RESUMEN

Despite the decrease in mortality and morbidity due to SARS-CoV-2 infection, the incidence of infections due to Omicron subvariants of SARS-CoV-2 remains high. The mutations acquired by these subvariants, mainly concentrated in the receptor-binding domain (RBD), have caused a shift in infectivity and transmissibility, leading to a loss of effectiveness of the first authorized COVID-19 vaccines, among other reasons, by neutralizing antibody evasion. Hence, the generation of new vaccine candidates adapted to Omicron subvariants is of special interest in an effort to overcome this immune evasion. Here, an optimized COVID-19 vaccine candidate, termed MVA-S(3P_BA.1), was developed using a modified vaccinia virus Ankara (MVA) vector expressing a full-length prefusion-stabilized SARS-CoV-2 spike (S) protein from the Omicron BA.1 variant. The immunogenicity and efficacy induced by MVA-S(3P_BA.1) were evaluated in mice in a head-to-head comparison with the previously generated vaccine candidates MVA-S(3P) and MVA-S(3Pbeta), which express prefusion-stabilized S proteins from Wuhan strain and Beta variant, respectively, and with a bivalent vaccine candidate composed of a combination of MVA-S(3P) and MVA-S(3P_BA.1). The results showed that all four vaccine candidates elicited, after a single intramuscular dose, protection of transgenic K18-hACE2 mice challenged with SARS-CoV-2 Omicron BA.1, reducing viral loads, histopathological lesions, and levels of proinflammatory cytokines in the lungs. They also elicited anti-S IgG and neutralizing antibodies against various Omicron subvariants, with MVA-S(3P_BA.1) and the bivalent vaccine candidate inducing higher titers. Additionally, an intranasal immunization in C57BL/6 mice with all four vaccine candidates induced systemic and mucosal S-specific CD4+ and CD8+ T-cell and humoral immune responses, and the bivalent vaccine candidate induced broader immune responses, eliciting antibodies against the ancestral Wuhan strain and different Omicron subvariants. These results highlight the use of MVA as a potent and adaptable vaccine vector against new emerging SARS-CoV-2 variants, as well as the promising feature of combining multivalent MVA vaccine candidates.

Asunto(s)

Anticuerpos Antivirales , Vacunas contra la COVID-19 , COVID-19 , Inmunidad Celular , Inmunidad Humoral , Ratones Transgénicos , SARS-CoV-2 , Glicoproteína de la Espiga del Coronavirus , Animales , Glicoproteína de la Espiga del Coronavirus/inmunología , Glicoproteína de la Espiga del Coronavirus/genética , SARS-CoV-2/inmunología , Vacunas contra la COVID-19/inmunología , COVID-19/prevención & control , COVID-19/inmunología , Ratones , Anticuerpos Antivirales/inmunología , Anticuerpos Antivirales/sangre , Humanos , Enzima Convertidora de Angiotensina 2/inmunología , Enzima Convertidora de Angiotensina 2/genética , Enzima Convertidora de Angiotensina 2/metabolismo , Anticuerpos Neutralizantes/inmunología , Anticuerpos Neutralizantes/sangre , Femenino , Vacunas de ADN/inmunología , Virus Vaccinia/inmunología , Virus Vaccinia/genética , Inmunogenicidad Vacunal

RESUMEN

The outbreak of coronavirus disease 19 (COVID-19) has highlighted the demand for vaccines that are safe and effective in inducing systemic and airway mucosal immunity against the aerosol transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In this study, we developed a novel helper-dependent adenoviral vector-based COVID-19 mucosal vaccine encoding a full-length SARS-CoV-2 spike protein (HD-Ad-FS). Through intranasal immunization (single-dose and prime-boost regimens), we demonstrated that the HD-Ad-FS was immunogenic and elicited potent systemic and airway mucosal protection in BALB/c mice, transgenic ACE2 (hACE2) mice, and hamsters. We detected high titers of neutralizing antibodies (NAbs) in sera and bronchoalveolar lavages (BALs) in the vaccinated animals. High levels of spike-specific secretory IgA (sIgA) and IgG were induced in the airway of the vaccinated animals. The single-dose HD-Ad-FS elicited a strong immune response and protected animals from SARS-CoV-2 infection. In addition, the prime-boost vaccination induced cross-reactive serum NAbs against variants of concern (VOCs; Beta, Delta, and Omicron). After challenge, VOC infectious viral particles were at undetectable or minimal levels in the lower airway. Our findings highlight the potential of airway delivery of HD-Ad-FS as a safe and effective vaccine platform for generating mucosal protection against SARS-CoV-2 and its VOCs.

Asunto(s)

Administración Intranasal , Anticuerpos Neutralizantes , Anticuerpos Antivirales , Vacunas contra la COVID-19 , COVID-19 , Inmunidad Mucosa , Ratones Endogámicos BALB C , SARS-CoV-2 , Glicoproteína de la Espiga del Coronavirus , Animales , SARS-CoV-2/inmunología , Vacunas contra la COVID-19/inmunología , Vacunas contra la COVID-19/administración & dosificación , Glicoproteína de la Espiga del Coronavirus/inmunología , Glicoproteína de la Espiga del Coronavirus/genética , COVID-19/prevención & control , COVID-19/inmunología , Ratones , Anticuerpos Antivirales/sangre , Anticuerpos Antivirales/inmunología , Anticuerpos Neutralizantes/inmunología , Anticuerpos Neutralizantes/sangre , Cricetinae , Femenino , Humanos , Ratones Transgénicos , Adenoviridae/genética , Adenoviridae/inmunología , Enzima Convertidora de Angiotensina 2/inmunología , Enzima Convertidora de Angiotensina 2/genética , Enzima Convertidora de Angiotensina 2/metabolismo , Mesocricetus

RESUMEN

The Omicron subvariants of SARS-CoV-2, especially for BA.2.86 and JN.1, have rapidly spread across multiple countries, posing a significant threat in the ongoing COVID-19 pandemic. Distinguished by 34 additional mutations on the Spike (S) protein compared to its BA.2 predecessor, the implications of BA.2.86 and its evolved descendant, JN.1 with additional L455S mutation in receptor-binding domains (RBDs), are of paramount concern. In this work, we systematically examine the neutralization susceptibilities of SARS-CoV-2 Omicron subvariants and reveal the enhanced antibody evasion of BA.2.86 and JN.1. We also determine the cryo-EM structures of the trimeric S proteins from BA.2.86 and JN.1 in complex with the host receptor ACE2, respectively. The mutations within the RBDs of BA.2.86 and JN.1 induce a remodeling of the interaction network between the RBD and ACE2. The L455S mutation of JN.1 further induces a notable shift of the RBD-ACE2 interface, suggesting the notably reduced binding affinity of JN.1 than BA.2.86. An analysis of the broadly neutralizing antibodies possessing core neutralizing epitopes reveals the antibody evasion mechanism underlying the evolution of Omicron BA.2.86 subvariant. In general, we construct a landscape of evolution in virus-receptor of the circulating Omicron subvariants.

