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1.
PLoS Pathog ; 19(11): e1011788, 2023 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-37943965

RESUMEN

The SARS-CoV-2 spike glycoprotein has 22 potential N-linked glycosylation sites per monomer that are highly conserved among diverse variants, but how individual glycans affect virus entry and neutralization of Omicron variants has not been extensively characterized. Here we compared the effects of specific glycan deletions or modifications in the Omicron BA.1 and D614G spikes on spike expression, processing, and incorporation into pseudoviruses, as well as on virus infectivity and neutralization by therapeutic antibodies. We found that loss of potential glycans at spike residues N717 and N801 each conferred a loss of pseudovirus infectivity for Omicron but not for D614G or Delta variants. This decrease in infectivity correlated with decreased spike processing and incorporation into Omicron pseudoviruses. Oligomannose-enriched Omicron pseudoviruses generated in GnTI- cells or in the presence of kifunensine were non-infectious, whereas D614G or Delta pseudoviruses generated under similar conditions remained infectious. Similarly, growth of live (authentic) SARS-CoV-2 in the presence of kifunensine resulted in a greater reduction of titers for the BA.1.1 variant than Delta or D614G variants relative to their respective, untreated controls. Finally, we found that loss of some N-glycans, including N343 and N234, increased the maximum percent neutralization by the class 3 S309 monoclonal antibody against D614G but not BA.1 variants, while these glycan deletions altered the neutralization potency of the class 1 COV2-2196 and Etesevimab monoclonal antibodies without affecting maximum percent neutralization. The maximum neutralization by some antibodies also varied with the glycan composition, with oligomannose-enriched pseudoviruses conferring the highest percent neutralization. These results highlight differences in the interactions between glycans and residues among SARS-CoV-2 variants that can affect spike expression, virus infectivity, and susceptibility of variants to antibody neutralization.


Asunto(s)
COVID-19 , Virosis , Humanos , SARS-CoV-2/genética , Anticuerpos Neutralizantes , Polisacáridos , Glicoproteína de la Espiga del Coronavirus/genética , Anticuerpos Antivirales
2.
PLoS Pathog ; 18(4): e1010468, 2022 04.
Artículo en Inglés | MEDLINE | ID: mdl-35385545

RESUMEN

An overreactive inflammatory response and coagulopathy are observed in patients with severe form of COVID-19. Since increased levels of D-dimer (DD) are associated with coagulopathy in COVID-19, we explored whether DD contributes to the aberrant cytokine responses. Here we show that treatment of healthy human monocytes with DD induced a dose dependent increase in production of pyrogenic mediator, Prostaglandin E2 (PGE2) and inflammatory cytokines, IL-6 and IL-8. The DD-induced PGE2 and inflammatory cytokines were enhanced significantly by co-treatment with immune complexes (IC) of SARS CoV-2 recombinant S protein or of pseudovirus containing SARS CoV-2 S protein (PVCoV-2) coated with spike-specific chimeric monoclonal antibody (MAb) containing mouse variable and human Fc regions. The production of PGE2 and cytokines in monocytes activated with DD and ICs was sensitive to the inhibitors of ß2 integrin and FcγRIIa, and to the inhibitors of calcium signaling, Mitogen-Activated Protein Kinase (MAPK) pathway, and tyrosine-protein kinase. Importantly, strong increase in PGE2 and in IL-6/IL-8/IL-1ß cytokines was observed in monocytes activated with DD in the presence of IC of PVCoV-2 coated with plasma from hospitalized COVID-19 patients but not from healthy donors. The IC of PVCoV-2 with convalescent plasma induced much lower levels of PGE2 and cytokines compared with plasma from hospitalized COVID-19 patients. PGE2 and IL-6/IL-8 cytokines produced in monocytes activated with plasma-containing IC, correlated well with the levels of spike binding antibodies and not with neutralizing antibody titers. Our study suggests that a combination of high levels of DD and high titers of spike-binding antibodies that can form IC with SARS CoV-2 viral particles might accelerate the inflammatory status of lung infiltrating monocytes leading to increased lung pathology in patients with severe form of COVID-19.


Asunto(s)
COVID-19 , Monocitos , Animales , Complejo Antígeno-Anticuerpo , COVID-19/terapia , Citocinas/metabolismo , Dinoprostona/metabolismo , Productos de Degradación de Fibrina-Fibrinógeno , Humanos , Inmunización Pasiva , Factores Inmunológicos/metabolismo , Interleucina-6/metabolismo , Interleucina-8/metabolismo , Ratones , Glicoproteína de la Espiga del Coronavirus/metabolismo , Sueroterapia para COVID-19
3.
J Infect Dis ; 228(4): 439-443, 2023 08 16.
Artículo en Inglés | MEDLINE | ID: mdl-37279924

RESUMEN

We compared neutralizing antibody responses to BA.4/5, BQ.1.1, XBB, and XBB.1.5 Omicron severe acute respiratory syndrome coronavirus 2 variants after a bivalent or ancestral coronavirus disease 2019 (COVID-19) messenger RNA booster vaccine or postvaccination infection. We found that the bivalent booster elicited moderately high antibody titers against BA.4/5 that were approximately 2-fold higher against all Omicron variants than titers elicited by the monovalent booster. The bivalent booster elicited low but similar titers against both XBB and XBB.1.5 variants. These findings inform risk assessments for future COVID-19 vaccine recommendations and suggest that updated COVID-19 vaccines containing matched vaccine antigens to circulating divergent variants may be needed.


