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1.
Vaccine ; 38(46): 7205-7212, 2020 10 27.
Artigo em Inglês | MEDLINE | ID: mdl-33010978

RESUMO

The development of an effective vaccine against SARS-CoV-2 is urgently needed. We generated SARS-CoV-2 RBD-Fc fusion protein and evaluated its potency to elicit neutralizing antibody response in mice. RBD-Fc elicited a higher neutralizing antibodies titer than RBD as evaluated by a pseudovirus neutralization assay and a live virus based microneutralization assay. Furthermore, RBD-Fc immunized sera better inhibited cell-cell fusion, as evaluated by a quantitative cell-cell fusion assay. The cell-cell fusion assay results correlated well with the virus neutralization potency and could be used for high-throughput screening of large panels of anti-SARS-CoV-2 antibodies and vaccines without the requirement of live virus infection in BSL3 containment. Moreover, the anti-RBD sera did not enhance the pseudotyped SARS-CoV-2 infection of K562 cells. These results demonstrate that Fc fusion can significantly improve the humoral immune response to recombinant RBD immunogen, and suggest that RBD-Fc could serve as a useful component of effective vaccines against SARS-CoV-2.


Assuntos
Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Infecções por Coronavirus/prevenção & controle , Fragmentos Fc das Imunoglobulinas/imunologia , Pandemias/prevenção & controle , Pneumonia Viral/prevenção & controle , Glicoproteína da Espícula de Coronavírus/imunologia , Vacinas Virais/imunologia , Animais , Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Betacoronavirus/imunologia , Fusão Celular , Linhagem Celular , Infecções por Coronavirus/imunologia , Ensaio de Imunoadsorção Enzimática , Feminino , Células HEK293 , Ensaios de Triagem em Larga Escala/métodos , Humanos , Imunidade Humoral/imunologia , Fragmentos Fc das Imunoglobulinas/genética , Camundongos , Camundongos Endogâmicos BALB C , Testes de Neutralização , Peptidil Dipeptidase A/genética , Domínios Proteicos/imunologia , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Glicoproteína da Espícula de Coronavírus/genética , Vacinas de Subunidades/imunologia
2.
Vaccine ; 38(42): 6487-6499, 2020 09 29.
Artigo em Inglês | MEDLINE | ID: mdl-32907757

RESUMO

The many carbohydrate chains on Covid-19 coronavirus SARS-CoV-2 and its S-protein form a glycan-shield that masks antigenic peptides and decreases uptake of inactivated virus or S-protein vaccines by APC. Studies on inactivated influenza virus and recombinant gp120 of HIV vaccines indicate that glycoengineering of glycan-shields to present α-gal epitopes (Galα1-3Galß1-4GlcNAc-R) enables harnessing of the natural anti-Gal antibody for amplifying vaccine efficacy, as evaluated in mice producing anti-Gal. The α-gal epitope is the ligand for the natural anti-Gal antibody which constitutes ~1% of immunoglobulins in humans. Upon administration of vaccines presenting α-gal epitopes, anti-Gal binds to these epitopes at the vaccination site and forms immune complexes with the vaccines. These immune complexes are targeted for extensive uptake by APC as a result of binding of the Fc portion of immunocomplexed anti-Gal to Fc receptors on APC. This anti-Gal mediated effective uptake of vaccines by APC results in 10-200-fold higher anti-viral immune response and in 8-fold higher survival rate following challenge with a lethal dose of live influenza virus, than same vaccines lacking α-gal epitopes. It is suggested that glycoengineering of carbohydrate chains on the glycan-shield of inactivated SARS-CoV-2 or on S-protein vaccines, for presenting α-gal epitopes, will have similar amplifying effects on vaccine efficacy. α-Gal epitope synthesis on coronavirus vaccines can be achieved with recombinant α1,3galactosyltransferase, replication of the virus in cells with high α1,3galactosyltransferase activity as a result of stable transfection of cells with several copies of the α1,3galactosyltransferase gene (GGTA1), or by transduction of host cells with replication defective adenovirus containing this gene. In addition, recombinant S-protein presenting multiple α-gal epitopes on the glycan-shield may be produced in glycoengineered yeast or bacteria expression systems containing the corresponding glycosyltransferases. Prospective Covid-19 vaccines presenting α-gal epitopes may provide better protection than vaccines lacking this epitope because of increased uptake by APC.


Assuntos
Antígenos Virais/genética , Betacoronavirus/efeitos dos fármacos , Infecções por Coronavirus/prevenção & controle , Pandemias/prevenção & controle , Pneumonia Viral/prevenção & controle , Glicoproteína da Espícula de Coronavírus/genética , Trissacarídeos/imunologia , Vacinas Virais/imunologia , Animais , Anticorpos Antivirais/biossíntese , Antígenos Virais/imunologia , Antígenos Virais/metabolismo , Betacoronavirus/imunologia , Betacoronavirus/patogenicidade , Infecções por Coronavirus/genética , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/virologia , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/imunologia , Células Dendríticas/virologia , Engenharia Genética , Proteína do Núcleo p24 do HIV/química , Proteína do Núcleo p24 do HIV/genética , Proteína do Núcleo p24 do HIV/imunologia , Proteína gp120 do Envelope de HIV/química , Proteína gp120 do Envelope de HIV/genética , Proteína gp120 do Envelope de HIV/imunologia , Humanos , Imunogenicidade da Vacina , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Macrófagos/virologia , Camundongos , Pneumonia Viral/imunologia , Pneumonia Viral/virologia , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Glicoproteína da Espícula de Coronavírus/metabolismo , Trissacarídeos/química , Vacinas Virais/administração & dosagem , Vacinas Virais/biossíntese , Vacinas Virais/genética
3.
Arch Virol ; 165(10): 2301-2309, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32757056

RESUMO

Porcine circovirus type 2 (PCV2) is a major pathogen associated with swine diseases. It is the smallest single-stranded DNA virus, and its genome contains four major open reading frames (ORFs). ORF2 encodes the major structural protein Cap, which can self-assemble into virus-like particles (VLPs) in vitro and contains the primary antigenic determinants. In this study, we developed a high-efficiency method for obtaining VLPs and optimized the purification conditions. In this method, we expressed the protein Cap with a 6× His tag using baculovirus-infected silkworm larvae as well as the E. coli BL21(DE3) prokaryotic expression system. The PCV2 Cap proteins produced by the silkworm larvae and E. coli BL21(DE3) were purified. Cap proteins purified from silkworm larvae self-assembled into VLPs in vitro, while the Cap proteins purified from bacteria were unable to self-assemble. Transmission electron microscopy confirmed the self-assembly of VLPs. The immunogenicity of the VLPs produced using the baculovirus system was demonstrated using an enzyme-linked immunosorbent assay (ELISA). Furthermore, the purification process was optimized. The results demonstrated that the expression system using baculovirus-infected silkworm larvae is a good choice for obtaining VLPs of PCV2 and has potential for the development of a low-cost and efficient vaccine.