Asunto(s)

Enzima Convertidora de Angiotensina 2 , Anticuerpos Neutralizantes , Anticuerpos Antivirales , COVID-19 , Microscopía por Crioelectrón , Evasión Inmune , Mutación , SARS-CoV-2 , Glicoproteína de la Espiga del Coronavirus , SARS-CoV-2/inmunología , SARS-CoV-2/genética , Glicoproteína de la Espiga del Coronavirus/inmunología , Glicoproteína de la Espiga del Coronavirus/química , Glicoproteína de la Espiga del Coronavirus/genética , Glicoproteína de la Espiga del Coronavirus/metabolismo , Humanos , COVID-19/inmunología , COVID-19/virología , Enzima Convertidora de Angiotensina 2/metabolismo , Enzima Convertidora de Angiotensina 2/química , Enzima Convertidora de Angiotensina 2/inmunología , Enzima Convertidora de Angiotensina 2/genética , Anticuerpos Neutralizantes/inmunología , Anticuerpos Neutralizantes/química , Anticuerpos Antivirales/inmunología , Anticuerpos Antivirales/química , Evolución Molecular , Unión Proteica , Modelos Moleculares

RESUMEN

The recently dominant SARS-CoV-2 Omicron JN.1 has evolved into multiple sublineages, with recurrent spike mutations R346T, F456L, and T572I, some of which exhibit growth advantages, such as KP.2 and KP.3. We investigated these mutations in JN.1, examining their individual and combined effects on immune evasion, ACE2 receptor affinity, and in vitro infectivity. F456L increased resistance to neutralization by human sera, including those after JN.1 breakthrough infections, and by RBD class-1 monoclonal antibodies, significantly altering JN.1 antigenicity. R346T enhanced ACE2-binding affinity and modestly boosted the infectivity of JN.1 pseudovirus, without a discernible effect on serum neutralization, while T572I slightly bolstered evasion of SD1-directed mAbs against JN.1's ancestor, BA.2, possibly by altering SD1 conformation. Importantly, expanding sublineages such as KP.2 containing R346T, F456L, and V1104L, showed similar neutralization resistance as JN.1 with R346T and F456L, suggesting V1104L does not appreciably affect antibody evasion. Furthermore, the hallmark mutation Q493E in KP.3 significantly reduced ACE2-binding affinity and viral infectivity, without noticeably impacting serum neutralization. Our findings illustrate how certain JN.1 mutations confer growth advantages in the population and could inform the design of the next COVID-19 vaccine booster.

Asunto(s)

COVID-19 , Evasión Inmune , Mutación , SARS-CoV-2 , Glicoproteína de la Espiga del Coronavirus , Glicoproteína de la Espiga del Coronavirus/genética , Glicoproteína de la Espiga del Coronavirus/inmunología , Glicoproteína de la Espiga del Coronavirus/química , Humanos , SARS-CoV-2/genética , SARS-CoV-2/inmunología , COVID-19/virología , COVID-19/inmunología , Anticuerpos Neutralizantes/inmunología , Enzima Convertidora de Angiotensina 2/metabolismo , Enzima Convertidora de Angiotensina 2/genética , Enzima Convertidora de Angiotensina 2/inmunología , Anticuerpos Antivirales/inmunología , Anticuerpos Monoclonales/inmunología

RESUMEN

COVID-19 has caused millions of deaths and many times more infections worldwide, emphasizing the unpreparedness of the global health system in the face of new infections and the key role for vaccines and therapeutics, including virus-neutralizing antibodies, in prevention and containment of the disease. Continuous evolution of the SARS-CoV-2 coronavirus has been causing its new variants to evade the action of the immune system, which highlighted the importance of detailed knowledge of the epitopes of already selected potent virus-neutralizing antibodies. A single-chain antibody ("nanobody") targeting the SARS-CoV-2 receptor-binding domain (RBD), clone P2C5, had exhibited robust virus-neutralizing activity against all SARS-CoV-2 variants and, being a major component of the anti-COVID-19 formulation "GamCoviMab", had successfully passed Phase I of clinical trials. However, after the emergence of the Delta and XBB variants, a decrease in the neutralizing activity of this nanobody was observed. Here we report on the successful crystal structure determination of the RBD:P2C5 complex at 3.1 Å, which revealed the intricate protein-protein interface, sterically occluding full ACE2 receptor binding by the P2C5-neutralized RBD. Moreover, the structure revealed the developed RBD:P2C5 interface centered around residues Leu452 and Phe490, thereby explaining the evasion of the Delta or Omicron XBB, but not Omicron B.1.1.529 variant, as a result of the single L452R or F490S mutations, respectively, from the action of P2C5. The structure obtained is expected to foster nanobody engineering in order to rescue neutralization activity and will facilitate epitope mapping for other neutralizing nanobodies by competition assays.

Asunto(s)

Anticuerpos Neutralizantes , SARS-CoV-2 , Anticuerpos de Dominio Único , Glicoproteína de la Espiga del Coronavirus , SARS-CoV-2/inmunología , SARS-CoV-2/efectos de los fármacos , Anticuerpos de Dominio Único/inmunología , Anticuerpos de Dominio Único/química , Anticuerpos de Dominio Único/farmacología , Anticuerpos Neutralizantes/inmunología , Anticuerpos Neutralizantes/química , Humanos , Glicoproteína de la Espiga del Coronavirus/inmunología , Glicoproteína de la Espiga del Coronavirus/química , Glicoproteína de la Espiga del Coronavirus/genética , Glicoproteína de la Espiga del Coronavirus/metabolismo , Anticuerpos Antivirales/inmunología , COVID-19/inmunología , COVID-19/virología , Dominios Proteicos , Unión Proteica , Epítopos/inmunología , Epítopos/química , Modelos Moleculares , Evasión Inmune , Enzima Convertidora de Angiotensina 2/metabolismo , Enzima Convertidora de Angiotensina 2/química , Enzima Convertidora de Angiotensina 2/inmunología , Sitios de Unión

