RESUMO
The need for improved dengue vaccines remains since the only licensed vaccine, Dengvaxia, shows variable efficacy depending on the infecting dengue virus (DENV) type, and increases the risk of hospitalization for severe dengue in children not exposed to DENV before vaccination. Here, we developed a tetravalent dengue purified and inactivated vaccine (DPIV) candidate and characterized, in rhesus macaques, its immunogenicity and efficacy to control DENV infection by analyzing, after challenge, both viral replication and changes in biological markers associated with dengue in humans. Although DPIV elicited cross-type and long-lasting DENV-neutralizing antibody responses, it failed to control DENV infection. Increased levels of viremia/RNAemia (correlating with serum capacity at enhancing DENV infection in vitro), AST, IL-10, IL-18 and IFN-γ, and decreased levels of IL-12 were detected in some vaccinated compared to non-vaccinated monkeys, indicating the vaccination may have triggered antibody-dependent enhancement of DENV infection. The dengue macaque model has been considered imperfect due to the lack of DENV-associated clinical signs. However, here we show that post-vaccination enhanced DENV infection can be detected in this model when integrating several parameters, including characterization of DENV-enhancing antibodies, viremia/RNAemia, and biomarkers relevant to dengue in humans. This improved dengue macaque model may be crucial for early assessment of efficacy and safety of future dengue vaccines.
Assuntos
Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Vacinas contra Dengue/imunologia , Vírus da Dengue/imunologia , Dengue/imunologia , Vacinas de Produtos Inativados/imunologia , Viremia/imunologia , Animais , Anticorpos Facilitadores , Dengue/prevenção & controle , Dengue/virologia , Vacinas contra Dengue/administração & dosagem , Modelos Animais de Doenças , Feminino , Macaca mulatta , Masculino , Vacinação , Viremia/virologiaRESUMO
Toward the goal of developing an effective HIV vaccine that can be administered in infancy to protect against postnatal and lifelong sexual HIV transmission risks, the current pilot study was designed to compare the effect of novel adjuvants on the induction of HIV Env-specific antibody responses in infant macaques. Aligning our studies with the adjuvanted proteins evaluated in a prime-boost schedule with ALVAC in the ongoing HVTN (HIV Vaccine Trials Network) 702 efficacy trial, we selected the bivalent clade C Env immunogens gp120 C.1086 and gp120 TV1 in combination with the MF59 adjuvant. However, we hypothesized that the adjuvant system AS01, that is included in the pediatric RTS,S malaria vaccine, would promote Env-specific antibody responses superior to those of the oil-in-water MF59 emulsion adjuvant. In a second study arm, we compared two emulsions, glucopyranosyl lipid adjuvant formulated in a stable emulsion (GLA-SE) and 3M-052-SE, containing Toll-like receptor 4 (TLR4) and TLR7/TLR8 (TLR7/8) ligand, respectively. The latter adjuvant had been previously demonstrated to be especially effective in activating neonatal antigen-presenting cells. Our results demonstrate that different adjuvants drive quantitatively or qualitatively distinct responses to the bivalent Env vaccine. AS01 induced higher Env-specific plasma IgG antibody levels than the antigen in MF59 and promoted improved antibody function in infants, and 3M-052-SE outperformed GLA-SE by inducing the highest breadth and functionality of antibody responses. Thus, distinct adjuvants are likely to be required for maximizing vaccine-elicited immune responses in infants, particularly when immunization in infancy aims to elicit both perinatal and lifelong immunity against challenging pathogens such as HIV.IMPORTANCE Alum remains the adjuvant of choice for pediatric vaccines. Yet the distinct nature of the developing immune system in infants likely requires novel adjuvants targeted specifically at the pediatric population to reach maximal vaccine efficacy with an acceptable safety profile. The current study supports the idea that additional adjuvants for pediatric vaccines should be, and need to be, tested in infants for their potential to enhance immune responses. Using an infant macaque model, our results suggest that both AS01 and 3M-052-SE can significantly improve and better sustain HIV Env-specific antibody responses than alum. Despite the limited number of animals, the results revealed interesting differences that warrant further testing of promising novel adjuvant candidates in larger preclinical and clinical studies to define the mechanisms leading to adjuvant-improved antibody responses and to identify targets for adjuvant and vaccine optimization.