Assuntos
Anticorpos Antivirais/biossíntese , Baculoviridae/genética , Bombyx/virologia , Proteínas do Capsídeo/imunologia , Circovirus/imunologia , Vacinas de Partículas Semelhantes a Vírus/biossíntese , Vacinas Virais/biossíntese , Animais , Antígenos Virais/química , Antígenos Virais/imunologia , Baculoviridae/imunologia , Proteínas do Capsídeo/biossíntese , Proteínas do Capsídeo/genética , Infecções por Circoviridae/imunologia , Infecções por Circoviridae/prevenção & controle , Infecções por Circoviridae/virologia , Circovirus/genética , Epitopos/química , Epitopos/imunologia , Escherichia coli/genética , Escherichia coli/metabolismo , Feminino , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Histidina/genética , Histidina/imunologia , Soros Imunes/química , Imunogenicidade da Vacina , Larva/virologia , Camundongos , Oligopeptídeos/genética , Oligopeptídeos/imunologia , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Suínos , Doenças dos Suínos/imunologia , Doenças dos Suínos/prevenção & controle , Doenças dos Suínos/virologia , Vacinas de Partículas Semelhantes a Vírus/administração & dosagem , Vacinas de Partículas Semelhantes a Vírus/genética , Vacinas de Partículas Semelhantes a Vírus/isolamento & purificação , Vacinas Virais/administração & dosagem , Vacinas Virais/genética , Vacinas Virais/isolamento & purificação
4.
Nat Struct Mol Biol ; 27(9): 846-854, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32661423

RESUMO

The SARS-CoV-2 virus is more transmissible than previous coronaviruses and causes a more serious illness than influenza. The SARS-CoV-2 receptor binding domain (RBD) of the spike protein binds to the human angiotensin-converting enzyme 2 (ACE2) receptor as a prelude to viral entry into the cell. Using a naive llama single-domain antibody library and PCR-based maturation, we have produced two closely related nanobodies, H11-D4 and H11-H4, that bind RBD (KD of 39 and 12 nM, respectively) and block its interaction with ACE2. Single-particle cryo-EM revealed that both nanobodies bind to all three RBDs in the spike trimer. Crystal structures of each nanobody-RBD complex revealed how both nanobodies recognize the same epitope, which partly overlaps with the ACE2 binding surface, explaining the blocking of the RBD-ACE2 interaction. Nanobody-Fc fusions showed neutralizing activity against SARS-CoV-2 (4-6 nM for H11-H4, 18 nM for H11-D4) and additive neutralization with the SARS-CoV-1/2 antibody CR3022.


Assuntos
Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Betacoronavirus/imunologia , Infecções por Coronavirus , Pandemias , Peptidil Dipeptidase A/metabolismo , Pneumonia Viral , Receptores Virais/metabolismo , Anticorpos de Domínio Único/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Sequência de Aminoácidos , Anticorpos Neutralizantes/metabolismo , Anticorpos Neutralizantes/ultraestrutura , Anticorpos Antivirais/metabolismo , Anticorpos Antivirais/ultraestrutura , Afinidade de Anticorpos , Reações Antígeno-Anticorpo/imunologia , Betacoronavirus/metabolismo , Ligação Competitiva , Microscopia Crioeletrônica , Cristalografia por Raios X , Epitopos/imunologia , Humanos , Fragmentos Fc das Imunoglobulinas/genética , Fragmentos Fc das Imunoglobulinas/imunologia , Modelos Moleculares , Biblioteca de Peptídeos , Peptidil Dipeptidase A/ultraestrutura , Ligação Proteica , Conformação Proteica , Receptores Virais/ultraestrutura , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/metabolismo , Homologia de Sequência de Aminoácidos , Anticorpos de Domínio Único/metabolismo , Anticorpos de Domínio Único/ultraestrutura , Glicoproteína da Espícula de Coronavírus/metabolismo , Glicoproteína da Espícula de Coronavírus/ultraestrutura
5.
Chem Commun (Camb) ; 56(61): 8683-8686, 2020 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-32613971

RESUMO

Herein, we report that a recombinant fusion protein, containing a 457 amino acid SARS-CoV-2 receptor binding domain (RBD, residues 319-541) and a mouse IgG1 Fc domain, could induce highly potent neutralizing antibodies and stimulate humoral and cellular immunity in mice. The antibodies also effectively suppressed SARS-CoV-2 RBD binding to soluble ACE2, indicating that RBD-mFc may be further developed as a safe and effective SARS-CoV-2 vaccine.