RESUMEN

Due to the pathogen-specific targeting, neutralization capabilities, and enduring efficacy, neutralizing antibodies (NAs) have received widespread attentions as a critical immunotherapeutic strategy against infectious viruses. However, because of the high variability and complexity of pathogens, rapid determination of neutralization activity of antiviral antibodies remains a challenge. Here, we report a new method, named as out-of-plane polarization imaging based single-particle rotational sensing, for rapid analysis of neutralization activity of antiviral antibody against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Using the spike protein functionalized gold nanorods (AuNRs) and angiotensin-converting enzyme 2 (ACE2) coated gold nanoparticles (AuNPs) as the rotational sensors and chaperone probes, we demonstrated the single-particle rotational sensing strategy for the measurement of rotational diffusion coefficient of the chaperone-bound rotational sensors caused by the specific spike protein-ACE2 interactions. This enables us to measure the neutralizing activity of neutralizing antibody from the analysis of dose-dependent changes in rotational diffusion coefficient (Dr) of the rotational sensors upon the treatment of SARS-CoV-2 antibody. With this technique, we achieved the quantitative determination of neutralization activity of a commercially available SARS-CoV-2 antibody (IC50, 294.1 ng/mL) with satisfying accuracy and anti-interference ability. This simple and robust method holds the potential for rapid and accurate evaluation of neutralization activity against different pathogenic viruses.

Asunto(s)

Enzima Convertidora de Angiotensina 2 , Anticuerpos Neutralizantes , Oro , Nanopartículas del Metal , Nanotubos , SARS-CoV-2 , Glicoproteína de la Espiga del Coronavirus , Oro/química , Anticuerpos Neutralizantes/inmunología , SARS-CoV-2/inmunología , Nanopartículas del Metal/química , Humanos , Nanotubos/química , Enzima Convertidora de Angiotensina 2/metabolismo , Enzima Convertidora de Angiotensina 2/inmunología , Glicoproteína de la Espiga del Coronavirus/inmunología , Antivirales/farmacología , Antivirales/inmunología , Anticuerpos Antivirales/inmunología , COVID-19/inmunología , COVID-19/virología , Pruebas de Neutralización/métodos

RESUMEN

Despite the high efficiency of current SARS-CoV-2 mRNA vaccines in reducing COVID-19 morbidity and mortality, waning immunity and the emergence of resistant variants underscore the need for novel vaccination strategies. This study explores a heterologous mRNA/Modified Vaccinia virus Ankara (MVA) prime/boost regimen employing a trimeric form of the receptor binding domain (RBD) of the SARS-CoV-2 spike (S) protein compared to a homologous MVA/MVA regimen. In C57BL/6 mice, the RBD was delivered during priming via an mRNA vector encapsulated in nanoemulsions (NE) or lipid nanoparticles (LNP), followed by a booster with a replication-deficient MVA-based recombinant virus (MVA-RBD). This heterologous mRNA/MVA regimen elicited strong anti-RBD binding and neutralizing antibodies (BAbs and NAbs) against both the ancestral SARS-CoV-2 strain and different variants of concern (VoCs). Additionally, this protocol induced robust and polyfunctional RBD-specific CD4 and CD8 T cell responses, particularly in animals primed with mLNP-RBD. In K18-hACE2 transgenic mice, the LNP-RBD/MVA combination provided complete protection from morbidity and mortality following a live SARS-CoV-2 challenge compared with the partial protection observed with mNE-RBD/MVA or MVA/MVA regimens. Although the mNE-RBD/MVA regimen only protects half of the animals, it was able to induce antibodies with Fc-mediated effector functions besides NAbs. Moreover, viral replication and viral load in the respiratory tract were markedly reduced and decreased pro-inflammatory cytokine levels were observed. These results support the efficacy of heterologous mRNA/MVA vaccine combinations over homologous MVA/MVA regimen, using alternative nanocarriers that circumvent intellectual property restrictions of current mRNA vaccine formulations.

Asunto(s)

Anticuerpos Neutralizantes , Anticuerpos Antivirales , Vacunas contra la COVID-19 , COVID-19 , Ratones Endogámicos C57BL , SARS-CoV-2 , Glicoproteína de la Espiga del Coronavirus , Virus Vaccinia , Animales , Vacunas contra la COVID-19/inmunología , Vacunas contra la COVID-19/administración & dosificación , SARS-CoV-2/inmunología , SARS-CoV-2/genética , Ratones , Glicoproteína de la Espiga del Coronavirus/inmunología , Glicoproteína de la Espiga del Coronavirus/genética , COVID-19/prevención & control , COVID-19/inmunología , Anticuerpos Antivirales/inmunología , Anticuerpos Antivirales/sangre , Anticuerpos Neutralizantes/inmunología , Virus Vaccinia/genética , Virus Vaccinia/inmunología , Humanos , Femenino , Nanopartículas/administración & dosificación , Vacunación , Vacunas de ARNm/administración & dosificación , Ratones Transgénicos , Vacunas Sintéticas/inmunología , Vacunas Sintéticas/administración & dosificación , Vacunas Sintéticas/genética , Linfocitos T CD8-positivos/inmunología , Enzima Convertidora de Angiotensina 2/inmunología , Enzima Convertidora de Angiotensina 2/genética , Liposomas

RESUMEN

Introduction: A potential overlap in symptoms between post-acute COVID-19 syndrome and post-COVID-19 vaccination syndrome has been noted. We report a paired description of patients presenting with similar manifestations involving the central (CNS) or peripheral nervous system (PNS) following SARS-CoV-2 infection or vaccination, suggesting that both may have triggered similar immune-mediated neurological disorders in the presence of anti-idiotype antibodies directed against the ACE2 protein. Patients and methods: Four patients exhibited overlapping neurological manifestations following SARS-CoV-2 infection or vaccination: radiculitis, Guillain-Barré syndrome, and MRI-negative myelitis, respectively, sharing positivity for anti-ACE2 antibodies. Autoantibodies against AQP-4, MOG, GlyR, GAD, and amphiphysin, onconeural antibodies for CNS syndromes, and anti-ganglioside antibodies for PNS syndromes tested negative in all patients. Discussion: Anti-idiotype antibodies against ACE2 have been detected in patients who recovered from COVID-19 infection, and it has been hypothesized that such antibodies may mediate adverse events following SARS-CoV-2 infection or vaccination, resulting in the activation of the immune system against cells expressing ACE2, such as neurons. Our data reveal clinically overlapping syndromes triggered by SARS-CoV-2 infection or vaccination, sharing positivity for anti-ACE2 antibodies. Their presence, in the absence of other classic autoimmune markers of CNS or PNS involvement, suggests that they might play an active role in the context of an aberrant immune response. Conclusion: Anti-idiotype antibodies directed against ACE2 may be triggered by both SARS-CoV-2 infection and vaccination, possibly contributing to neurological autoimmune manifestations. Their pathogenic role, however, remains to be demonstrated in large-scale, more structured studies.