Assuntos
Vacinas contra a AIDS/imunologia , Adjuvantes Imunológicos/administração & dosagem , Formação de Anticorpos , Anticorpos Anti-HIV/sangue , Proteína gp120 do Envelope de HIV/imunologia , Vacinas contra a AIDS/administração & dosagem , Animais , Animais Recém-Nascidos , Células Apresentadoras de Antígenos/efeitos dos fármacos , Células Apresentadoras de Antígenos/imunologia , Proteína gp120 do Envelope de HIV/administração & dosagem , Imunoglobulina G/sangue , Macaca mulattaRESUMO
Natural killer (NK) cells are lymphocytes that play a major role in the elimination of virally-infected cells and tumor cells. NK cells recognize and target abnormal cells through activation of stimulatory receptors such as NKG2D. NKG2D ligands are self-proteins, which are absent or expressed at low levels on healthy cells but are induced upon cellular stress, transformation, or viral infection. The exact molecular mechanisms driving expression of these ligands remain poorly understood. Here we show that murine cytomegalovirus (MCMV) infection activates the phosphatidylinositol-3-kinase (PI3K) pathway and that this activation is required for the induction of the RAE-1 family of mouse NKG2D ligands. Among the multiple PI3K catalytic subunits, inhibition of the p110α catalytic subunit blocks this induction. Similarly, inhibition of p110α PI3K reduces cell surface expression of RAE-1 on transformed cells. Many viruses manipulate the PI3K pathway, and tumors frequently mutate the p110α oncogene. Thus, our findings suggest that dysregulation of the PI3K pathway is an important signal to induce expression of RAE-1, and this may represent a commonality among various types of cellular stresses that result in the induction of NKG2D ligands.
Assuntos
Infecções por Citomegalovirus/fisiopatologia , Células Matadoras Naturais/metabolismo , Subfamília K de Receptores Semelhantes a Lectina de Células NK/biossíntese , Proteínas Associadas à Matriz Nuclear/biossíntese , Proteínas de Transporte Nucleocitoplasmático/biossíntese , Fosfatidilinositol 3-Quinase/fisiologia , Receptores de Células Matadoras Naturais/fisiologia , Animais , Domínio Catalítico/fisiologia , Linhagem Celular Tumoral , Transformação Celular Viral , Classe I de Fosfatidilinositol 3-Quinases , Fibroblastos/virologia , Células Matadoras Naturais/imunologia , Ligantes , Camundongos , Muromegalovirus/imunologia , Fosfatidilinositol 3-Quinases/fisiologiaRESUMO
Respiratory syncytial virus (RSV) causes a high disease burden in older adults. An effective vaccine for this RSV-primed population may need to boost/elicit robust RSV-neutralizing antibody responses and recall/induce RSV-specific T cell responses. To inform the selection of the vaccine formulation for older adults, RSVPreF3 (RSV fusion glycoprotein engineered to maintain the prefusion conformation) with/without AS01 adjuvant was evaluated in mice and bovine RSV infection-primed cattle. In mice, RSVPreF3/AS01 elicited robust RSV-A/B-specific neutralization titers and RSV F-specific polyfunctional CD4+ T cell responses exceeding those induced by non-adjuvanted RSVPreF3. In primed bovines, RSVPreF3/AS01 tended to induce higher pre-/post-vaccination fold-increases in RSV-A/B-specific neutralization titers relative to non-adjuvanted and Alum-adjuvanted RSVPreF3 formulations, and elicited higher RSV F-specific CD4+ T cell frequencies relative to the non-adjuvanted vaccine. Though AS01 adjuvanticity varied by animal species and priming status, RSVPreF3/AS01 elicited/boosted RSV-A/B-specific neutralization titers and RSV F-specific CD4+ T cell responses in both animal models, which supported its further clinical evaluation as prophylactic candidate vaccine for older adults.