Assuntos
Anticorpos Antivirais/imunologia , Betacoronavirus/imunologia , Infecções por Coronavirus/prevenção & controle , Pandemias/prevenção & controle , Pneumonia Viral/prevenção & controle , Proteínas Recombinantes de Fusão/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Vacinas de Subunidades/administração & dosagem , Vacinas Virais/administração & dosagem , Animais , Anticorpos Neutralizantes/imunologia , Betacoronavirus/efeitos dos fármacos , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/virologia , Feminino , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Peptidil Dipeptidase A/metabolismo , Pneumonia Viral/imunologia , Pneumonia Viral/virologia , Ligação Proteica , Glicoproteína da Espícula de Coronavírus/metabolismo
6.
Proc Natl Acad Sci U S A ; 117(30): 17757-17763, 2020 07 28.
Artigo em Inglês | MEDLINE | ID: mdl-32669430

RESUMO

Vaccination has been used to control the spread of seasonal flu; however, the virus continues to evolve and escape from host immune response through mutation and increasing glycosylation. Efforts have been directed toward development of a universal vaccine with broadly protective activity against multiple influenza strains and subtypes. Here we report the design and evaluation of various chimeric vaccines based on the most common avian influenza H5 and human influenza H1 sequences. Of these constructs, the chimeric HA (cHA) vaccine with consensus H5 as globular head and consensus H1 as stem was shown to elicit broadly protective CD4+ and CD8+ T cell responses. Interestingly, the monoglycosylated cHA (cHAmg) vaccine with GlcNAc on each glycosite induced more stem-specific antibodies, with higher antibody-dependent cellular cytotoxicity (ADCC), and better neutralizing and stronger cross-protection activities against H1, H3, H5, and H7 strains and subtypes. Moreover, the cHAmg vaccine combined with a glycolipid adjuvant designed for class switch further enhanced the vaccine efficacy with more IFN-γ, IL-4, and CD8+ memory T cells produced.


Assuntos
Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Proteção Cruzada/imunologia , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Vacinas contra Influenza/imunologia , Influenza Humana/prevenção & controle , Orthomyxoviridae/imunologia , Animais , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Citotoxicidade Celular Dependente de Anticorpos , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD8-Positivos/metabolismo , Modelos Animais de Doenças , Glicoproteínas de Hemaglutininação de Vírus da Influenza/química , Humanos , Influenza Humana/virologia , Camundongos , Modelos Moleculares , Testes de Neutralização , Orthomyxoviridae/classificação , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/imunologia , Relação Estrutura-Atividade , Vacinação
7.
Proc Natl Acad Sci U S A ; 117(31): 18832-18839, 2020 08 04.
Artigo em Inglês | MEDLINE | ID: mdl-32709746

RESUMO

Plant and animal intracellular nucleotide-binding, leucine-rich repeat (NLR) immune receptors detect pathogen-derived molecules and activate defense. Plant NLRs can be divided into several classes based upon their N-terminal signaling domains, including TIR (Toll-like, Interleukin-1 receptor, Resistance protein)- and CC (coiled-coil)-NLRs. Upon ligand detection, mammalian NAIP and NLRC4 NLRs oligomerize, forming an inflammasome that induces proximity of its N-terminal signaling domains. Recently, a plant CC-NLR was revealed to form an inflammasome-like hetero-oligomer. To further investigate plant NLR signaling mechanisms, we fused the N-terminal TIR domain of several plant NLRs to the N terminus of NLRC4. Inflammasome-dependent induced proximity of the TIR domain in planta initiated defense signaling. Thus, induced proximity of a plant TIR domain imposed by oligomerization of a mammalian inflammasome is sufficient to activate authentic plant defense. Ligand detection and inflammasome formation is maintained when the known components of the NLRC4 inflammasome is transferred across kingdoms, indicating that NLRC4 complex can robustly function without any additional mammalian proteins. Additionally, we found NADase activity of a plant TIR domain is necessary for plant defense activation, but NADase activity of a mammalian or a bacterial TIR is not sufficient to activate defense in plants.


Assuntos
Proteínas NLR , Imunidade Vegetal , Proteínas de Plantas , Proteínas Recombinantes de Fusão , Transdução de Sinais , Animais , Inflamassomos/genética , Inflamassomos/imunologia , Inflamassomos/metabolismo , Mamíferos , Proteínas NLR/química , Proteínas NLR/genética , Proteínas NLR/imunologia , Proteínas NLR/metabolismo , Imunidade Vegetal/genética , Imunidade Vegetal/imunologia , Proteínas de Plantas/química , Proteínas de Plantas/genética , Proteínas de Plantas/imunologia , Proteínas de Plantas/metabolismo , Domínios Proteicos/genética , Domínios Proteicos/fisiologia , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/metabolismo , Transdução de Sinais/genética , Transdução de Sinais/imunologia
8.
Cancer Immunol Immunother ; 69(11): 2291-2303, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32504247

RESUMO

Target expression heterogeneity and the presence of an immunosuppressive microenvironment can hamper severely the efficiency of immunotherapeutic approaches. We have analyzed the potential to encounter and overcome such conditions by a combinatory two-target approach involving a bispecific antibody retargeting T cells to tumor cells and tumor-directed antibody-fusion proteins with costimulatory members of the B7 and TNF superfamily. Targeting the tumor-associated antigens EpCAM and EGFR with the bispecific antibody and costimulatory fusion proteins, respectively, we analyzed the impact of target expression and the influence of the immunosuppressive factors IDO, IL-10, TGF-ß, PD-1 and CTLA-4 on the targeting-mediated stimulation of T cells. Here, suboptimal activity of the bispecific antibody at diverse EpCAM expression levels could be effectively enhanced by targeting-mediated costimulation by B7.1, 4-1BBL and OX40L in a broad range of EGFR expression levels. Furthermore, the benefit of combined costimulation by B7.1/4-1BBL and 4-1BBL/OX40L was demonstrated. In addition, the expression of immunosuppressive factors was shown in all co-culture settings, where blocking of prominent factors led to synergistic effects with combined costimulation. Thus, targeting-mediated costimulation showed general promise for a broad application covering diverse target expression levels, with the option for further selective enhancement by the identification and blockade of main immunosuppressive factors of the particular tumor environment.