Asunto(s)
Formación de Anticuerpos , COVID-19 , Humanos , COVID-19/prevención & control , Vacunas contra la COVID-19 , SARS-CoV-2/genética , Anticuerpos Neutralizantes , Anticuerpos Antivirales
4.
J Virol ; 96(1): e0111021, 2022 01 12.
Artículo en Inglés | MEDLINE | ID: mdl-34668774

RESUMEN

Mutations in the spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants can compromise the effectiveness of therapeutic antibodies. Most clinical-stage therapeutic antibodies target the spike receptor binding domain (RBD), but variants often have multiple mutations in several spike regions. To help predict antibody potency against emerging variants, we evaluated 25 clinical-stage therapeutic antibodies for neutralization activity against 60 pseudoviruses bearing spikes with single or multiple substitutions in several spike domains, including the full set of substitutions in B.1.1.7 (alpha), B.1.351 (beta), P.1 (gamma), B.1.429 (epsilon), B.1.526 (iota), A.23.1, and R.1 variants. We found that 14 of 15 single antibodies were vulnerable to at least one RBD substitution, but most combination and polyclonal therapeutic antibodies remained potent. Key substitutions in variants with multiple spike substitutions predicted resistance, but the degree of resistance could be modified in unpredictable ways by other spike substitutions that may reside outside the RBD. These findings highlight the importance of assessing antibody potency in the context of all substitutions in a variant and show that epistatic interactions in spike can modify virus susceptibility to therapeutic antibodies. IMPORTANCE Therapeutic antibodies are effective in preventing severe disease from SARS-CoV-2 infection (COVID-19), but their effectiveness may be reduced by virus variants with mutations affecting the spike protein. To help predict resistance to therapeutic antibodies in emerging variants, we profiled resistance patterns of 25 antibody products in late stages of clinical development against a large panel of variants that include single and multiple substitutions found in the spike protein. We found that the presence of a key substitution in variants with multiple spike substitutions can predict resistance against a variant but that other substitutions can affect the degree of resistance in unpredictable ways. These findings highlight complex interactions among substitutions in the spike protein affecting virus neutralization and, potentially, virus entry into cells.


Asunto(s)
Anticuerpos Monoclonales/inmunología , SARS-CoV-2/inmunología , Glicoproteína de la Espiga del Coronavirus/inmunología , Sustitución de Aminoácidos , Anticuerpos Neutralizantes/inmunología , Mutación , Unión Proteica , Dominios Proteicos , SARS-CoV-2/genética , Glicoproteína de la Espiga del Coronavirus/química , Glicoproteína de la Espiga del Coronavirus/genética
5.
J Virol ; 96(17): e0114022, 2022 09 14.
Artículo en Inglés | MEDLINE | ID: mdl-36000843

RESUMEN

The SARS-CoV-2 Omicron variants were first detected in November 2021, and several Omicron lineages (BA.1, BA.2, BA.3, BA.4, and BA.5) have since rapidly emerged. Studies characterizing the mechanisms of Omicron variant infection and sensitivity to neutralizing antibodies induced upon vaccination are ongoing by several groups. In the present study, we used pseudoviruses to show that the transmembrane serine protease 2 (TMPRSS2) enhances infection of BA.1, BA.1.1, BA.2, and BA.3 Omicron variants to a lesser extent than ancestral D614G. We further show that Omicron variants have higher sensitivity to inhibition by soluble angiotensin-converting enzyme 2 (ACE2) and the endosomal inhibitor chloroquine compared to D614G. The Omicron variants also more efficiently used ACE2 receptors from 9 out of 10 animal species tested, and unlike the D614G variant, used mouse ACE2 due to the Q493R and Q498R spike substitutions. Finally, neutralization of the Omicron variants by antibodies induced by three doses of Pfizer/BNT162b2 mRNA vaccine was 7- to 8-fold less potent than the D614G. These results provide insights into the transmissibility and immune evasion capacity of the emerging Omicron variants to curb their ongoing spread. IMPORTANCE The ongoing emergence of SARS-CoV-2 Omicron variants with an extensive number of spike mutations poses a significant public health and zoonotic concern due to enhanced transmission fitness and escape from neutralizing antibodies. We studied three Omicron lineage variants (BA.1, BA.2, and BA.3) and found that transmembrane serine protease 2 has less influence on Omicron entry into cells than on D614G, and Omicron exhibits greater sensitivity to endosomal entry inhibition compared to D614G. In addition, Omicron displays more efficient usage of diverse animal species ACE2 receptors than D614G. Furthermore, due to Q493R/Q498R substitutions in spike, Omicron, but not D614G, can use the mouse ACE2 receptor. Finally, three doses of Pfizer/BNT162b2 mRNA vaccination elicit high neutralization titers against Omicron variants, although the neutralization titers are still 7- to 8-fold lower those that against D614G. These results may give insights into the transmissibility and immune evasion capacity of the emerging Omicron variants to curb their ongoing spread.


