u domaćinu uspostavlja doživotnu latentnu infekciju. CMV se prenosi tjelesnim tekućinama i predstavlja najčešću prirođenu infekciju u ljudi. Infekcija ljudskim CMV-om (eng. Human CMV, HCMV) je kod zdravih ljudi obično asimptomatska, međutim kod osoba sa oslabljenim imunološkim sustavom te djece zaražene u prenatalnom razdoblju može izazvati kliničku manifestaciju bolesti. CMV inficira većinu staničnih tipova i posljedično većinu organa. Fuzija virusne i stanične membrane te ulazak virusa u stanicu su posredovani virusnim glikoproteinima. Specifičnost virusa za ulazak u različite tipove stanica je određena glikoproteinskom kompleksima gH/gL. Budući da je HCMV specifičan za vrstu, mišji CMV (eng. mouse CMV, MCMV) se koristi kao model za istraživanje infekcije ljudskim CMV-om. Dva glikoproteinska kompleksa gH/gL su karakterizirana u mišjem CMV-u: gH/gL/gO i gH/gL/MCK-2. Biološke funkcije i uloge ovih kompleksa su nepotpuno razjašnjene, djelomično zbog nedostatka specifičnih protutijela. U ovom radu sam karakterizirala protutijela specifična za proteine gO i MCK-2 koristeći protočnu citometriju, Western blot i enzimski vezani imunosorbentni test (ELISA). Protutijela su dobivena imunizacijom miševa proteinima proizvedenim u eukariotskim stanicama nakon čega je uslijedila fuzija B limfocita sa stanicama mijeloma. Dobiveni hibridomi preliminarno su analizirani uz pomoć ELISA-e te su pozitivna protutijela dalje analizirana u Western blotu i protočnoj citometriji. Sedam od ukupno pedeset dva testirana MCK-2 protutijela je bilo specifično za MCK-2 u protočnoj citometriji, a njih pet je također bilo pozitivno u Western blotu. Nadalje, pet od ukupno dvanaest testiranih gO protutijela bilo je specifično za gO u protočnoj citometriji, a dodatnih četiri u Western blotu. Identificirana protutijela će biti korištena u daljnim istraživanjima gH/gL kompleksa MCMV-a

Karakterizacija protutijela specifičnih za MCMV protein MCK-2 i gO

Cytomegalovirus (CMV) is β-herpesvirus, which establishes lifelong latent infection in host. CMV is transmitted through bodily fluids and represents the most frequent congenital infection in humans. Infection of healthy individuals with human CMV (HCMV) is usually asymptomatic, however, it can cause severe disease in immunocompromised individuals, as well as in infants infected in the prenatal period. CMV has broad tropism, infecting majority of cell types and organs. Viral glycoproteins mediate fusion with host cell membranes and entry of virus into target cells. Glycoprotein complexes gH/gL are determining virus specificity for infection of distinct cell types. Due to the species-specific nature of CMV, mouse CMV (MCMV) has been widely used as a model to study HCMV infection. Two gH/gL complexes have been identified in MCMV, gH/gL/gO and gH/gL/MCK-2. However, the biology and role of these complexes is still incompletely defined, in part due to the lack of antibodies specific for these proteins. Here I characterized new antibodies specific for gO and MCK-2 using flow cytometry, Western blot and enzyme-linked immunosorbent assay. Antibodies were generated by immunizing mice with proteins produced in eukaryotic cells and subsequent fusion of B cells with myeloma cells. Obtained hybridomas were preliminary screened by ELISA and positive antibodies were further tested in Western blot and flow cytometry. In total, seven out of fifty-one tested MCK-2 antibodies were specific for MCK-2 in flow cytometry. Five of them were positive in Western blot as well. Furthermore, five out of twelve tested gO antibodies specific for gO were identified by flow cytometry, and additional four by Western blot. Identified antibodies will be used in future studies of MCMV gH/gL complexes.Citomegalovirus (CMV) je β-herpesvirus koji u domaćinu uspostavlja doživotnu latentnu infekciju. CMV se prenosi tjelesnim tekućinama i predstavlja najčešću prirođenu infekciju u ljudi. Infekcija ljudskim CMV-om (eng. Human CMV, HCMV) je kod zdravih ljudi obično asimptomatska, međutim kod osoba sa oslabljenim imunološkim sustavom te djece zaražene u prenatalnom razdoblju može izazvati kliničku manifestaciju bolesti. CMV inficira većinu staničnih tipova i posljedično većinu organa. Fuzija virusne i stanične membrane te ulazak virusa u stanicu su posredovani virusnim glikoproteinima. Specifičnost virusa za ulazak u različite tipove stanica je određena glikoproteinskom kompleksima gH/gL. Budući da je HCMV specifičan za vrstu, mišji CMV (eng. mouse CMV, MCMV) se koristi kao model za istraživanje infekcije ljudskim CMV-om. Dva glikoproteinska kompleksa gH/gL su karakterizirana u mišjem CMV-u: gH/gL/gO i gH/gL/MCK-2. Biološke funkcije i uloge ovih kompleksa su nepotpuno razjašnjene, djelomično zbog nedostatka specifičnih protutijela. U ovom radu sam karakterizirala protutijela specifična za proteine gO i MCK-2 koristeći protočnu citometriju, Western blot i enzimski vezani imunosorbentni test (ELISA). Protutijela su dobivena imunizacijom miševa proteinima proizvedenim u eukariotskim stanicama nakon čega je uslijedila fuzija B limfocita sa stanicama mijeloma. Dobiveni hibridomi preliminarno su analizirani uz pomoć ELISA-e te su pozitivna protutijela dalje analizirana u Western blotu i protočnoj citometriji. Sedam od ukupno pedeset dva testirana MCK-2 protutijela je bilo specifično za MCK-2 u protočnoj citometriji, a njih pet je također bilo pozitivno u Western blotu. Nadalje, pet od ukupno dvanaest testiranih gO protutijela bilo je specifično za gO u protočnoj citometriji, a dodatnih četiri u Western blotu. Identificirana protutijela će biti korištena u daljnim istraživanjima gH/gL kompleksa MCMV-a