Assuntos
Anticorpos Biespecíficos/imunologia , Antígenos de Neoplasias/imunologia , Molécula de Adesão da Célula Epitelial/imunologia , Proteínas Recombinantes de Fusão/imunologia , Linfócitos T/imunologia , Anticorpos Biespecíficos/farmacologia , Linhagem Celular Tumoral , Receptores ErbB/imunologia , Humanos , Ativação Linfocitária/efeitos dos fármacos , Ativação Linfocitária/imunologia , Proteínas Recombinantes de Fusão/farmacologia , Linfócitos T/efeitos dos fármacos , Evasão Tumoral/efeitos dos fármacos , Evasão Tumoral/imunologia , Microambiente Tumoral/efeitos dos fármacos , Microambiente Tumoral/imunologia
9.
Sheng Wu Gong Cheng Xue Bao ; 36(6): 1223-1231, 2020 Jun 25.
Artigo em Chinês | MEDLINE | ID: mdl-32597072

RESUMO

In order to prepare human-mouse chimeric cytomegalovirus-immunoglobulin M (CMV-IgM) in vitro and study the effects of different signal peptides on the secretion of CMV-IgM, genes were amplified from hybridoma cell line using RLM-RACE to construct the expression vector of chimeric CMV-IgM. Then, the signal peptide of SigF itself was replaced by five different secreted signal peptides (SigA-SigE) by PCR method, and the CHO cell was chosen as host cell for in vitro expression. SDS-PAGE, SEC-HPLC and ELISA experiments were carried out to evaluate the protein expression level and immunoreactivity of the purified CMV-IgM. A 910 kDa recombinant protein was successfully prepared and signal peptides (SigA-SigE) had an increased expressed CMV-IgM, which were 6.72, 5.19, 1.44, 1.85 and 1.98 times higher than that of the CMV 6# cell signal peptide SigF. In summary, this work provides a theoretical basis for the development of human-mouse chimeric CMV-IgM, and a novel route to increase the expression level of CMV-IgM.


Assuntos
Anticorpos Antivirais , Citomegalovirus , Imunoglobulina M , Proteínas Recombinantes de Fusão , Animais , Anticorpos Antivirais/genética , Anticorpos Antivirais/imunologia , Cricetinae , Citomegalovirus/imunologia , Ensaio de Imunoadsorção Enzimática , Expressão Gênica , Humanos , Imunoglobulina M/imunologia , Camundongos , Sinais Direcionadores de Proteínas , Proteínas Recombinantes de Fusão/imunologia
10.
Cancer Immunol Immunother ; 69(10): 2147-2155, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32468232

RESUMO

A major factor impeding the success of numerous therapeutic approaches in cancer is the immunosuppressive nature of the tumor microenvironment (TME). Hence, methods capable of reverting tumor immunosuppression through depletion or reprogramming of myeloid-derived suppressive cells (MDSCs) and regulatory T cells (Tregs) are of great clinical need. Here, we explore NKG2D-Fc as a modality to modulate antitumor immunity through the depletion of immunosuppressive MDSCs and Tregs in the TME. We have generated the NKG2D-Fc fusion protein and characterized its potential to mediate tumor control and overall survival in LL2 and MC38 murine models. Upon treatment of LL2 or MC38 tumor-bearing mice with NKG2D-Fc, we observe significant tumor control and enhanced survival compared to Fc control. When characterizing MDCSs and Tregs from tumor-bearing mice, we observe clear expression of NKG2D-ligand RAE1γ and subsequent binding of NKG2D-Fc fusion protein to both MDSCs and Tregs. Examining the immune profile of mice treated with NKG2D-Fc reveals significant depletion of MDSCs and Tregs in the TME, as well as an increase in NK cells likely due to the reversed suppressive TME. In conclusion, NKG2D-Fc induces antitumor immunity and tumor control through the depletion of MDSCs and Tregs, subsequently providing a niche for the infiltration and expansion of proinflammatory cells, such as NK cells. Strategies capable of modulating the immunosuppressive state in cancer are in high clinical demand. NKG2D-Fc is a simple, single tool capable of depleting both MDSCs and Tregs and should be further investigated as a therapeutic agent for the treatment of cancer.


Assuntos
Neoplasias do Colo/imunologia , Fragmentos Fc das Imunoglobulinas/imunologia , Neoplasias Pulmonares/imunologia , Subfamília K de Receptores Semelhantes a Lectina de Células NK/imunologia , Proteínas Recombinantes de Fusão/imunologia , Linfócitos T Reguladores/imunologia , Microambiente Tumoral/imunologia , Animais , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Citotoxicidade Imunológica/imunologia , Feminino , Imunossupressão , Células Matadoras Naturais/imunologia , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Camundongos , Camundongos Endogâmicos C57BL , Células Supressoras Mieloides/imunologia , Células Tumorais Cultivadas
11.
Science ; 368(6492)2020 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-32409444

RESUMO

De novo protein design has been successful in expanding the natural protein repertoire. However, most de novo proteins lack biological function, presenting a major methodological challenge. In vaccinology, the induction of precise antibody responses remains a cornerstone for next-generation vaccines. Here, we present a protein design algorithm called TopoBuilder, with which we engineered epitope-focused immunogens displaying complex structural motifs. In both mice and nonhuman primates, cocktails of three de novo-designed immunogens induced robust neutralizing responses against the respiratory syncytial virus. Furthermore, the immunogens refocused preexisting antibody responses toward defined neutralization epitopes. Overall, our design approach opens the possibility of targeting specific epitopes for the development of vaccines and therapeutic antibodies and, more generally, will be applicable to the design of de novo proteins displaying complex functional motifs.


Assuntos
Anticorpos Neutralizantes/biossíntese , Biologia Computacional/métodos , Epitopos Imunodominantes/química , Engenharia de Proteínas/métodos , Proteínas Recombinantes de Fusão/química , Vacinas contra Vírus Sincicial Respiratório/química , Vírus Sincicial Respiratório Humano/imunologia , Motivos de Aminoácidos , Humanos , Epitopos Imunodominantes/imunologia , Conformação Proteica , Proteínas Recombinantes de Fusão/imunologia , Vacinas contra Vírus Sincicial Respiratório/imunologia , Anticorpos de Domínio Único/química , Anticorpos de Domínio Único/imunologia
12.
EBioMedicine ; 55: 102743, 2020 May.
Artigo em Inglês | MEDLINE | ID: covidwho-27911