Asunto(s)
Enzima Convertidora de Angiotensina 2 , Anticuerpos Neutralizantes , COVID-19 , Evasión Inmune , SARS-CoV-2 , Internalización del Virus , Enzima Convertidora de Angiotensina 2/química , Enzima Convertidora de Angiotensina 2/genética , Enzima Convertidora de Angiotensina 2/inmunología , Enzima Convertidora de Angiotensina 2/metabolismo , Animales , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , Vacuna BNT162/administración & dosificación , Vacuna BNT162/inmunología , COVID-19/inmunología , COVID-19/virología , Humanos , Evasión Inmune/inmunología , Ratones , SARS-CoV-2/química , SARS-CoV-2/genética , SARS-CoV-2/inmunología , SARS-CoV-2/metabolismo , Especificidad de la Especie , Glicoproteína de la Espiga del Coronavirus/química , Glicoproteína de la Espiga del Coronavirus/genética , Glicoproteína de la Espiga del Coronavirus/inmunología , Glicoproteína de la Espiga del Coronavirus/metabolismo
6.
Emerg Infect Dis ; 28(4): 828-832, 2022 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-35203111

RESUMEN

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibodies decay but persist 6 months postvaccination; lower levels of neutralizing titers persist against Delta than wild-type virus. Of 227 vaccinated healthcare workers tested, only 2 experienced outpatient symptomatic breakthrough infections, despite 59/227 exhibiting serologic evidence of SARS-CoV-2 infection, defined as presence of nucleocapsid protein antibodies.


Asunto(s)
COVID-19 , Anticuerpos Antivirales , Formación de Anticuerpos , COVID-19/epidemiología , COVID-19/prevención & control , Vacunas contra la COVID-19 , Personal de Salud , Humanos , SARS-CoV-2 , Vacunación
7.
Clin Infect Dis ; 73(11): e4312-e4320, 2021 12 06.
Artículo en Inglés | MEDLINE | ID: mdl-32898271

RESUMEN

BACKGROUND: Low vaccine effectiveness against A(H3N2) influenza in seasons with little antigenic drift has been attributed to substitutions in hemagglutinin (HA) acquired during vaccine virus propagation in eggs. Clinical trials comparing recombinant HA vaccine (rHA) and cell-derived inactivated influenza vaccine (IIV) to egg-derived IIVs provide opportunities to assess how egg-adaptive substitutions influence HA immunogenicity. METHODS: Neutralization titers in pre- and postimmunization sera from 133 adults immunized with 1 of 3 types of influenza vaccines in a randomized, open-label trial during the 2018-2019 influenza season were measured against egg- and cell-derived A/Singapore/INFIMH-16-0019/2016-like and circulating A(H3N2) influenza viruses using HA pseudoviruses. RESULTS: All vaccines elicited neutralizing antibodies to all H3 vaccine antigens, but the rHA vaccine elicited the highest titers and seroconversion rates against all strains tested. Egg- and cell-derived IIVs elicited responses similar to each other. Preimmunization titers against H3 HA pseudoviruses containing egg-adaptive substitutions T160K and L194P were high, but lower against H3 HA pseudoviruses without those substitutions. All vaccines boosted neutralization titers against HA pseudoviruses with egg-adaptive substitutions, but poorly neutralized wild-type 2019-2020 A/Kansas/14/2017 (H3N2) HA pseudoviruses. CONCLUSION: Egg- and cell-derived 2018-2019 season influenza vaccines elicited similar neutralization titers and response rates, indicating that the cell-derived vaccine did not improve immunogenicity against the A(H3N2) viruses. The higher responses after rHA vaccination may be due to its higher HA content. All vaccines boosted titers to HA with egg-adaptive substitutions, suggesting boosting from past antigens or better exposure of HA epitopes. Studies comparing immunogenicity and effectiveness of different influenza vaccines across many seasons are needed.


Asunto(s)
Vacunas contra la Influenza , Gripe Humana , Adulto , Anticuerpos Neutralizantes , Anticuerpos Antivirales , Glicoproteínas Hemaglutininas del Virus de la Influenza/genética , Hemaglutininas , Humanos , Subtipo H3N2 del Virus de la Influenza A , Estaciones del Año
8.
Clin Infect Dis ; 71(12): 3096-3102, 2020 12 15.
Artículo en Inglés | MEDLINE | ID: mdl-31840159

RESUMEN

BACKGROUND: Antibodies that inhibit hemagglutination have long been considered a correlate of protection against influenza, but these antibodies are only a subset of potentially protective antibodies. Neutralizing and neuraminidase antibodies may also contribute to protection, but data on their associations with protection are limited. METHODS: We measured preoutbreak hemagglutinin pseudovirus neutralization (PVN) and neuraminidase inhibition (NAI) antibody titers in unvaccinated military recruits who experienced an H3N2 influenza outbreak during training. We conducted a case-control study to investigate the association between titers and protection against influenza illness or H3N2-associated pneumonia using logistic regression. RESULTS: With every 2-fold increase in PVN titer, the odds of medically attended polymerase chain reaction-confirmed H3N2 infection (H3N2+) decreased by 41% (odds ratio [OR], 0.59; 95% confidence interval [CI], .45 to .77; P < .001). Among those who were H3N2+, the odds for pneumonia decreased by 52% (OR, 0.48; CI, .25 to .91; P = .0249). With every 2-fold increase in NAI titer, the odds of medically attended H3N2 infection decreased by 32% (OR, 0.68; 95% CI, .53 to .87; P = .0028), but there was no association between NAI titers and H3N2-associated pneumonia. There was also no synergistic effect of PVN and NAI antibodies. CONCLUSIONS: PVN and NAI titers were independently associated with reduced risk of influenza illness. NAI titers associated with protection had greater breadth of reactivity to drifted strains than PVN titers. These findings show that PVN and NAI titers are valuable biomarkers for assessing the odds of influenza infection.