Asunto(s)

Enzima Convertidora de Angiotensina 2 , Autoanticuerpos , COVID-19 , SARS-CoV-2 , Humanos , COVID-19/inmunología , Masculino , SARS-CoV-2/inmunología , Persona de Mediana Edad , Autoanticuerpos/inmunología , Autoanticuerpos/sangre , Femenino , Enzima Convertidora de Angiotensina 2/inmunología , Vacunas contra la COVID-19/efectos adversos , Vacunas contra la COVID-19/inmunología , Anciano , Anticuerpos Antiidiotipos/inmunología , Vacunación/efectos adversos , Adulto , Síndrome Post Agudo de COVID-19 , Enfermedades del Sistema Nervioso/inmunología , Enfermedades del Sistema Nervioso/etiología , Síndrome de Guillain-Barré/inmunología , Síndrome de Guillain-Barré/etiología

RESUMEN

Boosting with mRNA vaccines encoding variant-matched spike proteins has been implemented to mitigate their reduced efficacy against emerging SARS-CoV-2 variants. Nonetheless, in humans, it remains unclear whether boosting in the ipsilateral or contralateral arm with respect to the priming doses impacts immunity and protection. Here, we boosted K18-hACE2 mice with either monovalent mRNA-1273 (Wuhan-1 spike) or bivalent mRNA-1273.214 (Wuhan-1 + BA.1 spike) vaccine in the ipsilateral or contralateral leg after a two-dose priming series with mRNA-1273. Boosting in the ipsilateral or contralateral leg elicited equivalent levels of serum IgG and neutralizing antibody responses against Wuhan-1 and BA.1. While contralateral boosting with mRNA vaccines resulted in the expansion of spike-specific B and T cells beyond the ipsilateral draining lymph node (DLN) to the contralateral DLN, administration of a third mRNA vaccine dose at either site resulted in similar levels of antigen-specific germinal center B cells, plasmablasts/plasma cells, T follicular helper cells, and CD8+ T cells in the DLNs and the spleen. Furthermore, ipsilateral and contralateral boosting with mRNA-1273 or mRNA-1273.214 vaccines conferred similar homologous or heterologous immune protection against SARS-CoV-2 BA.1 virus challenge with equivalent reductions in viral RNA and infectious virus in the nasal turbinates and lungs. Collectively, our data show limited differences in B and T cell immune responses after ipsilateral and contralateral site boosting by mRNA vaccines that do not substantively impact protection against an Omicron strain.IMPORTANCESequential boosting with mRNA vaccines has been an effective strategy to overcome waning immunity and neutralization escape by emerging SARS-CoV-2 variants. However, it remains unclear how the site of boosting relative to the primary vaccination series shapes optimal immune responses or breadth of protection against variants. In K18-hACE2 transgenic mice, we observed that intramuscular boosting with historical monovalent or variant-matched bivalent vaccines in the ipsilateral or contralateral limb elicited comparable levels of serum spike-specific antibody and antigen-specific B and T cell responses. Moreover, boosting on either side conferred equivalent protection against a SARS-CoV-2 Omicron challenge strain. Our data in mice suggest that the site of intramuscular boosting with an mRNA vaccine does not substantially impact immunity or protection against SARS-CoV-2 infection.

Asunto(s)

Vacuna nCoV-2019 mRNA-1273 , Anticuerpos Neutralizantes , Anticuerpos Antivirales , Vacunas contra la COVID-19 , COVID-19 , Inmunización Secundaria , SARS-CoV-2 , Glicoproteína de la Espiga del Coronavirus , Vacunas de ARNm , Animales , SARS-CoV-2/inmunología , Ratones , COVID-19/prevención & control , COVID-19/inmunología , COVID-19/virología , Anticuerpos Antivirales/inmunología , Anticuerpos Antivirales/sangre , Anticuerpos Neutralizantes/inmunología , Anticuerpos Neutralizantes/sangre , Vacunas contra la COVID-19/inmunología , Vacunas contra la COVID-19/administración & dosificación , Glicoproteína de la Espiga del Coronavirus/inmunología , Glicoproteína de la Espiga del Coronavirus/genética , Vacuna nCoV-2019 mRNA-1273/inmunología , Vacunas de ARNm/inmunología , Femenino , Inmunoglobulina G/sangre , Inmunoglobulina G/inmunología , Humanos , Linfocitos B/inmunología , Linfocitos T/inmunología , Enzima Convertidora de Angiotensina 2/inmunología , Enzima Convertidora de Angiotensina 2/genética , Enzima Convertidora de Angiotensina 2/metabolismo

RESUMEN

The COVID-19 pandemic has uncovered the high genetic variability of the SARS-CoV-2 virus and its ability to evade the immune responses that were induced by earlier viral variants. Only a few monoclonal antibodies that have been reported to date are capable of neutralizing a broad spectrum of SARS-CoV-2 variants. Here, we report the isolation of a new broadly neutralizing human monoclonal antibody, iC1. The antibody was identified through sorting the SARS-CoV-1 RBD-stained individual B cells that were isolated from the blood of a vaccinated donor following a breakthrough infection. In vitro, iC1 potently neutralizes pseudoviruses expressing a wide range of SARS-CoV-2 Spike variants, including those of the XBB sublineage. In an hACE2-transgenic mouse model, iC1 provided effective protection against the Wuhan strain of the virus as well as the BA.5 and XBB.1.5 variants. Therefore, iC1 can be considered as a potential component of the broadly neutralizing antibody cocktails resisting the SARS-CoV-2 mutation escape.