RESUMO

BACKGROUND: Coronaviruses pose a serious threat to global health as evidenced by Severe Acute Respiratory Syndrome (SARS), Middle East Respiratory Syndrome (MERS), and COVID-19. SARS Coronavirus (SARS-CoV), MERS Coronavirus (MERS-CoV), and the novel coronavirus, previously dubbed 2019-nCoV, and now officially named SARS-CoV-2, are the causative agents of the SARS, MERS, and COVID-19 disease outbreaks, respectively. Safe vaccines that rapidly induce potent and long-lasting virus-specific immune responses against these infectious agents are urgently needed. The coronavirus spike (S) protein, a characteristic structural component of the viral envelope, is considered a key target for vaccines for the prevention of coronavirus infection. METHODS: We first generated codon optimized MERS-S1 subunit vaccines fused with a foldon trimerization domain to mimic the native viral structure. In variant constructs, we engineered immune stimulants (RS09 or flagellin, as TLR4 or TLR5 agonists, respectively) into this trimeric design. We comprehensively tested the pre-clinical immunogenicity of MERS-CoV vaccines in mice when delivered subcutaneously by traditional needle injection, or intracutaneously by dissolving microneedle arrays (MNAs) by evaluating virus specific IgG antibodies in the serum of vaccinated mice by ELISA and using virus neutralization assays. Driven by the urgent need for COVID-19 vaccines, we utilized this strategy to rapidly develop MNA SARS-CoV-2 subunit vaccines and tested their pre-clinical immunogenicity in vivo by exploiting our substantial experience with MNA MERS-CoV vaccines. FINDINGS: Here we describe the development of MNA delivered MERS-CoV vaccines and their pre-clinical immunogenicity. Specifically, MNA delivered MERS-S1 subunit vaccines elicited strong and long-lasting antigen-specific antibody responses. Building on our ongoing efforts to develop MERS-CoV vaccines, promising immunogenicity of MNA-delivered MERS-CoV vaccines, and our experience with MNA fabrication and delivery, including clinical trials, we rapidly designed and produced clinically-translatable MNA SARS-CoV-2 subunit vaccines within 4 weeks of the identification of the SARS-CoV-2 S1 sequence. Most importantly, these MNA delivered SARS-CoV-2 S1 subunit vaccines elicited potent antigen-specific antibody responses that were evident beginning 2 weeks after immunization. INTERPRETATION: MNA delivery of coronaviruses-S1 subunit vaccines is a promising immunization strategy against coronavirus infection. Progressive scientific and technological efforts enable quicker responses to emerging pandemics. Our ongoing efforts to develop MNA-MERS-S1 subunit vaccines enabled us to rapidly design and produce MNA SARS-CoV-2 subunit vaccines capable of inducing potent virus-specific antibody responses. Collectively, our results support the clinical development of MNA delivered recombinant protein subunit vaccines against SARS, MERS, COVID-19, and other emerging infectious diseases.


Assuntos
Betacoronavirus/imunologia , Coronavírus da Síndrome Respiratória do Oriente Médio/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Vacinas Virais/administração & dosagem , Adjuvantes Imunológicos/administração & dosagem , Animais , Anticorpos Antivirais/biossíntese , Anticorpos Antivirais/sangue , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/prevenção & controle , Feminino , Imunização Secundária , Imunoglobulina G/biossíntese , Imunoglobulina G/sangue , Injeções Subcutâneas , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Proteínas Recombinantes de Fusão/administração & dosagem , Proteínas Recombinantes de Fusão/imunologia , Organismos Livres de Patógenos Específicos , Fatores de Tempo , Vacinas de Subunidades/administração & dosagem , Vacinas Virais/imunologia
13.
Biotechnol Bioeng ; 117(7): 1990-2007, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32297972

RESUMO

High-quality antibody (Ab) production depends on the availability of immunologically relevant antigens. We present a potentially universal platform for generating soluble antigens from bacterial hosts, tailored to immunized animals for Ab production. A novel RNA-dependent chaperone, in which the target antigen is genetically fused with an RNA-interacting domain (RID) docking tag derived from the immunized host, promotes the solubility and robust folding of the target antigen. We selected the N-terminal tRNA-binding domain of lysyl-tRNA synthetase (LysRS) as the RID for fusion with viral proteins and demonstrated the expression of the RID fusion proteins in their soluble and native conformations; immunization predominantly elicited Ab responses to the target antigen, whereas the "self" RID tag remained nonimmunogenic. Differential immunogenicity of the fusion proteins greatly enriched and simplified the screening of hybridoma clones of monoclonal antibodies (mAbs), enabling specific and sensitive serodiagnosis of MERS-CoV infection. Moreover, mAbs against the consensus influenza hemagglutinin stalk domain enabled a novel assay for trivalent seasonal influenza vaccines. The Fc-mediated effector function was demonstrated, which could be harnessed for the design of next-generation "universal" influenza vaccines. The nonimmunogenic built-in antigen folding module tailored to a repertoire of immunized animal hosts will drive immunochemical diagnostics, therapeutics, and designer vaccines.


Assuntos
Anticorpos Monoclonais/biossíntese , Anticorpos Antivirais/biossíntese , Antígenos Virais/química , Infecções por Coronavirus/diagnóstico , Hibridomas/imunologia , Coronavírus da Síndrome Respiratória do Oriente Médio/imunologia , Chaperonas Moleculares , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/isolamento & purificação , Anticorpos Antivirais/imunologia , Anticorpos Antivirais/isolamento & purificação , Antígenos Virais/genética , Antígenos Virais/imunologia , Infecções por Coronavirus/sangue , Ensaio de Imunoadsorção Enzimática , Feminino , Glicoproteínas de Hemaglutininação de Vírus da Influenza/química , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Humanos , Imunização , Vacinas contra Influenza , Lisina-tRNA Ligase/química , Lisina-tRNA Ligase/genética , Camundongos , Camundongos Endogâmicos BALB C , Conformação Proteica , Domínios Proteicos , Dobramento de Proteína , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Testes Sorológicos , Solubilidade
14.
EBioMedicine ; 55: 102743, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32249203