Asunto(s)
Vacunas contra la Influenza , Gripe Humana , Personal Militar , Anticuerpos Neutralizantes , Anticuerpos Antivirales , Estudios de Casos y Controles , Brotes de Enfermedades , Pruebas de Inhibición de Hemaglutinación , Humanos , Subtipo H3N2 del Virus de la Influenza A , Gripe Humana/epidemiología , Gripe Humana/prevención & control , Neuraminidasa , Estaciones del Año
9.
J Virol ; 93(11)2019 06 01.
Artículo en Inglés | MEDLINE | ID: mdl-30894471

RESUMEN

Binding of the gp120 surface subunit of the envelope glycoprotein (Env) of HIV-1 to CD4 and chemokine receptors on target cells triggers refolding of the gp41 transmembrane subunit into a six-helix bundle (6HB) that promotes fusion between virus and host cell membranes. To elucidate details of Env entry and potential differences between viruses that use CXCR4 (X4) or CCR5 (R5) coreceptors, we generated viruses that are resistant to peptide fusion inhibitors corresponding to the first heptad repeat region (HR1) of gp41 that target fusion-intermediate conformations of Env. Previously we reported that an R5 virus selected two resistance pathways, each defined by an early gp41 resistance mutation in either HR1 or the second heptad repeat (HR2), to escape inhibition by an HR1 peptide, but preferentially selected the HR1 pathway to escape inhibition by a trimer-stabilized HR1 peptide. Here, we report that an X4 virus selected the same HR1 and HR2 resistance pathways as the R5 virus to escape inhibition by the HR1 peptide. However, in contrast to the R5 virus, the X4 virus selected a unique mutation in HR2 to escape inhibition by the trimer-stabilized peptide. Significantly, both of these X4 and R5 viruses acquired gp41 resistance mutations that improved the thermostability of the six-helix bundle, but they selected different gp120 adaptive mutations. These findings show that these X4 and R5 viruses use a similar resistance mechanism to escape from HR1 peptide inhibition but different gp120-gp41 interactions to regulate Env conformational changes.IMPORTANCE HIV-1 fuses with cells when the gp41 subunit of Env refolds into a 6HB after binding to cellular receptors. Peptides corresponding to HR1 or HR2 interrupt gp41 refolding and inhibit HIV infection. Previously, we found that a CCR5 coreceptor-tropic HIV-1 acquired a key HR1 or HR2 resistance mutation to escape HR1 peptide inhibitors but only the key HR1 mutation to escape a trimer-stabilized HR1 peptide inhibitor. Here, we report that a CXCR4 coreceptor-tropic HIV-1 selected the same key HR1 or HR2 mutations to escape inhibition by the HR1 peptide but different combinations of HR1 and HR2 mutations to escape the trimer-stabilized HR1 peptide. All gp41 mutations enhance 6HB stability to outcompete inhibitors, but gp120 adaptive mutations differed between these R5 and X4 viruses, providing new insights into gp120-gp41 functional interactions affecting Env refolding during HIV entry.


Asunto(s)
Proteína gp120 de Envoltorio del VIH/genética , Proteína gp41 de Envoltorio del VIH/metabolismo , VIH-1/genética , Antirretrovirales/farmacología , Farmacorresistencia Viral/efectos de los fármacos , Glicoproteínas/genética , Células HEK293 , Proteína gp120 de Envoltorio del VIH/metabolismo , Proteína gp41 de Envoltorio del VIH/fisiología , Inhibidores de Fusión de VIH/farmacología , Infecciones por VIH/genética , Seropositividad para VIH , VIH-1/fisiología , Humanos , Mutación , Conformación Proteica , Receptores CCR5/genética , Receptores CXCR4/genética
10.
Clin Infect Dis ; 68(12): 2067-2078, 2019 05 30.
Artículo en Inglés | MEDLINE | ID: mdl-30256912

RESUMEN

BACKGROUND: Prior influenza immunity influences the homologous neutralizing antibody responses elicited by inactivated influenza vaccines (IIV), but neutralizing antibody responses to heterologous strains have not been extensively characterized. METHODS: We analyzed neutralizing antibody titers in individuals aged 1-88 who received the 2009-2010 season IIV before infection by or vaccination against the 2009 pandemic H1N1 virus. Neutralization titers to homologous and heterologous past, recent, and advanced H1 and H3 strains, as well as H2, H5, and H7 strains, were measured using influenza hemagglutinin pseudoviruses. We performed exploratory analyses based on age, prior-year IIV, and prevaccination titer, without controlling for Type I errors. RESULTS: IIV elicited neutralizing antibodies to past and advanced H1 and H3 strains, as well as to an H2 strain in individuals who were likely infected early in life. The neutralization of avian subtype viruses was rare, and there was no imprinting of neutralization responses to novel avian subtype viruses based on the influenza group. Compared to adults, children had higher seroresponse rates to homologous and heterologous strains, and their sera generated larger antigenic distances among strains. Seroresponse rates to homologous and heterologous strains were lower in subjects vaccinated with prior-year IIV, though postimmunization titers were generally high. CONCLUSIONS: IIV elicited neutralizing antibodies to heterologous H1 and H3 strains in all ages groups, but titers and seroresponse rates were usually higher in children. Prior-year vaccination with the same strains tended to blunt IIV neutralization responses to all strains in young and old age groups, yet postimmunization titers were high.


