RESUMO
The rabbit is a commonly used animal model in studying antibody responses in HIV/AIDS vaccine development. However, no rabbit monoclonal antibodies (MAbs) have been developed previously to study the epitope-specific antibody responses against HIV-1 envelope (Env) glycoproteins, and little is known about how the rabbit immune system can mimic the human immune system in eliciting such antibodies. Here we present structural analyses of two rabbit MAbs, R56 and R20, against the third variable region (V3) of HIV-1 gp120. R56 recognizes the well-studied immunogenic region in the V3 crown, while R20 targets a less-studied region at the C terminus of V3. By comparison of the Fab/epitope complex structures of these two antibodies raised by immunization with that of the corresponding human antibodies derived from patients chronically infected with HIV-1, we found that rabbit antibodies can recognize immunogenic regions of gp120 and mimic the binding modes of human antibodies. This result can provide new insight into the use of the rabbit as an animal model in AIDS vaccine development.
Assuntos
Anticorpos Monoclonais/metabolismo , Anticorpos Anti-HIV/metabolismo , Proteína gp120 do Envelope de HIV/metabolismo , HIV-1/imunologia , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/imunologia , Cristalografia por Raios X , Epitopos/química , Epitopos/imunologia , Epitopos/metabolismo , Anticorpos Anti-HIV/química , Anticorpos Anti-HIV/imunologia , Proteína gp120 do Envelope de HIV/química , Proteína gp120 do Envelope de HIV/imunologia , HIV-1/química , Humanos , Modelos Moleculares , Ligação Proteica , Conformação Proteica , CoelhosRESUMO
The majority of available monoclonal antibodies (MAbs) in the current HIV vaccine field are generated from HIV-1-infected people. In contrast, preclinical immunogenicity studies have mainly focused on polyclonal antibody responses in experimental animals. Although rabbits have been widely used for antibody studies, there has been no report of using rabbit MAbs to dissect the specificity of antibody responses for AIDS vaccine development. Here we report on the production of a panel of 12 MAbs from a New Zealand White (NZW) rabbit that was immunized with an HIV-1 JR-FL gp120 DNA prime and protein boost vaccination regimen. These rabbit MAbs recognized a diverse repertoire of envelope (Env) epitopes ranging from the highly immunogenic V3 region to several previously underappreciated epitopes in the C1, C4, and C5 regions. Nine MAbs showed cross-reactivity to gp120s of clades other than clade B. Increased somatic mutation and extended CDR3 were observed with Ig genes of several molecularly cloned rabbit MAbs. Phylogenic tree analysis showed that the heavy chains of MAbs recognizing the same region on gp120 tend to segregate into an independent subtree. At least three rabbit MAbs showed neutralizing activities with various degrees of breadth and potency. The establishment of this rabbit MAb platform will significantly enhance our ability to test optimal designs of Env immunogens to gain a better understanding of the structural specificity and evolution process of Env-specific antibody responses elicited by candidate AIDS vaccines.
Assuntos
Anticorpos Monoclonais/imunologia , Epitopos/imunologia , Anticorpos Anti-HIV/imunologia , Proteína gp120 do Envelope de HIV/imunologia , HIV-1/imunologia , Animais , Reações Cruzadas , Testes de Neutralização , CoelhosRESUMO
The COVID-19 pandemic has led to the deaths of millions of people and severe global economic impacts. Small molecule therapeutics have played an important role in the fight against SARS-CoV-2, the virus responsible for COVID-19, but their efficacy has been limited in scope and availability, with many people unable to access their benefits, and better options are needed. EDP-235 is specifically designed to inhibit the SARS-CoV-2 3CLpro, with potent nanomolar activity against all SARS-CoV-2 variants to date, as well as clinically relevant human and zoonotic coronaviruses. EDP-235 maintains potency against variants bearing mutations associated with nirmatrelvir resistance. Additionally, EDP-235 demonstrates a ≥ 500-fold selectivity index against multiple host proteases. In a male Syrian hamster model of COVID-19, EDP-235 suppresses SARS-CoV-2 replication and viral-induced hamster lung pathology. In a female ferret model, EDP-235 inhibits production of SARS-CoV-2 infectious virus and RNA at multiple anatomical sites. Furthermore, SARS-CoV-2 contact transmission does not occur when naïve ferrets are co-housed with infected, EDP-235-treated ferrets. Collectively, these results demonstrate that EDP-235 is a broad-spectrum coronavirus inhibitor with efficacy in animal models of primary infection and transmission.
