RESUMEN
There is a great need for the development of vaccines for preventing SARS-CoV-2 infection and mitigating the COVID-19 pandemic. Here, we developed two modified vaccinia Ankara (MVA) based vaccines which express either a membrane anchored full-length spike protein (MVA/S) stabilized in a prefusion state or the S1 region of the spike (MVA/S1) which forms trimers and is secreted. Both immunogens contained the receptor-binding domain (RBD) which is a known target of antibody-mediated neutralization. Following immunizations with MVA/S or MVA/S1, both spike protein recombinants induced strong IgG antibodies to purified full-length SARS-CoV-2 spike protein. The MVA/S induced a robust antibody response to purified RBD, S1 and S2 whereas MVA/S1 induced an antibody response to the S1 region outside of the RBD region. Both vaccines induced an antibody response in the lung and that was associated with induction of bronchus-associated lymphoid tissue. MVA/S but not MVA/S1 vaccinated mice generated robust neutralizing antibody responses against SARS-CoV-2 that strongly correlated with RBD antibody binding titers. Mechanistically, S1 binding to ACE-2 was strong but reduced following prolonged pre-incubation at room temperature suggesting confirmation changes in RBD with time. These results demonstrate MVA/S is a potential vaccine candidate against SARS-CoV-2 infection.Competing Interest StatementThe authors have declared no competing interest.View Full Text
RESUMEN
Extensive glycosylation and variable loops of the HIV envelope protein (Env) are reported to shield some neutralizing epitopes. Here, we investigated the immunogenicity of mutated HIV Envs presented in virus-like particles (VLPs). We immunized mice with simian human immunodeficiency virus (SHIV) VLPs containing mutant HIV Env with reduced glycosylation (3G), variable loop-deleted mutations (dV1V2), or combinations of both types of mutations (3G-dV2-1G), and evaluated immune responses. Immune sera from mice that received VLPs with modified HIV Envs (3G or dV1V2) showed higher neutralizing activities against the homologous HIV 89.6 virus as well as heterologous viruses when compared with wild type SHIV VLP-immunized mice. Lymphocytes from immunized mice produced HIV Env-specific cytokines, with the 3G-dV2-1G mutant producing high levels of cytokines. Interestingly, both dendritic cells and B cells were found to interact with VLPs suggesting that VLPs are effective immunogens. Therefore, this study suggests that VLPs containing modified HIV Env have the potential to be developed as candidate vaccines capable of inducing cellular and humoral immune responses including neutralizing activities.
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Vacunas contra el SIDA/administración & dosificación , Vacunas contra el SIDA/inmunología , Productos del Gen env/inmunología , Anticuerpos Anti-VIH/sangre , Infecciones por VIH/prevención & control , Mutación , Virión/inmunología , Animales , Citocinas/metabolismo , Femenino , Productos del Gen env/genética , Infecciones por VIH/inmunología , Infecciones por VIH/virología , VIH-1/genética , VIH-1/inmunología , Humanos , Inmunidad Celular , Inmunización , Ratones , Ratones Endogámicos BALB C , Pruebas de Neutralización , Virus de la Inmunodeficiencia de los Simios/genética , Virus de la Inmunodeficiencia de los Simios/inmunologíaRESUMEN
The rapid spread of human immunodeficiency virus (HIV) worldwide makes it a high priority to develop an effective vaccine. Since live attenuated or inactivated HIV is not likely to be approved as a vaccine due to safety concerns, HIV virus like particles (VLPs) offer an attractive alternative because they are safe due to the lack of a viral genome. Although HIV VLPs have been shown to induce humoral and cellular immune responses, it is important to understand the mechanisms by which they induce such responses and to improve their immunogenicity. We generated HIV VLPs, and VLPs containing Flt3 ligand (FL), a dendritic cell growth factor, to target VLPs to dendritic cells, and investigated the roles of these VLPs in the initiation of adaptive immune responses in vitro and in vivo. We found that HIV-1 VLPs induced maturation of dendritic cells and monocyte/macrophage populations in vitro and in vivo, with enhanced expression of maturation markers and cytokines. Dendritic cells pulsed with VLPs induced activation of splenocytes resulting in increased production of cytokines. VLPs containing FL were found to increase dendritic cells and monocyte/macrophage populations in the spleen when administered to mice. Administration of VLPs induced acute activation of multiple types of cells including T and B cells as indicated by enhanced expression of the early activation marker CD69 and down-regulation of the homing receptor CD62L. VLPs containing FL were an effective form of antigen in activating immune cells via dendritic cells, and immunization with HIV VLPs containing FL resulted in enhanced T helper type 2-like immune responses.