Asunto(s)
Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , Virus de la Influenza A/inmunología , Vacunas contra la Influenza/inmunología , Gripe Humana/inmunología , Gripe Humana/prevención & control , Vacunación , Vacunas de Productos Inactivados/inmunología , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Niño , Preescolar , Femenino , Glicoproteínas Hemaglutininas del Virus de la Influenza/inmunología , Humanos , Virus de la Influenza A/clasificación , Virus de la Influenza A/genética , Vacunas contra la Influenza/administración & dosificación , Gripe Humana/virología , Masculino , Persona de Mediana Edad , Adulto Joven
11.
J Virol ; 92(16)2018 08 15.
Artículo en Inglés | MEDLINE | ID: mdl-29875245

RESUMEN

Entry of human immunodeficiency virus type 1 (HIV-1) into host cells is mediated by conformational changes in the envelope glycoprotein (Env) that are triggered by Env binding to cellular CD4 and chemokine receptors. These conformational changes involve the opening of the gp120 surface subunit, exposure of the fusion peptide in the gp41 transmembrane subunit, and refolding of the gp41 N- and C-terminal heptad repeat regions (HR1 and HR2) first into an extended prehairpin intermediate and then into a compact 6-helix bundle (6HB) that facilitates fusion between viral and host cell membranes. Previously, we reported that Envs resistant to HR1 peptide fusion inhibitors acquired key resistance mutations in either HR1 or HR2 that increased 6HB stability. Here, we identify residues in HR1 that contribute not only to fusion inhibitor resistance and 6HB stability but also to reduced reactivity to CD4-induced conformational changes that lead to 6HB formation. While all Envs show increased neutralization sensitivity to mimetic CD4 (mCD4), Envs with either the E560K or Q577R HR1 mutation reduced conformational reactivity to CD4 that resisted viral inactivation and triggering to the 6HB. Using a panel of monoclonal antibodies (mAbs), we further determined that Envs from both HR1 and HR2 resistance pathways exhibit a relaxed trimer conformation due to gp120 adaptive mutations in different regions of Env that segregate by resistance pathway. These findings highlight regions of cross talk between gp120 and gp41 and identify HR1 residues that play important roles in regulating CD4-induced conformational changes in Env.IMPORTANCE Binding of the HIV envelope glycoprotein (Env) to cellular CD4 and chemokine receptors triggers conformational changes in Env that mediate virus entry, but premature triggering of Env conformational changes leads to virus inactivation. Currently, we have a limited understanding of the network of residues that regulate Env conformational changes. Here, we identify residues in HR1 of gp41 that modulate conformational changes in response to gp120 binding to CD4 and show that the mutations in HR1 and HR2 that confer resistance to fusion inhibitors are associated with gp120 mutations in different regions of Env that confer a more open conformation. These findings contribute to our understanding of the regulation of Env conformational changes and efforts to design new entry inhibitors and stable Env vaccine immunogens.


Asunto(s)
Linfocitos T CD4-Positivos/metabolismo , Proteína gp120 de Envoltorio del VIH/metabolismo , Proteína gp41 de Envoltorio del VIH/metabolismo , VIH-1/fisiología , Internalización del Virus , Línea Celular , Proteína gp120 de Envoltorio del VIH/química , Proteína gp41 de Envoltorio del VIH/química , Proteína gp41 de Envoltorio del VIH/genética , VIH-1/genética , Humanos , Unión Proteica , Conformación Proteica
12.
J Virol ; 92(12)2018 06 15.
Artículo en Inglés | MEDLINE | ID: mdl-29593038

RESUMEN

Vaccines that elicit broadly neutralizing antibodies to the conserved stem of hemagglutinin (HA) are being developed as universal influenza vaccines that protect against influenza across multiple years. However, different influenza virus strains, even those in the same subtype with identical stem sequences, can vary in susceptibility to broadly neutralizing stem antibodies, and the reasons are not understood. Here we studied potential mechanisms underlying the differing sensitivities of a panel of H5N1 HA pseudoviruses to broadly neutralizing stem antibodies. We found that greater HA conformational stability, as measured by thermal inactivation and pH triggering of conformational changes, correlates with reduced neutralization sensitivity and antibody binding to HA under neutral- and low-pH conditions. Our data indicate that the conformational stability of HA is an important attribute of susceptibility to broadly neutralizing stem antibodies and is influenced by residues outside the stem antibody epitopes.IMPORTANCE The influenza virus surface glycoprotein hemagglutinin (HA) mediates virus attachment and membrane fusion between virus and host cells, allowing the viral core to enter the host cell cytoplasm for replication. Fusion occurs when HA undergoes low-pH-induced-conformational changes during endocytosis. Broadly neutralizing antibodies targeted to the conserved stem region of HA interfere with conformational changes required for fusion. Vaccines that elicit such antibodies are being developed as novel universal influenza vaccines for multiyear protection. We investigated why H5N1 HAs from different strains differ in their sensitivity to broadly neutralizing stem antibodies despite having conserved epitopes. We report that HA conformational stability due to residues outside the antibody binding site accounted for much of the variation in susceptibility to neutralization by stem antibodies. These findings highlight the importance of nonepitope residues in influencing neutralization sensitivity to stem antibodies and the complexities in developing universal vaccines targeting conserved epitopes in the HA stem.