Assuntos
Antivirais , COVID-19 , Proteases 3C de Coronavírus , SARS-CoV-2 , Replicação Viral , Animais , Cricetinae , Feminino , Humanos , Masculino , Antivirais/farmacologia , Chlorocebus aethiops , Proteases 3C de Coronavírus/antagonistas & inibidores , Proteases 3C de Coronavírus/metabolismo , COVID-19/virologia , COVID-19/transmissão , Tratamento Farmacológico da COVID-19 , Modelos Animais de Doenças , Furões , Lactamas , Leucina , Pulmão/virologia , Pulmão/efeitos dos fármacos , Pulmão/patologia , Mesocricetus , Nitrilas , Compostos Orgânicos , Pandemias/prevenção & controle , Pneumonia Viral/tratamento farmacológico , Pneumonia Viral/virologia , Pneumonia Viral/transmissão , Pneumonia Viral/prevenção & controle , Prolina , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/genética , SARS-CoV-2/fisiologia , Células Vero , Replicação Viral/efeitos dos fármacosRESUMO
Identification of immunogens capable of eliciting broadly neutralizing antibody (NAb) responses against HIV-1 is a major goal toward the development of an AIDS vaccine. Despite significant progress in understanding the structural features of the HIV-1 envelope glycoprotein (Env) and the discovery of multiple broadly neutralizing monoclonal antibodies with defined antigenic structures, the design of optimal Env immunogens to elicit broad NAbs remains a major challenge. As the structural determinants of Env immunogenicity remain unclear, we assessed two closely related Env antigens isolated from the same HIV-1-infected patient with different phenotypic features to identify what may result in a favorable immunogenic profile. One Env, B33, isolated from brain, was highly macrophage tropic with a high CD4 affinity, while the other, LN40, isolated from the lymph nodes, was poorly macrophage tropic with a low CD4 affinity. Using a DNA prime-protein boost approach, rabbits primed with LN40 Env antigen had a NAb response against heterologous primary isolates, while B33 Env antigens were capable of eliciting NAbs against only homologous and sensitive viral isolates. Further analysis revealed that the specificity of NAbs elicited by the LN40 antigen mapped to limited residues within or flanking the CD4 binding site. Certain key structural determinants were identified that could differentiate primary Env immunogens based on their potential to elicit broader NAbs. This progress will facilitate the rational design of effective HIV-1 vaccine formulations with optimal Env antigens.
Assuntos
Anticorpos Neutralizantes/sangue , Anticorpos Anti-HIV/sangue , Infecções por HIV/imunologia , Infecções por HIV/virologia , HIV-1/imunologia , HIV-1/isolamento & purificação , Produtos do Gene env do Vírus da Imunodeficiência Humana/imunologia , Animais , Encéfalo/virologia , Humanos , Imunização/métodos , Imunização Secundária/métodos , Linfonodos/virologia , Coelhos , Tropismo Viral , Produtos do Gene env do Vírus da Imunodeficiência Humana/administração & dosagemRESUMO
A major challenge in human immunodeficiency virus type 1 (HIV-1) vaccine development is to elicit potent and broadly neutralizing antibodies that are effective against primary viral isolates. Previously, we showed that DNA prime-protein boost vaccination using HIV-1 gp120 antigens was more effective in eliciting neutralizing antibodies against primary HIV-1 isolates than was a recombinant gp120 protein-only vaccination approach. In the current study, we analyzed the difference in antibody specificities in rabbit sera elicited by these two immunization regimens using peptide enzyme-linked immunosorbent assay and a competitive virus capture assay. Our results indicate that a DNA prime-protein boost regimen is more effective than a protein-alone vaccination approach in inducing antibodies that target two key neutralizing domains: the V3 loop and the CD4 binding site. In particular, positive antibodies targeting several peptides that overlap with the known CD4 binding area were detected only in DNA-primed sera. Different profiles of antibody specificities provide insight into the mechanisms behind the elicitation of better neutralizing antibodies with the DNA prime-protein boost approach, and our results support the use of this approach to further optimize Env formulations for HIV vaccine development.