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Vacunas contra el SIDA/inmunología , VIH-1/inmunología , Vacunas de Virosoma/inmunología , Animales , Antígenos CD/biosíntesis , Antígenos de Diferenciación de Linfocitos T/biosíntesis , Proliferación Celular , Células Cultivadas , Citocinas/biosíntesis , Células Dendríticas/inmunología , Citometría de Flujo , Productos del Gen env/genética , Productos del Gen env/inmunología , Productos del Gen gag/genética , Genes env , Anticuerpos Anti-VIH/sangre , Antígenos VIH/genética , Antígenos VIH/inmunología , VIH-1/genética , Humanos , Selectina L/biosíntesis , Lectinas Tipo C , Subgrupos Linfocitarios/inmunología , Macrófagos/inmunología , Proteínas de la Membrana/inmunología , Ratones , Ratones Endogámicos BALB C , Modelos Animales , Bazo/citología , Bazo/inmunología , Spodoptera/citología , Virosomas/inmunología , Tirosina Quinasa 3 Similar a fms/genética , Tirosina Quinasa 3 Similar a fms/inmunologíaRESUMEN
In this study, we have taken advantage of the unique property of a potent dendritic cell (DC) growth factor, Flt-3 ligand (FL), which could act as a vaccine adjuvant. Accordingly, a single injection of plasmid DNA coding for soluble FL (FLex) was shown to induce large numbers of DCs in various tissue compartments and was critical for generating high frequencies of antigen-specific (HIV gp120 and LCMV NP) immune responses in mice. Interestingly, this enhanced level of immune response is strictly dependent on the co-delivery (i.m.) of the DNA vaccines and hFLex DNA to mice harboring large numbers of DCs. The high frequencies of antigen-specific CD8(+) T cells were largely associated with the expansion phase of DCs in vivo. However, DC expansion and immune enhancement have not reciprocally maintained a linear correlation, suggesting that other factors, cytokines/chemokines, which have the potential to modulate the microenvironment of DCs, could influence immunological outcome in this vaccination modality.
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Proteínas de la Membrana/genética , Proteínas de la Membrana/inmunología , Vacunas de ADN/genética , Vacunas de ADN/inmunología , Adyuvantes Inmunológicos/genética , Animales , Secuencia de Bases , Linfocitos T CD8-positivos/inmunología , Diferenciación Celular , Proliferación Celular , ADN Viral/genética , Células Dendríticas/citología , Células Dendríticas/inmunología , Femenino , Proteína gp120 de Envoltorio del VIH/genética , Proteína gp120 de Envoltorio del VIH/inmunología , VIH-1/genética , VIH-1/inmunología , Humanos , Memoria Inmunológica , Virus de la Coriomeningitis Linfocítica/genética , Virus de la Coriomeningitis Linfocítica/inmunología , Ratones , Ratones Endogámicos BALB C , Nucleoproteínas/genética , Nucleoproteínas/inmunología , Plásmidos/genética , Proteínas Virales/genética , Proteínas Virales/inmunologíaRESUMEN
DNA vaccines exploit the inherent abilities of professional antigen-presenting cells to prime the immune system and to elicit immunity against diverse pathogens. In this study, we explored the possibility of augmenting human immunodeficiency virus type 1 gp120-specific immune responses by a DNA vaccine coding for a fusion protein, CTLA4:gp120, in mice. In vitro binding studies revealed that secreted CTLA4:gp120 protein induced a mean florescence intensity shift, when incubated with Raji B cells, indicating its binding to B7 proteins on Raji B cells. Importantly, we instituted three different vaccination regimens to test the efficacy of DNA vaccines encoding gp120 and CTLA4:gp120 in the induction of both cellular (CD8(+)) and antibody responses. Each of the vaccination regimens incorporated a single intramuscular (i.m.) injection of the DNA vaccines to prime the immune system, followed by two booster injections. The i.m.