Asunto(s)

Enzima Convertidora de Angiotensina 2 , Anticuerpos Monoclonales , Anticuerpos Neutralizantes , Anticuerpos Antivirales , COVID-19 , Ratones Transgénicos , SARS-CoV-2 , Animales , SARS-CoV-2/inmunología , Humanos , COVID-19/inmunología , COVID-19/prevención & control , COVID-19/virología , Enzima Convertidora de Angiotensina 2/inmunología , Enzima Convertidora de Angiotensina 2/genética , Enzima Convertidora de Angiotensina 2/metabolismo , Ratones , Anticuerpos Antivirales/inmunología , Anticuerpos Monoclonales/inmunología , Anticuerpos Neutralizantes/inmunología , Glicoproteína de la Espiga del Coronavirus/inmunología , Glicoproteína de la Espiga del Coronavirus/genética , Pandemias/prevención & control , Betacoronavirus/inmunología , Betacoronavirus/genética , Anticuerpos ampliamente neutralizantes/inmunología , Modelos Animales de Enfermedad , Neumonía Viral/inmunología , Neumonía Viral/virología , Neumonía Viral/prevención & control , Infecciones por Coronavirus/inmunología , Infecciones por Coronavirus/virología , Infecciones por Coronavirus/prevención & control

RESUMEN

The upper respiratory tract is the initial site of SARS-CoV-2 infection. Nasal spike-specific secretory immunoglobulin A (sIgA) correlates with protection against Omicron breakthrough infection. We report that intranasal vaccination using human adenovirus serotype 5 (Ad5) vectored Omicron spike in people who previously vaccinated with ancestral vaccine could induce robust neutralizing sIgA in the nasal passage. Nasal sIgA was predominantly present in dimeric and multimeric forms and accounted for nearly 40% of total proteins in nasal mucosal lining fluids (NMLFs). A low-level IgG could also be detected in NMLFs but not IgM, IgD, and IgE. After a complete nasal wash, sIgA in the nasal passage could be replenished rapidly within a few hours. A comparison of purified paired serum IgA, serum IgG, and nasal sIgA from the same individuals showed that sIgA was up to 3-logs more potent than serum antibodies in binding to spikes and in neutralizing Omicron subvariants. Serum IgG and IgA failed to neutralize XBB and BA.2.86, while nasal sIgA retained potent neutralization against these newly emerged variants. Further analysis showed that sIgA was more effective than IgG or IgA in blocking spike-mediated cell-to-cell transmission and protecting hACE2 mice from XBB challenge. Using a sIgA monoclonal antibody as a reference, we estimated that the total nasal sIgA contains about 2.6-3.9% spike-specific sIgA in NMLFs collected approximately one month after intranasal vaccination. Our study provided insights for developing intranasal vaccines that can induce sIgA to build an effective and mutation-resistant first-line immune barrier against constantly emerging variants.

Asunto(s)

Administración Intranasal , Anticuerpos Neutralizantes , Anticuerpos Antivirales , Vacunas contra la COVID-19 , COVID-19 , SARS-CoV-2 , Glicoproteína de la Espiga del Coronavirus , Humanos , SARS-CoV-2/inmunología , SARS-CoV-2/genética , COVID-19/prevención & control , COVID-19/inmunología , Anticuerpos Neutralizantes/inmunología , Anticuerpos Neutralizantes/sangre , Animales , Ratones , Glicoproteína de la Espiga del Coronavirus/inmunología , Glicoproteína de la Espiga del Coronavirus/genética , Anticuerpos Antivirales/inmunología , Anticuerpos Antivirales/sangre , Vacunas contra la COVID-19/inmunología , Vacunas contra la COVID-19/genética , Vacunas contra la COVID-19/administración & dosificación , Inmunoglobulina A/inmunología , Inmunoglobulina A/sangre , Inmunoglobulina A/genética , Mucosa Nasal/inmunología , Mucosa Nasal/virología , Femenino , Vectores Genéticos/inmunología , Vectores Genéticos/genética , Enzima Convertidora de Angiotensina 2/genética , Enzima Convertidora de Angiotensina 2/inmunología , Inmunoglobulina A Secretora/inmunología , Adenoviridae/genética , Adenoviridae/inmunología , Inmunoglobulina G/inmunología , Inmunoglobulina G/sangre , Masculino

RESUMEN

Neutralizing antibodies (nAbs) produced from infection or vaccination play an important role in acquired immunity. Determining virus-specific nAb titers is a useful tool for measuring aquired immunity in an individual. The standard methods to do so rely on titrating serum samples against live virus and monitoring viral infection in cultured cells which requires high biosafety level containment. The surrogate virus neutralization test (sVNT) reduces the biohazards and it is suitable for designing rapid test device in a lateral flow assay (LFA) format. Here, we introduce the fabrication and development of a unique paper-based LFA device for determining the level of SARS-CoV-2 nAb in a sample with a semiquantitative direct colorimetric readout. A LFA-based gradient assay design was used to facilitate the sVNT, where the spike glycoprotein receptor binding domain (RBD) and angiotensin-converting enzyme 2 (ACE2) stand in as proxies for viruses and cells, respectively. The gradient assay employed multiple test dots of ACE2 spotted in increasing concentration along the sample flow path and gold nanoparticle-conjugated RBD for readout. In this way, the number of developed spots is inversely proportional to the concentration of nAbs present in the sample. The assay was tested with both standard solutions of nAb as well as human serum samples. We have demonstrated that the device can effectively provide semiquantitative test results of nAbs by direct instrument-free colorimetric detection.