Assuntos
Vacinas contra a AIDS/administração & dosagem , Vacinas contra a AIDS/imunologia , Anticorpos Antivirais/sangue , Proteína gp120 do Envelope de HIV/imunologia , Vacinas de DNA/administração & dosagem , Vacinas de DNA/imunologia , Animais , Ensaio de Imunoadsorção Enzimática/métodos , Epitopos/imunologia , Feminino , HIV-1/imunologia , Imunização Secundária , Modelos Moleculares , Testes de Neutralização , Coelhos , Vacinas de Subunidades Antigênicas/imunologia , Vacinas Sintéticas/imunologiaRESUMO
This article was originally published under a CC BY-NC-SA License, but has now been made available under a CC BY 4.0 License.
RESUMO
Studies using the European rabbit Oryctolagus cuniculus contributed to elucidating numerous fundamental aspects of antibody structure and diversification mechanisms and continue to be valuable for the development and testing of therapeutic humanized polyclonal and monoclonal antibodies. Additionally, during the last two decades, the use of the European rabbit as an animal model has been increasingly extended to many human diseases. This review documents the continuing wide utility of the rabbit as a reliable disease model for development of therapeutics and vaccines and studies of the cellular and molecular mechanisms underlying many human diseases. Examples include syphilis, tuberculosis, HIV-AIDS, acute hepatic failure and diseases caused by noroviruses, ocular herpes, and papillomaviruses. The use of rabbits for vaccine development studies, which began with Louis Pasteur's rabies vaccine in 1881, continues today with targets that include the potentially blinding HSV-1 virus infection and HIV-AIDS. Additionally, two highly fatal viral diseases, rabbit hemorrhagic disease and myxomatosis, affect the European rabbit and provide unique models to understand co-evolution between a vertebrate host and viral pathogens.
Assuntos
Modelos Animais de Doenças , Animais , Evolução Biológica , Humanos , Sistema Imunitário/fisiologia , Imunidade , CoelhosRESUMO
In order to identify novel genes associated with the initiation of programmed cell death during development, we employed a differential screening protocol to isolate cDNAs that were induced when the intersegmental muscles (ISM) of the moth Manduca sexta become committed to die at the end of metamorphosis. In this report we provide the first description of Acheron (Achn), a novel protein that was isolated in this screen. Acheron contains three Lupus antigen (La) repeats, nuclear localization and export (NLS and NES) signals, and an RNA recognition motif. Achn defines a new subfamily of La proteins that appears to have branched from authentic La protein relatively late in metazoan evolution. Achn is widely expressed in various insect, mouse and human tissues. Consistent with its expression during ISM death, Achn has been shown in separate studies to control muscle differentiation and apoptosis in both mice and zebrafish. These data define Achn as a newly discovered regulatory molecule that presumably mediates a variety of developmental and homeostatic processes in animals.