RESUMO

BACKGROUND: Coronaviruses pose a serious threat to global health as evidenced by Severe Acute Respiratory Syndrome (SARS), Middle East Respiratory Syndrome (MERS), and COVID-19. SARS Coronavirus (SARS-CoV), MERS Coronavirus (MERS-CoV), and the novel coronavirus, previously dubbed 2019-nCoV, and now officially named SARS-CoV-2, are the causative agents of the SARS, MERS, and COVID-19 disease outbreaks, respectively. Safe vaccines that rapidly induce potent and long-lasting virus-specific immune responses against these infectious agents are urgently needed. The coronavirus spike (S) protein, a characteristic structural component of the viral envelope, is considered a key target for vaccines for the prevention of coronavirus infection. METHODS: We first generated codon optimized MERS-S1 subunit vaccines fused with a foldon trimerization domain to mimic the native viral structure. In variant constructs, we engineered immune stimulants (RS09 or flagellin, as TLR4 or TLR5 agonists, respectively) into this trimeric design. We comprehensively tested the pre-clinical immunogenicity of MERS-CoV vaccines in mice when delivered subcutaneously by traditional needle injection, or intracutaneously by dissolving microneedle arrays (MNAs) by evaluating virus specific IgG antibodies in the serum of vaccinated mice by ELISA and using virus neutralization assays. Driven by the urgent need for COVID-19 vaccines, we utilized this strategy to rapidly develop MNA SARS-CoV-2 subunit vaccines and tested their pre-clinical immunogenicity in vivo by exploiting our substantial experience with MNA MERS-CoV vaccines. FINDINGS: Here we describe the development of MNA delivered MERS-CoV vaccines and their pre-clinical immunogenicity. Specifically, MNA delivered MERS-S1 subunit vaccines elicited strong and long-lasting antigen-specific antibody responses. Building on our ongoing efforts to develop MERS-CoV vaccines, promising immunogenicity of MNA-delivered MERS-CoV vaccines, and our experience with MNA fabrication and delivery, including clinical trials, we rapidly designed and produced clinically-translatable MNA SARS-CoV-2 subunit vaccines within 4 weeks of the identification of the SARS-CoV-2 S1 sequence. Most importantly, these MNA delivered SARS-CoV-2 S1 subunit vaccines elicited potent antigen-specific antibody responses that were evident beginning 2 weeks after immunization. INTERPRETATION: MNA delivery of coronaviruses-S1 subunit vaccines is a promising immunization strategy against coronavirus infection. Progressive scientific and technological efforts enable quicker responses to emerging pandemics. Our ongoing efforts to develop MNA-MERS-S1 subunit vaccines enabled us to rapidly design and produce MNA SARS-CoV-2 subunit vaccines capable of inducing potent virus-specific antibody responses. Collectively, our results support the clinical development of MNA delivered recombinant protein subunit vaccines against SARS, MERS, COVID-19, and other emerging infectious diseases.


Assuntos
Betacoronavirus/imunologia , Coronavírus da Síndrome Respiratória do Oriente Médio/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Vacinas Virais/administração & dosagem , Adjuvantes Imunológicos/administração & dosagem , Animais , Anticorpos Antivirais/biossíntese , Anticorpos Antivirais/sangue , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/prevenção & controle , Feminino , Imunização Secundária , Imunoglobulina G/biossíntese , Imunoglobulina G/sangue , Injeções Subcutâneas , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Proteínas Recombinantes de Fusão/administração & dosagem , Proteínas Recombinantes de Fusão/imunologia , Organismos Livres de Patógenos Específicos , Fatores de Tempo , Vacinas de Subunidades/administração & dosagem , Vacinas Virais/imunologia
15.
PLoS Negl Trop Dis ; 14(3): e0008093, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32176691

RESUMO

Human leishmaniasis is a public health problem worldwide for which the development of a vaccine remains a challenge. T cell-mediated immune responses are crucial for protection. Peptide vaccines based on the identification of immunodominant T cell epitopes able to induce T cell specific immune responses constitute a promising strategy. Here, we report the identification of human leukocyte antigen class-I (HLA-I) and -II (HLA-II)-restricted multi-epitope peptides from Leishmania proteins that we have previously described as vaccine candidates. Promastigote Surface Antigen (PSA), LmlRAB (L. major large RAB GTPase) and Histone (H2B) were screened, in silico, for T cell epitopes. 6 HLA-I and 5 HLA-II-restricted multi-epitope peptides, able to bind to the most frequent HLA molecules, were designed and used as pools to stimulate PBMCs from individuals with healed cutaneous leishmaniasis. IFN-γ, IL-10, TNF-α and granzyme B (GrB) production was evaluated by ELISA/CBA. The frequency of IFN-γ-producing T cells was quantified by ELISpot. T cells secreting cytokines and memory T cells were analyzed by flow cytometry. 16 of 25 peptide pools containing HLA-I, HLA-II or HLA-I and -II peptides were able to induce specific and significant IFN-γ levels. No IL-10 was detected. 6 peptide pools were selected among those inducing the highest IFN-γ levels for further characterization. 3/6 pools were able to induce a significant increase of the percentages of CD4+IFN-γ+, CD8+IFN-γ+ and CD4+GrB+ T cells. The same pools also induced a significant increase of the percentages of bifunctional IFN-γ+/TNF-α+CD4+ and/or central memory T cells. We identified highly promiscuous HLA-I and -II restricted epitope combinations from H2B, PSA and LmlRAB proteins that stimulate both CD4+ and CD8+ T cell responses in recovered individuals. These multi-epitope peptides could be used as potential components of a polytope vaccine for human leishmaniasis.