Asunto(s)
Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , Epítopos/inmunología , Glicoproteínas Hemaglutininas del Virus de la Influenza/inmunología , Subtipo H5N1 del Virus de la Influenza A/inmunología , Vacunas contra la Influenza/inmunología , Gripe Humana/prevención & control , Células HEK293 , Glicoproteínas Hemaglutininas del Virus de la Influenza/metabolismo , Humanos , Gripe Humana/inmunología , Gripe Humana/virología , Conformación Proteica , Acoplamiento Viral
13.
J Infect Dis ; 213(3): 403-6, 2016 Feb 01.
Artículo en Inglés | MEDLINE | ID: mdl-26243315

RESUMEN

We examined serum samples from adults ages 48-64 who received multiple seasonal influenza vaccines from 2004 to 2009 for cross-neutralizing antibodies to potential pandemic strains. Using pseudoviruses bearing various hemagglutinins (HA-pseudoviruses), we found serum neutralization titers (≥160) in 100% against A/Japan/305/1957 (H2N2), 53% against A/Hong Kong/1073/99 (H9N2), 56% against the H3N2 variant A/Indiana/08/11 (H3N2v), 11% against A/Hong Kong/G9/97 (H9N2), and 36% A/chicken/Hong Kong/SF4/01 (H6N1). None had titers >160 to A/Shanghai/2/13 (H7N9) or A/Netherlands/219/03 (H7N7). Thirty-six percent to 0% had neutralization titers to various H5N1 strains. Titers to H9, H6, and H5 HA-pseudoviruses correlated with each other, but not with H3N2v, suggesting group-specific cross-neutralization.


Asunto(s)
Anticuerpos Antivirales/sangre , Virus de la Influenza A/inmunología , Vacunas contra la Influenza/inmunología , Gripe Humana/prevención & control , Gripe Humana/virología , Humanos , Vacunas contra la Influenza/administración & dosificación , Gripe Humana/sangre , Persona de Mediana Edad , Pandemias
14.
J Virol ; 89(20): 10602-11, 2015 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-26269180

RESUMEN

UNLABELLED: Influenza virus hemagglutinin (HA) mediates virus entry by binding to cell surface receptors and fusing the viral and endosomal membranes following uptake by endocytosis. The acidic environment of endosomes triggers a large-scale conformational change in the transmembrane subunit of HA (HA2) involving a loop (B loop)-to-helix transition, which releases the fusion peptide at the HA2 N terminus from an interior pocket within the HA trimer. Subsequent insertion of the fusion peptide into the endosomal membrane initiates fusion. The acid stability of HA is influenced by residues in the fusion peptide, fusion peptide pocket, coiled-coil regions of HA2, and interactions between the surface (HA1) and HA2 subunits, but details are not fully understood and vary among strains. Current evidence suggests that the HA from the circulating pandemic 2009 H1N1 influenza A virus [A(H1N1)pdm09] is less stable than the HAs from other seasonal influenza virus strains. Here we show that residue 205 in HA1 and residue 399 in the B loop of HA2 (residue 72, HA2 numbering) in different monomers of the trimeric A(H1N1)pdm09 HA are involved in functionally important intermolecular interactions and that a conserved histidine in this pair helps regulate HA stability. An arginine-lysine pair at this location destabilizes HA at acidic pH and mediates fusion at a higher pH, while a glutamate-lysine pair enhances HA stability and requires a lower pH to induce fusion. Our findings identify key residues in HA1 and HA2 that interact to help regulate H1N1 HA stability and virus infectivity. IMPORTANCE: Influenza virus hemagglutinin (HA) is the principal antigen in inactivated influenza vaccines and the target of protective antibodies. However, the influenza A virus HA is highly variable, necessitating frequent vaccine changes to match circulating strains. Sequence changes in HA affect not only antigenicity but also HA stability, which has important implications for vaccine production, as well as viral adaptation to hosts. HA from the pandemic 2009 H1N1 influenza A virus is less stable than other recent seasonal influenza virus HAs, but the molecular interactions that contribute to HA stability are not fully understood. Here we identify molecular interactions between specific residues in the surface and transmembrane subunits of HA that help regulate the HA conformational changes needed for HA stability and virus entry. These findings contribute to our understanding of the molecular mechanisms controlling HA function and antigen stability.


Asunto(s)
Glicoproteínas Hemaglutininas del Virus de la Influenza/química , Subtipo H1N1 del Virus de la Influenza A/química , Subtipo H1N1 del Virus de la Influenza A/patogenicidad , Subunidades de Proteína/química , Internalización del Virus , Secuencia de Aminoácidos , Animales , Perros , Eritrocitos/virología , Células HEK293 , Pruebas de Hemaglutinación , Glicoproteínas Hemaglutininas del Virus de la Influenza/metabolismo , Caballos , Humanos , Subtipo H1N1 del Virus de la Influenza A/fisiología , Células de Riñón Canino Madin Darby , Modelos Moleculares , Datos de Secuencia Molecular , Dominios y Motivos de Interacción de Proteínas , Estabilidad Proteica , Estructura Secundaria de Proteína , Subunidades de Proteína/metabolismo , Alineación de Secuencia , Ovinos , Pavos
15.
J Virol ; 89(4): 1975-85, 2015 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-25473053