Assuntos
Apoptose , Autoantígenos/genética , Autoantígenos/metabolismo , Manduca/metabolismo , Músculo Esquelético/citologia , Músculo Esquelético/metabolismo , Ribonucleoproteínas/genética , Ribonucleoproteínas/metabolismo , Sequência de Aminoácidos , Animais , Autoantígenos/química , Linhagem Celular Tumoral , Mapeamento Cromossômico , Clonagem Molecular , DNA Complementar/genética , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Genoma Humano/genética , Humanos , Camundongos , Dados de Sequência Molecular , Filogenia , Ribonucleoproteínas/química , Análise de Sequência de DNA , Antígeno SS-BRESUMO
The RepliVax vaccine platform(RV) is based on flavivirus genomes that are rationally attenuated by deletion. The self-limiting infection provided by RV has been demonstrated to be safe, highly immunogenic and efficacious for several vaccine candidates against flaviviruses. Here respiratory syncytial virus (RSV) F, influenza virus HA, and simian immunodeficiency virus (SIV) Env proteins were expressed in place of either prM-E or C-prM-E gene deletions of the West Nile (WN) virus genome. The resulting RV-RSV, -influenza and -SIV vaccine prototypes replicated efficiently in complementing helper cells expressing the WN structural proteins in trans. Expressed antigens exhibited correct post-translational processing and the RV recombinants were shown to be highly attenuated and immunogenic in mice, eliciting strong antigen-specific antibodies as well as detectable T-cell responses. These data support the utility of RV vectors for development of vaccines against non-flavivirus targets including rabies and HIV.
Assuntos
Vírus Defeituosos/genética , Portadores de Fármacos , Vetores Genéticos , Vacinas Virais/imunologia , Vírus do Nilo Ocidental/genética , Animais , Anticorpos Antivirais/sangue , Citomegalovirus/genética , Citomegalovirus/imunologia , Expressão Gênica , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Camundongos Endogâmicos BALB C , Orthomyxoviridae/genética , Orthomyxoviridae/imunologia , Vírus da Imunodeficiência Símia/genética , Vírus da Imunodeficiência Símia/imunologia , Linfócitos T/imunologia , Linfócitos T Auxiliares-Indutores , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologia , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/imunologia , Proteínas Virais de Fusão/genética , Proteínas Virais de Fusão/imunologia , Vacinas Virais/administração & dosagem , Vacinas Virais/genética , Replicação ViralRESUMO
The fourth conserved region (C4) in the HIV-1 envelope glycoprotein (Env) gp120 is a structural element that is important for its function, as it binds to both the receptor CD4 and the co-receptor CCR5/CXCR4. It has long been known that this region is highly immunogenic and that it harbors B-cell as well as T-cell epitopes. It is the target of a number of antibodies in animal studies, which are called CD4-blockers. However, the mechanism by which the virus shields itself from such antibody responses is not known. Here, we determined the crystal structure of R53 in complex with its epitope peptide using a novel anti-C4 rabbit monoclonal antibody R53. Our data show that although the epitope of R53 covers a highly conserved sequence (433)AMYAPPI(439), it is not available in the gp120 trimer and in the CD4-bound conformation. Our results suggest a masking mechanism to explain how HIV-1 protects this critical region from the human immune system.
Assuntos
Anticorpos Monoclonais/química , Anticorpos Monoclonais/imunologia , Antígenos CD4/imunologia , Epitopos/imunologia , Proteína gp120 do Envelope de HIV/imunologia , Animais , Anticorpos Monoclonais/metabolismo , Sítios de Ligação de Anticorpos/imunologia , Ligação Competitiva , Antígenos CD4/metabolismo , Cristalização , Epitopos/química , Epitopos/metabolismo , Proteína gp120 do Envelope de HIV/metabolismo , Fragmentos Fab das Imunoglobulinas/isolamento & purificação , Fragmentos Fab das Imunoglobulinas/metabolismo , Coelhos , Receptores CCR5/imunologia , Receptores CCR5/metabolismo , Receptores CXCR4/imunologia , Receptores CXCR4/metabolismo , OvinosRESUMO
Using a gp120 envelope glycoprotein from the JR-FL strain of human immunodeficiency virus-1 (HIV-1) as a model antigen, the goal of the current study was to evaluate the level and quality of antibody responses elicited by different prime-boost vaccination regimens (protein only, DNA only, DNA plus protein) in rabbits. Our data demonstrated that incorporating DNA immunization as a prime in a heterologous prime-boost regimen was able to elicit a more diverse and conformational epitope profile, higher antibody avidity, and improved neutralizing activity than immunization with only protein. Additionally, this improved neutralizing activity was observed in spite of similar antibody specificities and avidities seen when only DNA vaccination was used, providing additional evidence that the use of a combination immunization regimen increases the protective antibody response. Insights gained from the current study confirmed that the heterologous DNA prime-protein boost approach is effective in eliciting not only high level but also improved quality of antigen-specific antibody responses, and thus may offer a new technology platform to develop better and safer subunit vaccines.