RESUMO
Dengue virus (DENV) is transmitted by infectious mosquitoes during blood-feeding via saliva containing biologically-active proteins. Here, we examined the effect of varying DENV infection modality in rhesus macaques in order to improve the DENV nonhuman primate (NHP) challenge model. NHPs were exposed to DENV-1 via subcutaneous or intradermal inoculation of virus only, intradermal inoculation of virus and salivary gland extract, or infectious mosquito feeding. The infectious mosquito feeding group exhibited delayed onset of viremia, greater viral loads, and altered clinical and immune responses compared to other groups. After 15 months, NHPs in the subcutaneous and infectious mosquito feeding groups were re-exposed to either DENV-1 or DENV-2. Viral replication and neutralizing antibody following homologous challenge were suggestive of sterilizing immunity, whereas heterologous challenge resulted in productive, yet reduced, DENV-2 replication and boosted neutralizing antibody. These results show that a more transmission-relevant exposure modality resulted in viral replication closer to that observed in humans.
Assuntos
Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Dengue/imunologia , Animais , Dengue/virologia , Vírus da Dengue/fisiologia , Modelos Animais de Doenças , Feminino , Cinética , Macaca mulatta/imunologia , Mosquitos Vetores/virologia , RNA Viral/sangue , Glândulas Salivares/virologia , Vacinação , Carga Viral , Viremia/prevenção & controle , Replicação ViralRESUMO
The macaque is widely accepted as a suitable model for preclinical characterization of dengue vaccine candidates. However, the only vaccine for which both preclinical and clinical efficacy results were reported so far showed efficacy levels that were substantially different between macaques and humans. We hypothesized that this model's predictive capacity may be improved using recent and minimally passaged dengue virus isolates, and by assessing vaccine efficacy by characterizing not only the post-dengue virus challenge viremia/RNAemia but also the associated-cytokine profile. Ten recent and minimally passaged Brazilian clinical isolates from the four dengue virus serotypes were tested for their infectivity in rhesus macaques. For the strains showing robust replication capacity, the associated-changes in soluble mediator levels, and the elicited dengue virus-neutralizing antibody responses, were also characterized. Three isolates from dengue virus serotypes 1, 2 and 4 induced viremia of high magnitude and longer duration relative to previously reported viremia kinetics in this model, and robust dengue virus-neutralizing antibody responses. Consistent with observations in humans, increased MCP-1, IFN-γ and VEGF-A levels, and transiently decreased IL-8 levels were detected after infection with the selected isolates. These results may contribute to establishing a dengue macaque model showing a higher predictability for vaccine efficacy in humans.
Assuntos
Vírus da Dengue/imunologia , Dengue/patologia , Animais , Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Brasil , Quimiocina CCL2/metabolismo , Chlorocebus aethiops , Dengue/veterinária , Vírus da Dengue/isolamento & purificação , Regulação para Baixo , Interferon gama/metabolismo , Interleucina-8 , Macaca mulatta , Sorogrupo , Regulação para Cima , Fator A de Crescimento do Endotélio Vascular/metabolismo , Células VeroRESUMO
Two HIV-1 subtype C gp120 protein candidates were the selected antigens for several experimental vaccine regimens now under evaluation in HVTN 100 Phase I/II clinical trial aiming to support the start of the HVTN 702 Phase IIb/III trial in southern Africa, which is designed to confirm and extend the partial protection seen against HIV-1 infection in the RV144 Thai trial. Here, we report the comprehensive physicochemical characterization of the gp120 reference materials that are representative of the clinical trial materials. Gp120 proteins were stably expressed in Chinese Hamster Ovary (CHO) cells and subsequently purified and formulated. A panel of analytical techniques was used to characterize the physicochemical properties of the two protein molecules. When formulated in the AS01 Adjuvant System, the bivalent subtype C gp120 antigens elicited 1086.C- and TV1.C-specific binding antibody and CD4+ T cell responses in mice. All the characteristics were highly representative of the Clinical Trial Materials (CTM). Data from this report demonstrate the immunogenicity of the gp120 antigens, provide comprehensive characterization of the molecules, set the benchmark for assessment of current and future CTM lots, and lay the physicochemical groundwork for interpretation of future clinical trial data.