RESUMEN

UNLABELLED: The influenza virus hemagglutinin (HA) envelope protein mediates virus entry by first binding to cell surface receptors and then fusing viral and endosomal membranes during endocytosis. Cleavage of the HA precursor (HA0) into a surface receptor-binding subunit (HA1) and a fusion-inducing transmembrane subunit (HA2) by host cell enzymes primes HA for fusion competence by repositioning the fusion peptide to the newly created N terminus of HA2. We previously reported that the influenza virus M2 protein enhances pandemic 2009 influenza A virus [(H1N1)pdm09] HA-pseudovirus infectivity, but the mechanism was unclear. In this study, using cell-cell fusion and HA-pseudovirus infectivity assays, we found that the ion channel function of M2 was required for enhancement of HA fusion and HA-pseudovirus infectivity. The M2 activity was needed only during HA biosynthesis, and proteolysis experiments indicated that M2 proton channel activity helped to protect (H1N1)pdm09 HA from premature conformational changes as it traversed low-pH compartments during transport to the cell surface. While M2 has previously been shown to protect avian influenza virus HA proteins of the H5 and H7 subtypes that have polybasic cleavage motifs, this study demonstrates that M2 can protect HA proteins from human H1N1 strains that lack a polybasic cleavage motif. This finding suggests that M2 proton channel activity may play a wider role in preserving HA fusion competence among a variety of HA subtypes, including HA proteins from emerging strains that may have reduced HA stability. IMPORTANCE: Influenza virus infects cells when the hemagglutinin (HA) surface protein undergoes irreversible pH-induced conformational changes after the virus is taken into the cell by endocytosis. HA fusion competence is primed when host cell enzymes cleave the HA precursor. The proton channel function of influenza virus M2 protein has previously been shown to protect avian influenza virus HA proteins that contain a polybasic cleavage site from pH-induced conformational changes during biosynthesis, but this effect is less well understood for human influenza virus HA proteins that lack polybasic cleavage sites. Using assays that focus on HA entry and fusion, we found that the M2 protein also protects (H1N1)pdm09 influenza A virus HA from premature conformational changes as it transits low-pH compartments during biosynthesis. This work suggests that M2 may play a wider role in preserving HA function in a variety of influenza virus subtypes that infect humans and may be especially important for HA proteins that are less stable.


Asunto(s)
Glicoproteínas Hemaglutininas del Virus de la Influenza/metabolismo , Subtipo H1N1 del Virus de la Influenza A/fisiología , Proteínas de la Matriz Viral/metabolismo , Acoplamiento Viral , Internalización del Virus , Línea Celular , Humanos , Transporte de Proteínas
16.
Retrovirology ; 11: 86, 2014 Oct 02.
Artículo en Inglés | MEDLINE | ID: mdl-25274545

RESUMEN

BACKGROUND: The HIV-1 envelope glycoprotein (Env) undergoes conformational changes that mediate fusion between virus and host cell membranes. These changes involve transient exposure of two heptad-repeat domains (HR1 and HR2) in the gp41 subunit and their subsequent self-assembly into a six-helix bundle (6HB) that drives fusion. Env residues and features that influence conformational changes and the rate of virus entry, however, are poorly understood. Peptides corresponding to HR1 and HR2 (N and C peptides, respectively) interrupt formation of the 6HB by binding to the heptad repeats of a fusion-intermediate conformation of Env, making the peptides valuable probes for studying Env conformational changes. RESULTS: Using a panel of Envs that are resistant to N-peptide fusion inhibitors, we investigated relationships between virus entry kinetics, 6HB stability, and resistance to peptide fusion inhibitors to elucidate how HR1 and HR2 mutations affect Env conformational changes and virus entry. We found that gp41 resistance mutations increased 6HB stability without increasing entry kinetics. Similarly, we show that increased 6HB thermodynamic stability does not correlate with increased entry kinetics. Thus, N-peptide fusion inhibitors do not necessarily select for Envs with faster entry kinetics, nor does faster entry kinetics predict decreased potency of peptide fusion inhibitors. CONCLUSIONS: These findings provide new insights into the relationship between 6HB stability and viral entry kinetics and mechanisms of resistance to inhibitors targeting fusion-intermediate conformations of Env. These studies further highlight how residues in HR1 and HR2 can influence virus entry by altering stability of the 6HB and possibly other conformations of Env that affect rate-limiting steps in HIV entry.


Asunto(s)
Farmacorresistencia Viral , Proteína gp41 de Envoltorio del VIH/química , Inhibidores de Fusión de VIH/metabolismo , VIH-1/efectos de los fármacos , Complejos Multiproteicos/química , Multimerización de Proteína , Internalización del Virus/efectos de los fármacos , Línea Celular , Proteína gp41 de Envoltorio del VIH/genética , Proteína gp41 de Envoltorio del VIH/metabolismo , VIH-1/genética , VIH-1/fisiología , Humanos , Cinética , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Conformación Proteica , Estabilidad Proteica , Termodinámica
17.
bioRxiv ; 2024 Apr 06.
Artículo en Inglés | MEDLINE | ID: mdl-38712124

RESUMEN

Antigenic assessments of SARS-CoV-2 variants inform decisions to update COVID-19 vaccines. Primary infection sera are often used for assessments, but such sera are rare due to population immunity from SARS-CoV-2 infections and COVID-19 vaccinations. Here, we show that neutralization titers and breadth of matched human and hamster pre-Omicron variant primary infection sera correlate well and generate similar antigenic maps. The hamster antigenic map shows modest antigenic drift among XBB sub-lineage variants, with JN.1 and BA.4/BA.5 variants within the XBB cluster, but with five to six-fold antigenic differences between these variants and XBB.1.5. Compared to sera following only ancestral or bivalent COVID-19 vaccinations, or with post-vaccination infections, XBB.1.5 booster sera had the broadest neutralization against XBB sub-lineage variants, although a five-fold titer difference was still observed between JN.1 and XBB.1.5 variants. These findings suggest that antibody coverage of antigenically divergent JN.1 could be improved with a matched vaccine antigen.