Assuntos
Vacinas contra a AIDS/imunologia , Afinidade de Anticorpos , Anticorpos Anti-HIV/sangue , Proteína gp120 do Envelope de HIV/imunologia , Imunização Secundária/métodos , Vacinação/métodos , Vacinas de DNA/imunologia , Animais , Anticorpos Neutralizantes/sangue , Proteína gp120 do Envelope de HIV/genética , CoelhosRESUMO
In the current report, we compared the specificities of antibody responses in sera from volunteers enrolled in three US NIH-supported HIV vaccine trials using different immunization regimens. HIV-1 Env-specific binding antibody, neutralizing antibody, antibody-dependent cell-mediated cytotoxicity (ADCC), and profiles of antibody specificity were analyzed for human immune sera collected from vaccinees enrolled in the NIH HIV Vaccine Trial Network (HVTN) Study #041 (recombinant protein alone), HVTN Study #203 (poxviral vector prime-protein boost), and the DP6-001 study (DNA prime-protein boost). Vaccinees from HVTN Study #041 had the highest neutralizing antibody activities against the sensitive virus along with the highest binding antibody responses, particularly those directed toward the V3 loop. DP6-001 sera showed a higher frequency of positive neutralizing antibody activities against more resistant viral isolate with a significantly higher CD4 binding site (CD4bs) antibody response compared to both HVTN studies #041 and #203. No differences were found in CD4-induced (CD4i) antibody responses, ADCC activity, or complement activation by Env-specific antibody among these sera. Given recent renewed interest in realizing the importance of antibody responses for next generation HIV vaccine development, different antibody profiles shown in the current report, based on the analysis of a wide range of antibody parameters, provide critical biomarker information for the selection of HIV vaccines for more advanced human studies and, in particular, those that can elicit antibodies targeting conformational-sensitive and functionally conserved epitopes.
Assuntos
Vacinas contra a AIDS/imunologia , Anticorpos Anti-HIV/imunologia , Proteína gp120 do Envelope de HIV/imunologia , Infecções por HIV/imunologia , Vacinas contra a AIDS/administração & dosagem , Anticorpos Neutralizantes/sangue , Anticorpos Neutralizantes/imunologia , Especificidade de Anticorpos/imunologia , Citotoxicidade Celular Dependente de Anticorpos/imunologia , Ensaio de Imunoadsorção Enzimática , Anticorpos Anti-HIV/sangue , Infecções por HIV/sangue , Infecções por HIV/prevenção & controle , HIV-1/imunologia , HumanosRESUMO
Infection with HIV type 1 (HIV-1), the causative agent of AIDS, is one of the most catastrophic pandemics to affect human healthcare in the latter 20th century. The best hope of controlling this pandemic is the development of a successful prophylactic vaccine. However, to date, this goal has proven to be exceptionally elusive. The recent failure of an experimental vaccine in a phase IIb study, named the STEP trial, intended solely to elicit cell-mediated immune responses against HIV-1, has highlighted the need for a balanced immune response consisting of not only cellular immunity but also a broad and potent humoral antibody response that can prevent infection with HIV-1. This article reviews the efforts made up to this point to elicit such antibody responses, especially with regard to the use of a DNA prime-protein boost regimen, which has been proven to be a highly effective platform for the induction of neutralizing antibodies in both animal and early-phase human studies.