Assuntos
Antígenos de Protozoários/imunologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Epitopos de Linfócito T/imunologia , Leishmania/imunologia , Leishmaniose Cutânea/imunologia , Proteínas Recombinantes de Fusão/imunologia , Adulto , Antígenos de Protozoários/genética , Ensaio de Imunoadsorção Enzimática , Epitopos de Linfócito T/genética , Feminino , Citometria de Fluxo , Granzimas/análise , Antígenos de Histocompatibilidade Classe I/metabolismo , Antígenos de Histocompatibilidade Classe II/metabolismo , Humanos , Interferon gama/análise , Interleucina-10/análise , Masculino , Pessoa de Meia-Idade , Ligação Proteica , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Fator de Necrose Tumoral alfa/análise , Voluntários , Adulto Jovem
16.
Nat Commun ; 11(1): 1137, 2020 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-32111835

RESUMO

The interaction between immune cells and phosphatidylserine (PS) molecules exposed on the surface of apoptotic-tumor bodies, such as those induced by chemotherapies, contributes to the formation of an immunosuppressive tumor microenvironment (TME). Annexin A5 (AnxA5) binds with high affinity to PS externalized by apoptotic cells, thereby hindering their interaction with immune cells. Here, we show that AnxA5 administration rescue the immunosuppressive state of the TME induced by chemotherapy. Due to the preferential homing of AnxA5 to the TME enriched with PS+ tumor cells, we demonstrate in vivo that fusing tumor-antigen peptide to AnxA5 significantly enhances its immunogenicity and antitumor efficacy when administered after chemotherapy. Also, the therapeutic antitumor effect of an AnxA5-peptide fusion can be further enhanced by administration of other immune checkpoint inhibitors. Our findings support the administration of AnxA5 following chemotherapy as a promising immune checkpoint inhibitor for cancer treatment.


Assuntos
Anexina A5/uso terapêutico , Vacinas Anticâncer/uso terapêutico , Fatores Imunológicos/uso terapêutico , Neoplasias/terapia , Animais , Anexina A5/genética , Anexina A5/metabolismo , Anticorpos Bloqueadores/uso terapêutico , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/imunologia , Antígenos de Neoplasias/uso terapêutico , Vacinas Anticâncer/genética , Vacinas Anticâncer/imunologia , Vacinas Anticâncer/metabolismo , Linhagem Celular Tumoral , Cisplatino/efeitos adversos , Cisplatino/uso terapêutico , Modelos Animais de Doenças , Feminino , Humanos , Fatores Imunológicos/metabolismo , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Neoplasias/imunologia , Proteínas E7 de Papillomavirus/genética , Proteínas E7 de Papillomavirus/imunologia , Proteínas E7 de Papillomavirus/uso terapêutico , Fosfatidilserinas/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/uso terapêutico , Fator de Crescimento Transformador beta/imunologia , Fator de Crescimento Transformador beta/metabolismo , Microambiente Tumoral/efeitos dos fármacos , Microambiente Tumoral/imunologia , Fator de Necrose Tumoral alfa/imunologia , Fator de Necrose Tumoral alfa/metabolismo
17.
PLoS One ; 15(3): e0230138, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32176708

RESUMO

Surface-expressed colonization factors and their subunits are promising candidates for inclusion into a multivalent vaccine targeting enterotoxigenic Escherichia coli (ETEC), a leading cause of acute bacterial diarrhea in developing regions. However, soluble antigens are often poorly immunogenic in the absence of an adjuvant. We show here that the serum immune response to CfaE, the adhesin of the ETEC colonization factor CFA/I, can be enhanced in BALB/c mice by immunization with a chimeric antigen containing CfaE and pentameric cholera toxin B subunit (CTB) of cholera toxin from Vibrio cholerae. We constructed this antigen by replacing the coding sequence for the A1 domain of the cholera toxin A subunit (CTA) with the sequence of donor strand complemented CfaE (dscCfaE) within the cholera toxin operon, resulting in a dscCfaE-CTA2 fusion. After expression, via non-covalent interactions between CTA2 and CTB, the fusion and CTB polypeptides assemble into a complex containing a single dscCfaE-CTA2 protein bound to pentameric CTB (dscCfaE-CTA2/CTB). This holotoxin-like chimera retained the GM1 ganglioside binding activity of CTB, as well as the ability of CfaE to mediate the agglutination of bovine red blood cells when adsorbed to polystyrene beads. When administered intranasally to mice, the presence of CTB in the chimera significantly increased the serum immune response to CfaE compared to dscCfaE alone, stimulating a response similar to that obtained with a matched admixture of dscCfaE and CTB. However, by the orogastric route, immunization with the chimera elicited a superior functional immune response compared to an equivalent admixture of dscCfaE and CTB, supporting further investigation of the chimera as an ETEC vaccine candidate.


Assuntos
Toxina da Cólera , Escherichia coli Enterotoxigênica/imunologia , Vacinas contra Escherichia coli/imunologia , Proteínas de Fímbrias , Proteínas Recombinantes de Fusão , Adesinas Bacterianas/imunologia , Adesinas Bacterianas/metabolismo , Adjuvantes Imunológicos/administração & dosagem , Animais , Quimera/genética , Toxina da Cólera/genética , Toxina da Cólera/imunologia , Toxina da Cólera/metabolismo , Feminino , Proteínas de Fímbrias/genética , Proteínas de Fímbrias/imunologia , Proteínas de Fímbrias/metabolismo , Imunização , Imunogenicidade da Vacina , Camundongos , Camundongos Endogâmicos BALB C , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/metabolismo
18.
Mol Immunol ; 121: 118-126, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32199211

RESUMO

Foot-and-mouth disease (FMD) is an acute, severe, and highly contagious disease that affects cloven-hoofed animals and can lead to serious economic losses and social effects. Therefore, a safe and effective subunit vaccine is required to prevent and control FMD. Dendritic cells (DCs) are a type of professional antigen presenting cell (APC). Immature DCs are typically stimulated by various adjuvants via immune receptors (e.g., toll-like receptor 4 [TLR4]), which activate DCs to induce their maturation. TLR4 has been well-established to induce both innate and adaptive immune responses to various external microbial or internal damage-related molecular patterns. In this study, the multi-epitope immunogen, HAO, of foot-and-mouth disease virus (FMDV) serotypes A and O was fused with the recombinant protein, heparin-binding hemagglutinin (HBHA), a novel TLR4 agonist, to obtain a new recombinant fusion protein, termed HAO-HBHA. HAO-HBHA was found to be highly efficient at activating murine DCs by the TLR4 pathway, both in vitro and in vivo. HAO-HBHA elicited strong specific humoral immune responses detected with an ELISA and virus neutralizing antibody test (VNT). HAO-HBHA also elevated the cellular immune responses, as indicated by intracellular cytokine (e.g., IFN-γ, TNF-α, IL-4, IL-6, IL-10, and IL-12p70) expression in Th1 and Th2 cells. As a TLR4 agonist, HBHA has significant advantages for enhancing the immune efficacy of a FMDV serotype A and O bivalent multi-epitope vaccine. These findings provide a novel strategy for the development of a safe and effective multi-epitope vaccine candidate against FMDV and further extends the application of TLR agonist-based vaccine platforms.