Asunto(s)

Anticuerpos Neutralizantes , COVID-19 , Pruebas de Neutralización , Papel , SARS-CoV-2 , Anticuerpos Neutralizantes/inmunología , Anticuerpos Neutralizantes/sangre , SARS-CoV-2/inmunología , SARS-CoV-2/aislamiento & purificación , Humanos , Pruebas de Neutralización/métodos , COVID-19/diagnóstico , COVID-19/virología , COVID-19/inmunología , COVID-19/sangre , Anticuerpos Antivirales/inmunología , Anticuerpos Antivirales/sangre , Colorimetría/métodos , Enzima Convertidora de Angiotensina 2/metabolismo , Enzima Convertidora de Angiotensina 2/inmunología , Glicoproteína de la Espiga del Coronavirus/inmunología

RESUMEN

We generated a replication-competent OC43 human seasonal coronavirus (CoV) expressing the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike in place of the native spike (rOC43-CoV2 S). This virus is highly attenuated relative to OC43 and SARS-CoV-2 in cultured cells and animals and is classified as a biosafety level 2 (BSL-2) agent by the NIH biosafety committee. Neutralization of rOC43-CoV2 S and SARS-CoV-2 by S-specific monoclonal antibodies and human sera is highly correlated, unlike recombinant vesicular stomatitis virus-CoV2 S. Single-dose immunization with rOC43-CoV2 S generates high levels of neutralizing antibodies against SARS-CoV-2 and fully protects human ACE2 transgenic mice from SARS-CoV-2 lethal challenge, despite nondetectable replication in respiratory and nonrespiratory organs. rOC43-CoV2 S induces S-specific serum and airway mucosal immunoglobulin A and IgG responses in rhesus macaques. rOC43-CoV2 S has enormous value as a BSL-2 agent to measure S-specific antibodies in the context of a bona fide CoV and is a candidate live attenuated SARS-CoV-2 mucosal vaccine that preferentially replicates in the upper airway.

Asunto(s)

Anticuerpos Neutralizantes , Anticuerpos Antivirales , COVID-19 , Pruebas de Neutralización , SARS-CoV-2 , Glicoproteína de la Espiga del Coronavirus , Animales , Glicoproteína de la Espiga del Coronavirus/inmunología , Glicoproteína de la Espiga del Coronavirus/genética , SARS-CoV-2/inmunología , SARS-CoV-2/genética , Humanos , Anticuerpos Neutralizantes/inmunología , Ratones , COVID-19/inmunología , COVID-19/virología , COVID-19/prevención & control , Anticuerpos Antivirales/inmunología , Pruebas de Neutralización/métodos , Ratones Transgénicos , Coronavirus Humano OC43/inmunología , Coronavirus Humano OC43/genética , Vacunas contra la COVID-19/inmunología , Vacunas contra la COVID-19/administración & dosificación , Enzima Convertidora de Angiotensina 2/metabolismo , Enzima Convertidora de Angiotensina 2/inmunología , Chlorocebus aethiops , Células Vero , Macaca mulatta

RESUMEN

BACKGROUND: SARS-CoV2 virus, responsible for the COVID-19 pandemic, has four structural proteins and 16 nonstructural proteins. S-protein is one of the structural proteins exposed on the virus surface and is the main target for producing neutralizing antibodies and vaccines. The S-protein forms a trimer that can bind the angiotensin-converting enzyme 2 (ACE2) through its receptor binding domain (RBD) for cell entry. AIMS: The goal of this study was to express in HEK293 cells a new RBD recombinant protein in a constitutive and stable manner in order to use it as an alternative immunogen and diagnostic tool for COVID-19. MATERIALS & METHODS: The protein was designed to contain an immunoglobulin signal sequence, an explanded C-terminal section of the RBD, a region responsible for the bacteriophage T4 trimerization inducer, and six histidines in the pCDNA-3.1 plasmid. Following transformation, the cells were selected with geneticin-G418 and purified from serum-fre culture supernatants using Ni2+-agarand size exclusion chromatography. The protein was structurally identified by cross-linking and circular dichroism experiments, and utilized to immunize mice in conjuction with AS03 or alum adjuvants. The mice sera were examined for antibody recognition, receptor-binding inhibition, and virus neutralization, while spleens were evaluated for γ-interferon production in the presence of RBD. RESULTS: The protein released in the culture supernatant of cells, and exhibited a molecular mass of 135 kDa with a secondary structure like the monomeric and trimeric RBD. After purification, it formed a multimeric structure comprising trimers and hexamers, which were able to bind the ACE2 receptor. It generated high antibody titers in mice when combined with AS03 adjuvant (up to 1:50,000). The sera were capable of inhibiting binding of biotin-labeled ACE2 to the virus S1 subunit and could neutralize the entry of the Wuhan virus strain into cells at dilutions up to 1:2000. It produced specific IFN-γ producing cells in immunized mouse splenocytes. DISCUSSION: Our data describe a new RBD containing protein, forming trimers and hexamers, which are able to induce a protective humoral and cellular response against SARS-CoV2. CONCLUSION: These results add a new arsenal to combat COVID-19, as an alternative immunogen or antigen for diagnosis.

Asunto(s)

Enzima Convertidora de Angiotensina 2 , Anticuerpos Neutralizantes , Anticuerpos Antivirales , COVID-19 , Proteínas Recombinantes , SARS-CoV-2 , Glicoproteína de la Espiga del Coronavirus , Animales , Humanos , Glicoproteína de la Espiga del Coronavirus/inmunología , Glicoproteína de la Espiga del Coronavirus/genética , Glicoproteína de la Espiga del Coronavirus/química , Ratones , Anticuerpos Neutralizantes/inmunología , SARS-CoV-2/inmunología , COVID-19/inmunología , COVID-19/prevención & control , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/genética , Proteínas Recombinantes/química , Células HEK293 , Enzima Convertidora de Angiotensina 2/metabolismo , Enzima Convertidora de Angiotensina 2/inmunología , Anticuerpos Antivirales/inmunología , Vacunas contra la COVID-19/inmunología , Ratones Endogámicos BALB C , Femenino , Multimerización de Proteína , Dominios Proteicos/inmunología , Unión Proteica

RESUMEN

Background: In early infected or severe coronavirus disease 2019 (COVID-19) patients, circulating NK cells are consistently reduced, despite being highly activated or exhausted. The aim of this paper was to establish whether severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike glycoprotein (SP) may directly trigger NK cells and through which receptor(s). Methods: SP-stimulated human NK cells have been evaluated for the expression of activation markers, cytokine release, and cytotoxic activity, as well as for gene expression profiles and NF-kB phosphorylation, and they have been silenced with specific small interfering RNAs. Results: SPs from the Wuhan strain and other variants of concern (VOCs) directly bind and stimulate purified NK cells by increasing activation marker expression, cytokine release, and cytolytic activity, prevalently in the CD56brightNK cell subset. VOC-SPs differ in their ability to activate NK cells, G614, and Delta-Plus strains providing the strongest activity in the majority of donors. While VOC-SPs do not trigger ACE2, which is not expressed on NK cells, or other activating receptors, they directly and variably bind to both Toll-like receptor 2 (TLR2) and TLR4. Moreover, SP-driven NK cell functions are inhibited upon masking such receptors or silencing the relative genes. Lastly, VOC-SPs upregulate CD56dimNK cell functions in COVID-19 recovered, but not in non-infected, individuals. Conclusions: TLR2 and TLR4 are novel activating receptors for SP in NK cells, suggesting a new role of these cells in orchestrating the pathophysiology of SARS-CoV-2 infection. The pathogenic relevance of this finding is highlighted by the fact that free SP providing NK cell activation is frequently detected in a SARS-CoV-2 inflamed environment and in plasma of infected and long-COVID-19 subjects.