Dissecting the cytomegalovirus CC chemokine: Chemokine activity and gHgLchemokine-dependent cell tropism are independent players in CMV infection

Like all herpesviruses, cytomegaloviruses (CMVs) code for many immunomodulatory proteins including chemokines. The human cytomegalovirus (HCMV) CC chemokine pUL128 has a dual role in the infection cycle. On one hand, it forms the pentameric receptor-binding complex gHgLpUL(128,130,131A), which is crucial for the broad cell tropism of HCMV. On the other hand, it is an active chemokine that attracts leukocytes and shapes their activation. All animal CMVs studied so far have functionally homologous CC chemokines. In murine cytomegalovirus (MCMV), the CC chemokine is encoded by the m131/m129 reading frames. The MCMV CC chemokine is called MCK2 and forms a trimeric gHgLMCK2 entry complex. Here, we have generated MCK2 mutant viruses either unable to form gHgLMCK2 complexes, lacking the chemokine function or lacking both functions. By using these viruses, we could demonstrate that gHgLMCK2-dependent entry and MCK2 chemokine activity are independent functions of MCK2 in vitro and in vivo. The gHgLMCK2 complex promotes the tropism for leukocytes like macrophages and dendritic cells and secures high titers in salivary glands in MCMV-infected mice independent of the chemokine activity of MCK2. In contrast, reduced early antiviral T cell responses in MCMV-infected mice are dependent on MCK2 being an active chemokine and do not require the formation of gHgLMCK2 complexes. High levels of CCL2 and IFN-gamma in spleens of infected mice and MCMV virulence depend on both, the formation of gHgLMCK2 complexes and the MCK2 chemokine activity. Thus, independent and concerted functions of MCK2 serving as chemokine and part of a gHgL entry complex shape antiviral immunity and virus dissemination. Studies on immunomodulatory proteins of herpesviruses have strongly contributed to understanding antiviral immune responses elicited during infection and to defining targets for intervention during herpesvirus infections. CC chemokines of cytomegaloviruses have been shown to shape the viral cell tropism and antiviral immune responses, yet, their modes of action are not really understood. Here, we used well-defined mutants of the MCMV chemokine MCK2 to exemplarily dissect the role of a CMV CC chemokine in viral dissemination and antiviral innate and adaptive immune responses. We could show that the chemokine activity and the entry function of MCK2 are independent players in the MCMV infection of the mouse. As cytomegaloviruses are discussed as potential vaccine vectors which may be programmed to elicit specific CD8+ T cell responses and combat specific pathogens, it is important to understand the contribution of immunomodulatory proteins to vector-induced immune responses. Specifically, in infections of Rhesus macaques with Rhesus CMV (RhCMV), the viral CC chemokine has been shown to drastically shape the immune response to vaccine antigens. Our detailed analysis of the dual roles of a CMV CC chemokine may further help to decide whether CMV vaccine vectors should express an intact viral CC chemokine or not

ACE2-binding exposes the SARS-CoV-2 fusion peptide to broadly neutralizing coronavirus antibodies