Assuntos
Vírus da Febre Aftosa/imunologia , Febre Aftosa/prevenção & controle , Lectinas/farmacologia , Vacinas Virais/administração & dosagem , Adjuvantes Imunológicos/farmacologia , Animais , Anticorpos Neutralizantes/sangue , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/sangue , Anticorpos Antivirais/imunologia , Células Dendríticas/imunologia , Epitopos de Linfócito B/genética , Epitopos de Linfócito B/imunologia , Epitopos de Linfócito T/genética , Epitopos de Linfócito T/imunologia , Feminino , Febre Aftosa/sangue , Febre Aftosa/imunologia , Febre Aftosa/virologia , Vírus da Febre Aftosa/genética , Imunidade Celular , Imunogenicidade da Vacina , Lectinas/imunologia , Camundongos , Mycobacterium tuberculosis/imunologia , Proteínas Recombinantes de Fusão/administração & dosagem , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Sorogrupo , Receptor 4 Toll-Like/agonistas , Receptor 4 Toll-Like/imunologia , Vacinas de Subunidades/administração & dosagem , Vacinas de Subunidades/genética , Vacinas de Subunidades/imunologia , Vacinas Virais/genética , Vacinas Virais/imunologia
19.
Sci Rep ; 10(1): 1664, 2020 02 03.
Artigo em Inglês | MEDLINE | ID: mdl-32015441

RESUMO

The use of monoclonal antibodies is ubiquitous in science and biomedicine but the generation and validation process of antibodies is nevertheless complicated and time-consuming. To address these issues we developed a novel selective technology based on an artificial cell surface construct by which secreted antibodies were connected to the corresponding hybridoma cell when they possess the desired antigen-specificity. Further the system enables the selection of desired isotypes and the screening for potential cross-reactivities in the same context. For the design of the construct we combined the transmembrane domain of the EGF-receptor with a hemagglutinin epitope and a biotin acceptor peptide and performed a transposon-mediated transfection of myeloma cell lines. The stably transfected myeloma cell line was used for the generation of hybridoma cells and an antigen- and isotype-specific screening method was established. The system has been validated for globular protein antigens as well as for haptens and enables a fast and early stage selection and validation of monoclonal antibodies in one step.


Assuntos
Anticorpos Monoclonais/biossíntese , Células Produtoras de Anticorpos/imunologia , Hibridomas/imunologia , Animais , Especificidade de Anticorpos , Antígenos/genética , Antígenos/imunologia , Biotinilação , Fusão Celular , Linhagem Celular Tumoral , Epitopos/genética , Epitopos/imunologia , Receptores ErbB/genética , Receptores ErbB/imunologia , Hemaglutininas/genética , Hemaglutininas/imunologia , Humanos , Hibridomas/citologia , Camundongos , Mieloma Múltiplo/genética , Mieloma Múltiplo/imunologia , Ovalbumina/imunologia , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Transfecção
20.
J Immunol ; 204(6): 1543-1561, 2020 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-32066595

RESUMO

Elicitation of broadly neutralizing Ab (bNAb) responses toward the conserved HIV-1 envelope (Env) CD4 binding site (CD4bs) by vaccination is an important goal for vaccine development and yet to be achieved. The outcome of previous immunogenicity studies suggests that the limited accessibility of the CD4bs and the presence of predominant nonneutralizing determinants (nND) on Env may impede the elicitation of bNAbs and their precursors by vaccination. In this study, we designed a panel of novel immunogens that 1) preferentially expose the CD4bs by selective elimination of glycosylation sites flanking the CD4bs, and 2) minimize the nND immune response by engineering fusion proteins consisting of gp120 Core and one or two CD4-induced (CD4i) mAbs for masking nND epitopes, referred to as gp120-CD4i fusion proteins. As expected, the fusion proteins possess improved antigenicity with retained affinity for VRC01-class, CD4bs-directed bNAbs and dampened affinity for nonneutralizing Abs. We immunized C57BL/6 mice with these fusion proteins and found that overall the fusion proteins elicit more focused CD4bs Ab response than prototypical gp120 Core by serological analysis. Consistently, we found that mice immunized with selected gp120-CD4i fusion proteins have higher frequencies of germinal center-activated B cells and CD4bs-directed memory B cells than those inoculated with parental immunogens. We isolated three mAbs from mice immunized with selected gp120-CD4i fusion proteins and found that their footprints on Env are similar to VRC01-class bNAbs. Thus, using gp120-CD4i fusion proteins with selective glycan deletion as immunogens could focus Ab response toward CD4bs epitope.


Assuntos
Vacinas contra a AIDS/imunologia , Anticorpos Neutralizantes/sangue , Anticorpos Anti-HIV/sangue , Proteína gp120 do Envelope de HIV/imunologia , Infecções por HIV/prevenção & controle , HIV-1/imunologia , Vacinas contra a AIDS/administração & dosagem , Vacinas contra a AIDS/genética , Animais , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Sítios de Ligação de Anticorpos/genética , Sítios de Ligação de Anticorpos/imunologia , Antígenos CD4/imunologia , Antígenos CD4/metabolismo , Epitopos de Linfócito T/genética , Epitopos de Linfócito T/imunologia , Feminino , Anticorpos Anti-HIV/imunologia , Proteína gp120 do Envelope de HIV/genética , Infecções por HIV/sangue , Infecções por HIV/imunologia , Infecções por HIV/virologia , HIV-1/genética , Humanos , Imunogenicidade da Vacina , Camundongos , Modelos Animais , Proteínas Recombinantes de Fusão/administração & dosagem , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologia
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