Asunto(s)

COVID-19 , Células Asesinas Naturales , SARS-CoV-2 , Glicoproteína de la Espiga del Coronavirus , Receptor Toll-Like 2 , Receptor Toll-Like 4 , Humanos , Células Asesinas Naturales/inmunología , Células Asesinas Naturales/metabolismo , Glicoproteína de la Espiga del Coronavirus/inmunología , Glicoproteína de la Espiga del Coronavirus/metabolismo , SARS-CoV-2/inmunología , SARS-CoV-2/fisiología , COVID-19/inmunología , COVID-19/virología , Receptor Toll-Like 4/metabolismo , Receptor Toll-Like 4/inmunología , Receptor Toll-Like 2/metabolismo , Receptor Toll-Like 2/inmunología , Activación de Linfocitos/inmunología , Citocinas/metabolismo , Citocinas/inmunología , Enzima Convertidora de Angiotensina 2/metabolismo , Enzima Convertidora de Angiotensina 2/genética , Enzima Convertidora de Angiotensina 2/inmunología

RESUMEN

Vaccines and antibodies that specifically target or neutralize components of the SARS-CoV-2 virus are effective in prevention and treatment of human patients with SARS-CoV-2 infection. However, vaccines and SARS-CoV-2 neutralization antibodies target a subset of epitopes of viral proteins, and the fast evolution of the SARS-CoV-2 virus and the continuing emergence of SARS-CoV-2 variants confer SARS-CoV-2 immune escape from these therapies. ACE2 is the human cell receptor that serves as the entry point for SARS-CoV-2 into human cells and thus is the gatekeeper for SARS-CoV-2 infection of humans. We report here the development of 4G8C11, an anti-human ACE2 receptor monoclonal antibody that recognizes ACE2 on human cell surfaces. We determined that 4G8C11 blocks SARS-CoV-2 and variant infection of ACE2+ human cells. Furthermore, 4G8C11 has minimal effects on ACE2 receptor activity. 4G8C11 is therefore a monoclonal antibody for ACE2 receptor detection and potentially an effective immunotherapeutic agent for SARS-CoV-2 and variants.

Asunto(s)

Enzima Convertidora de Angiotensina 2 , Anticuerpos Monoclonales , Anticuerpos Neutralizantes , Anticuerpos Antivirales , COVID-19 , SARS-CoV-2 , Humanos , Enzima Convertidora de Angiotensina 2/metabolismo , Enzima Convertidora de Angiotensina 2/inmunología , SARS-CoV-2/inmunología , COVID-19/inmunología , Anticuerpos Monoclonales/farmacología , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/uso terapéutico , Anticuerpos Antivirales/inmunología , Anticuerpos Neutralizantes/inmunología , Internalización del Virus/efectos de los fármacos , Células HEK293 , Animales , Chlorocebus aethiops

RESUMEN

The Omicron BA.5 variant of SARS-CoV-2 is known for its high transmissibility and its capacity to evade immunity provided by vaccine protection against the (original) Wuhan strain. In our prior research, we successfully produced the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein in an E. coli expression system. Extensive biophysical characterization indicated that, even without glycosylation, the RBD maintained native-like conformational and biophysical properties. The current study explores the immunogenicity and neutralization capacity of the E. coli-expressed Omicron BA.5 RBD using a mouse model. Administration of three doses of the RBD without any adjuvant elicited high titer antisera of up to 7.3 × 105 and up to 1.6 × 106 after a booster shot. Immunization with RBD notably enhanced the population of CD44+CD62L+ T cells, indicating the generation of T cell memory. The in vitro assays demonstrated the antisera's protective efficacy through significant inhibition of the interaction between SARS-CoV-2 and its human receptor, ACE2, and through potent neutralization of a pseudovirus. These findings underscore the potential of our E. coli-expressed RBD as a viable vaccine candidate against the Omicron variant of SARS-CoV-2.

Asunto(s)

Enzima Convertidora de Angiotensina 2 , Anticuerpos Neutralizantes , Vacunas contra la COVID-19 , COVID-19 , SARS-CoV-2 , Glicoproteína de la Espiga del Coronavirus , Animales , SARS-CoV-2/inmunología , Glicoproteína de la Espiga del Coronavirus/inmunología , Glicoproteína de la Espiga del Coronavirus/química , Glicoproteína de la Espiga del Coronavirus/metabolismo , Ratones , Anticuerpos Neutralizantes/inmunología , COVID-19/prevención & control , COVID-19/inmunología , Enzima Convertidora de Angiotensina 2/metabolismo , Enzima Convertidora de Angiotensina 2/inmunología , Enzima Convertidora de Angiotensina 2/química , Humanos , Vacunas contra la COVID-19/inmunología , Vacunas contra la COVID-19/química , Anticuerpos Antivirales/inmunología , Modelos Animales de Enfermedad , Dominios Proteicos , Glicosilación , Unión Proteica , Femenino , Escherichia coli/metabolismo , Linfocitos T/inmunología

RESUMEN

Coronavirus disease 2019 (COVID-19), the global pandemic caused by severe acute respiratory syndrome 2 virus (SARS-CoV-2) infection, has caused millions of infections and fatalities worldwide. Extensive SARS-CoV-2 research has been conducted to develop therapeutic drugs and prophylactic vaccines, and even though some drugs have been approved to treat SARS-CoV-2 infection, treatment efficacy remains limited. Therefore, preventive vaccination has been implemented on a global scale and represents the primary approach to combat the COVID-19 pandemic. Approved vaccines vary in composition, although vaccine design has been based on either the key viral structural (spike) protein or viral components carrying this protein. Therefore, mutations of the virus, particularly mutations in the S protein, severely compromise the effectiveness of current vaccines and the ability to control COVID-19 infection. This review begins by describing the SARS-CoV-2 viral composition, the mechanism of infection, the role of angiotensin-converting enzyme 2, the host defence responses against infection and the most common vaccine designs. Next, this review summarizes the common mutations of SARS-CoV-2 and how these mutations change viral properties, confer immune escape and influence vaccine efficacy. Finally, this review discusses global strategies that have been employed to mitigate the decreases in vaccine efficacy encountered against new variants.