18.
Front Immunol ; 15: 1287504, 2024.
Artículo en Inglés | MEDLINE | ID: mdl-38566991

RESUMEN

Introduction: We sought to determine pre-infection correlates of protection against SARS-CoV-2 post-vaccine inzfections (PVI) acquired during the first Omicron wave in the United States. Methods: Serum and saliva samples from 176 vaccinated adults were collected from October to December of 2021, immediately before the Omicron wave, and assessed for SARS-CoV-2 Spike-specific IgG and IgA binding antibodies (bAb). Sera were also assessed for bAb using commercial assays, and for neutralization activity against several SARS-CoV-2 variants. PVI duration and severity, as well as risk and precautionary behaviors, were assessed by questionnaires. Results: Serum anti-Spike IgG levels assessed by research assay, neutralization titers against Omicron subvariants, and low home risk scores correlated with protection against PVIs after multivariable regression analysis. Commercial assays did not perform as well as research assay, likely due to their lower dynamic range. Discussion: In the 32 participants that developed PVI, anti-Spike IgG bAbs correlated with lower disease severity and shorter duration of illness.


Asunto(s)
COVID-19 , Adulto , Humanos , COVID-19/prevención & control , SARS-CoV-2 , Vacunas contra la COVID-19 , Anticuerpos Antivirales , Inmunoglobulina G
19.
Sci Transl Med ; 16(747): eadl1722, 2024 May 15.
Artículo en Inglés | MEDLINE | ID: mdl-38748773

RESUMEN

The evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) requires ongoing monitoring to judge the ability of newly arising variants to escape the immune response. A surveillance system necessitates an understanding of differences in neutralization titers measured in different assays and using human and animal serum samples. We compared 18 datasets generated using human, hamster, and mouse serum and six different neutralization assays. Datasets using animal model serum samples showed higher titer magnitudes than datasets using human serum samples in this comparison. Fold change in neutralization of variants compared to ancestral SARS-CoV-2, immunodominance patterns, and antigenic maps were similar among serum samples and assays. Most assays yielded consistent results, except for differences in fold change in cytopathic effect assays. Hamster serum samples were a consistent surrogate for human first-infection serum samples. These results inform the transition of surveillance of SARS-CoV-2 antigenic variation from dependence on human first-infection serum samples to the utilization of serum samples from animal models.


Asunto(s)
Anticuerpos Neutralizantes , Anticuerpos Antivirales , COVID-19 , Pruebas de Neutralización , SARS-CoV-2 , Animales , Humanos , SARS-CoV-2/inmunología , COVID-19/inmunología , COVID-19/sangre , COVID-19/virología , Ratones , Anticuerpos Neutralizantes/sangre , Anticuerpos Neutralizantes/inmunología , Cricetinae , Anticuerpos Antivirales/sangre , Anticuerpos Antivirales/inmunología , Modelos Animales de Enfermedad
20.
J Biol Chem ; 287(11): 8297-309, 2012 Mar 09.
Artículo en Inglés | MEDLINE | ID: mdl-22235115

RESUMEN

Peptides corresponding to N- and C-terminal heptad repeat regions (HR1 and HR2, respectively) of viral fusion proteins can block infection of viruses in a dominant negative manner by interfering with refolding of the viral HR1 and HR2 to form a six-helix bundle (6HB) that drives fusion between viral and host cell membranes. The 6HB of the HIV gp41 (endogenous bundle) consists of an HR1 coiled-coil trimer with grooves lined by antiparallel HR2 helices. HR1 peptides form coiled-coil oligomers that may bind to gp41 HR2 as trimers to form a heterologous 6HB (inhibitor bundle) or to gp41 HR1 as monomers or dimers to form a heterologous coiled coil. To gain insights into mechanisms of Env entry and inhibition by HR1 peptides, we compared resistance to a peptide corresponding to 36 residues in gp41 HR1 (N36) and the same peptide with a coiled-coil trimerization domain fused to its N terminus (IZN36) that stabilizes the trimer and increases inhibitor potency (Eckert, D. M., and Kim, P. S. (2001) Proc. Nat. Acad. Sci. U.S.A. 98, 11187-11192). Whereas N36 selected two genetic pathways with equal probability, each defined by an early mutation in either HR1 or HR2, IZN36 preferentially selected the HR1 pathway. Both pathways conferred cross-resistance to both peptides. Each HR mutation enhanced the thermostability of the endogenous 6HB, potentially allowing the virus to simultaneously escape inhibitors targeting either gp41 HR1 or HR2. These findings inform inhibitor design and identify regions of plasticity in the highly conserved gp41 that modulate virus entry and escape from HR1 peptide inhibitors.


Asunto(s)
Diseño de Fármacos , Farmacorresistencia Viral/fisiología , Proteína gp41 de Envoltorio del VIH/antagonistas & inhibidores , Inhibidores de Fusión de VIH/química , VIH-1/metabolismo , Péptidos/química , Internalización del Virus , Línea Celular , Farmacorresistencia Viral/efectos de los fármacos , Proteína gp41 de Envoltorio del VIH/genética , Proteína gp41 de Envoltorio del VIH/metabolismo , Inhibidores de Fusión de VIH/farmacología , VIH-1/genética , Humanos , Mutación , Péptidos/genética , Péptidos/farmacología , Estructura Secundaria de Proteína
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