The coronavirus spike (S) glycoprotein attaches to host receptors and mediates viral fusion. Using a broad screening approach, we isolated from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) immune donors seven monoclonal antibodies (mAbs) that bind to all human-infecting coronavirus S proteins. This class of mAbs recognize the fusion peptide and acquire affinity and breadth through somatic mutations. Despite targeting a conserved motif, only some mAbs show broad neutralizing activity in vitro against alpha- and beta-coronaviruses, including animal coronavirus WIV-1 and PDF-2180. Two selected mAbs also neutralize Omicron BA.1 and BA.2 authentic viruses and reduce viral burden and pathology in vivo. Structural and functional analyses show that the fusion peptide-specific mAbs bind with different modalities to a cryptic epitope, which is hidden in prefusion stabilized S, and becomes exposed upon binding of angiotensin-converting enzyme 2 (ACE2) or ACE2-mimicking mAbs.ISSN:0036-8075ISSN:1095-920

SARS-CoV-2 spike conformation determines plasma neutralizing activity elicited by a wide panel of human vaccines

Numerous safe and effective coronavirus disease 2019 vaccines have been developed worldwide that use various delivery technologies and engineering strategies. We show here that vaccines containing prefusion-stabilizing S mutations elicit antibody responses in humans with enhanced recognition of S and the S1 subunit relative to postfusion S as compared with vaccines lacking these mutations or natural infection. Prefusion S and S1 antibody binding titers positively and equivalently correlated with neutralizing activity, and depletion of S1-directed antibodies completely abrogated plasma neutralizing activity. We show that neutralizing activity is almost entirely directed to the S1 subunit and that variant cross-neutralization is mediated solely by receptor binding domain–specific antibodies. Our data provide a quantitative framework for guiding future S engineering efforts to develop vaccines with higher resilience to the emergence of variants than current technologies.ISSN:2470-946

SARS-CoV-2 spike conformation determines plasma neutralizing activity elicited by a wide panel of human vaccines

Numerous safe and effective coronavirus disease 2019 vaccines have been developed worldwide that use various delivery technologies and engineering strategies. We show here that vaccines containing prefusion-stabilizing S mutations elicit antibody responses in humans with enhanced recognition of S and the S1 subunit relative to postfusion S as compared with vaccines lacking these mutations or natural infection. Prefusion S and S1 antibody binding titers positively and equivalently correlated with neutralizing activity, and depletion of S1-directed antibodies completely abrogated plasma neutralizing activity. We show that neutralizing activity is almost entirely directed to the S1 subunit and that variant cross-neutralization is mediated solely by receptor binding domain–specific antibodies. Our data provide a quantitative framework for guiding future S engineering efforts to develop vaccines with higher resilience to the emergence of variants than current technologies.Fil: Bowen, John E.. University of Washington; Estados UnidosFil: Park, Young Jun. University of Washington; Estados UnidosFil: Stewart, Cameron. University of Washington; Estados UnidosFil: Brown, Jack T.. University of Washington; Estados UnidosFil: Sharkey, William K.. University of Washington; Estados UnidosFil: Walls, Alexandra C.. University of Washington; Estados UnidosFil: Joshi, Anshu. University of Washington; Estados UnidosFil: Sprouse, Kaitlin R.. University of Washington; Estados UnidosFil: McCallum, Matthew. University of Washington; Estados UnidosFil: Tortorici, M. Alejandra. University of Washington; Estados UnidosFil: Franko, Nicholas M.. University of Washington; Estados UnidosFil: Logue, Jennifer K.. University of Washington; Estados UnidosFil: Mazzitelli, Ignacio Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas en Retrovirus y Sida. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Biomédicas en Retrovirus y Sida; ArgentinaFil: Nguyen, Annalee W.. University of Texas at Austin; Estados UnidosFil: Silva, Rui P.. University of Texas at Austin; Estados UnidosFil: Huang, Yimin. University of Texas at Austin; Estados UnidosFil: Low, Jun Siong. Università della Svizzera Italiana; SuizaFil: Jerak, Josipa. Università della Svizzera Italiana; SuizaFil: Tiles, Sasha W.. University of Washington. School of Medicine; Estados UnidosFil: Ahmed, Kumail. The Aga Khan University; PakistánFil: Shariq, Asefa. The Aga Khan University; PakistánFil: Dan, Jennifer M.. La Jolla Institute for Allergy and Immunology; Estados Unidos. University of California; Estados UnidosFil: Zhang, Zeli. La Jolla Institute for Allergy and Immunology; Estados Unidos. University of California; Estados UnidosFil: Weiskopf, Daniela. University of California; Estados Unidos. La Jolla Institute for Allergy and Immunology; Estados UnidosFil: Sette, Alessandro. University of California; Estados Unidos. La Jolla Institute for Allergy and Immunology; Estados UnidosFil: Snell, Gyorgy. Vir Biotechnology; Estados UnidosFil: Posavad, Christine M.. Fred Hutchinson Cancer Center; Estados UnidosFil: Iqbal, Najeeha Talat. The Aga Khan University; PakistánFil: Geffner, Jorge Raúl. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas en Retrovirus y Sida. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Biomédicas en Retrovirus y Sida; ArgentinaFil: Bandera, Alessandra. Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico; Itali