Asunto(s)

Vacunas contra la COVID-19 , COVID-19 , Mutación , SARS-CoV-2 , Desarrollo de Vacunas , Humanos , SARS-CoV-2/inmunología , SARS-CoV-2/genética , Vacunas contra la COVID-19/inmunología , COVID-19/prevención & control , COVID-19/inmunología , COVID-19/virología , Glicoproteína de la Espiga del Coronavirus/inmunología , Glicoproteína de la Espiga del Coronavirus/genética , Enzima Convertidora de Angiotensina 2/metabolismo , Enzima Convertidora de Angiotensina 2/genética , Enzima Convertidora de Angiotensina 2/inmunología

RESUMEN

Since the SARS-CoV-2 Omicron virus has gained dominance worldwide, its continual evolution with unpredictable mutations and patterns has revoked all authorized immunotherapeutics. Rapid viral evolution has also necessitated several rounds of vaccine updates in order to provide adequate immune protection. It remains imperative to understand how Omicron evolves into different subvariants and causes immune escape as this could help reevaluate the current intervention strategies mostly implemented in the clinics as emergency measures to counter the pandemic and, importantly, develop new solutions. Here, we provide a review focusing on the major events of Omicron viral evolution, including the features of spike mutation that lead to immune evasion against monoclonal antibody (mAb) therapy and vaccination, and suggest alternative durable options such as the ACE2-based experimental therapies superior to mAbs to address this unprecedented evolution of Omicron virus. In addition, this type of unique ACE2-based virus-trapping molecules can counter all zoonotic SARS coronaviruses, either from unknown animal hosts or from established wild-life reservoirs of SARS-CoV-2, and even seasonal alpha coronavirus NL63 that depends on human ACE2 for infection.

Asunto(s)

COVID-19 , Evasión Inmune , SARS-CoV-2 , Glicoproteína de la Espiga del Coronavirus , SARS-CoV-2/inmunología , SARS-CoV-2/genética , Humanos , COVID-19/inmunología , COVID-19/virología , Glicoproteína de la Espiga del Coronavirus/inmunología , Glicoproteína de la Espiga del Coronavirus/genética , Glicoproteína de la Espiga del Coronavirus/metabolismo , Enzima Convertidora de Angiotensina 2/metabolismo , Enzima Convertidora de Angiotensina 2/inmunología , Animales , Evolución Molecular , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/uso terapéutico , Mutación , Vacunas contra la COVID-19/inmunología , Anticuerpos Antivirales/inmunología

RESUMEN

Recent progress on chimeric antigen receptor (CAR)-NK cells has shown promising results in treating CD19-positive lymphoid tumors with minimal toxicities [including graft versus host disease (GvHD) and cytokine release syndrome (CRS) in clinical trials. Nevertheless, the use of CAR-NK cells in combating viral infections has not yet been fully explored. Previous studies have shown that CAR-NK cells expressing S309 single-chain fragment variable (scFv), hereinafter S309-CAR-NK cells, can bind to SARS-CoV-2 wildtype pseudotyped virus (PV) and effectively kill cells expressing wild-type spike protein in vitro. In this study, we further demonstrate that the S309-CAR-NK cells can bind to different SARS-CoV-2 variants, including the B.1.617.2 (Delta), B.1.621 (Mu), and B.1.1.529 (Omicron) variants in vitro. We also show that S309-CAR-NK cells reduce virus loads in the NOD/SCID gamma (NSG) mice expressing the human angiotensin-converting enzyme 2 (hACE2) receptor challenged with SARS-CoV-2 wild-type (strain USA/WA1/2020). Our study demonstrates the potential use of S309-CAR-NK cells for inhibiting infection by SARS-CoV-2 and for the potential treatment of COVID-19 patients unresponsive to otherwise currently available therapeutics. IMPORTANCE: Chimeric antigen receptor (CAR)-NK cells can be "off-the-shelf" products that treat various diseases, including cancer, infections, and autoimmune diseases. In this study, we engineered natural killer (NK) cells to express S309 single-chain fragment variable (scFv), to target the Spike protein of SARS-CoV-2, hereinafter S309-CAR-NK cells. Our study shows that S309-CAR-NK cells are effective against different SARS-CoV-2 variants, including the B.1.617.2 (Delta), B.1.621 (Mu), and B.1.1.529 (Omicron) variants. The S309-CAR-NK cells can (i) directly bind to SARS-CoV-2 pseudotyped virus (PV), (ii) competitively bind to SARS-CoV-2 PV with 293T cells expressing the human angiotensin-converting enzyme 2 (hACE2) receptor (293T-hACE2 cells), (iii) specifically target and lyse A549 cells expressing the spike protein, and (iv) significantly reduce the viral loads of SARS-CoV-2 wild-type (strain USA/WA1/2020) in the lungs of NOD/SCID gamma (NSG) mice expressing hACE2 (hACE2-NSG mice). Altogether, the current study demonstrates the potential use of S309-CAR-NK immunotherapy as an alternative treatment for COVID-19 patients.

Asunto(s)

Enzima Convertidora de Angiotensina 2 , COVID-19 , Células Asesinas Naturales , Receptores Quiméricos de Antígenos , SARS-CoV-2 , Glicoproteína de la Espiga del Coronavirus , Carga Viral , Animales , SARS-CoV-2/inmunología , Células Asesinas Naturales/inmunología , Enzima Convertidora de Angiotensina 2/metabolismo , Enzima Convertidora de Angiotensina 2/genética , Enzima Convertidora de Angiotensina 2/inmunología , Ratones , Humanos , Receptores Quiméricos de Antígenos/inmunología , Receptores Quiméricos de Antígenos/genética , Receptores Quiméricos de Antígenos/metabolismo , COVID-19/inmunología , COVID-19/virología , COVID-19/terapia , Glicoproteína de la Espiga del Coronavirus/inmunología , Glicoproteína de la Espiga del Coronavirus/genética , Glicoproteína de la Espiga del Coronavirus/metabolismo , Anticuerpos de Cadena Única/inmunología , Anticuerpos de Cadena Única/genética , Ratones SCID , Ratones Endogámicos NOD