-i.m.-i.m. regimen induced only modest levels of gp120-specific CD8(+) T cells, but the antibody response by CTLA4:gp120 DNA was nearly 16-fold higher than that induced by gp120 DNA. In contrast, using the i.m.-subcutaneous (s.c.)-i.m. regimen, it was found that gp120 and CTLA4:gp120 DNAs were capable of inducing significant levels of gp120-specific CD8(+) T cells (3.5 and 11%), with antibody titers showing a modest twofold increase for CTLA4:gp120 DNA. In the i.m.-gene gun (g.g.)-g.g. regimen, the mice immunized with gp120 and CTLA4:gp120 harbored gp120-specific CD8(+) T cells at frequencies of 0.9 and 2.9%, with the latter showing an eightfold increase in antibody titers. Thus, covalent antigen modification and the routes of genetic vaccination have the potential to modulate antigen-specific immune responses in mice.
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Vacunas contra el SIDA/inmunología , Antígenos de Diferenciación/inmunología , Proteína gp120 de Envoltorio del VIH/inmunología , VIH-1/inmunología , Proteínas Recombinantes de Fusión/inmunología , Vacunas de ADN/inmunología , Vacunas contra el SIDA/administración & dosificación , Animales , Antígenos CD/análisis , Antígenos de Diferenciación/genética , Antígeno B7-1/análisis , Antígeno B7-2 , Linfocitos T CD8-positivos/inmunología , Antígeno CTLA-4 , Femenino , Anticuerpos Anti-VIH/sangre , Proteína gp120 de Envoltorio del VIH/genética , Inmunización Secundaria , Glicoproteínas de Membrana/análisis , Ratones , Ratones Endogámicos BALB C , Vacunación , Vacunas de ADN/administración & dosificaciónRESUMEN
DNA vaccines target dendritic cells (DC) to induce Ag-specific immune responses in animals. Potent HIV-specific immunity could be achieved by efficient priming of the immune system by DNA vaccines. We investigated a novel DNA vaccine approach based on the role of growth factors in DC expansion and differentiation. To this end, we constructed chimeric genes encoding the HIV envelope glycoproteins physically linked to the extracellular domain of Fms-like tyrosine kinase receptor-3 ligand (FLex; a DC growth factor; both mouse (m)FLex and human (h)FLex). These chimeric gene constructs synthesized biologically active, oligomeric FLex:gp120 fusion proteins and induced DC expansion (CD11c(+)CD11b(+)) when injected i.v. into mice. This DC expansion is comparable to that achieved by FLex DNA encoding native FLex protein. When delivered intramuscularly as DNA vaccines, hFLex:gp120 induced high frequencies of gp120-specific CD8(+) T cells in the presence or absence of FLex DNA-induced DC expansion, but gp120 and mFLex:gp120 elicited only low to moderate levels of Ag-specific CD8(+) T cells. In contrast, mFLex:gp120 induced high levels of anti-gp120 Abs under identical conditions of DNA vaccination. However, the Ab levels in mice immunized with DNA vaccines encoding hFLex:gp120 and gp120 proteins were low without DC expansion, but reached high levels comparable to that elicited by mFLex:gp120 only after the second boost in the presence of DC expansion. Importantly, the gp120-specific CD8(+) T cells persisted at high frequency for 114 days (16 wk) after a booster injection. These experiments provide insight into the importance of modulating DC function in vivo for effective genetic vaccination in animals.