RESUMO
Live attenuated RNA viruses make highly efficient vaccines. Among them, measles virus (MV) vaccine has been given to a very large number of children and shown to be highly effective and safe. MV vaccine induces a life-long immunity after a single or two low-dose injections. It is easily produced on a large scale in most countries and can be distributed at low cost. Reversion to pathogenicity has never been observed with this vaccine. Because of all these characteristics, MV vaccine might be a very promising vector to immunise children against both measles and other infectious agents, such as HIV or flaviviruses, in the developing world. In this article, we describe recent data that we obtained showing the capacity of recombinant Schwarz MVs to express proteins from human immunodeficiency or West Nile viruses, and to induce specific immune responses able, in the case of West Nile virus, to protect from an experimental challenge.
Assuntos
Vacina contra Sarampo/uso terapêutico , Criança , Clonagem Molecular , HIV-1/imunologia , Humanos , Sarampo/prevenção & controle , Sarampo/virologia , Vacinas Atenuadas , Vacinas Sintéticas/uso terapêutico , Proteínas do Envelope Viral/imunologiaRESUMO
HIV-1-specific CD4+ and CD8+ T lymphocytes are important for HIV-1 replication control. F4/AS01 consists of F4 recombinant fusion protein (containing clade B Gag/p24, Pol/RT, Nef and Gag/p17) formulated in AS01 Adjuvant System, and was shown to induce F4-specific polyfunctional CD4+ T-cell responses in humans. While replication-incompetent recombinant HIV-1/SIV antigen-expressing human adenoviral vectors can elicit high-frequency antigen-specific CD8+ T-cell responses, their use is hampered by widespread pre-existing immunity to human serotypes. Non-human adenovirus serotypes associated with lower prevalence may offer an alternative strategy. We evaluated the immunogenicity of AdC7-GRN ('A'), a recombinant chimpanzee adenovirus type 7 vector expressing clade B Gag, RT and Nef, and F4/AS01 ('P'), when delivered intramuscularly in homologous (PP or AA) and heterologous (AAPP or PPAA) prime-boost regimens, in macaques and mice. Vaccine-induced HIV-1-antigen-specific T cells in peripheral blood (macaques), liver, spleen, and intestinal and genital mucosa (mice) were characterized by intracellular cytokine staining. Vaccine-specific IgG antibodies (macaques) were detected using ELISA. In macaques, only the heterologous prime-boost regimens induced polyfunctional, persistent and balanced CD4+ and CD8+ T-cell responses specific to each HIV-1 vaccine antigen. AdC7-GRN priming increased the polyfunctionality of F4/AS01-induced CD4+ T cells. Approximately 50% of AdC7-GRN-induced memory CD8+ T cells exhibited an effector-memory phenotype. HIV-1-specific antibodies were detected with each regimen. In mice, antigen-specific CD4+ and CD8+ T-cell responses were detected in the mucosal and systemic anatomical compartments assessed. When administered in heterologous prime-boost regimens, AdC7-GRN and F4/AS01 candidate vaccines acted complementarily in inducing potent and persistent peripheral blood HIV-1-specific CD4+ and CD8+ T-cell responses and antibodies in macaques. Besides, adenoviral vector priming modulated the cytokine-expression profile of the protein-induced CD4+ T cells. Each regimen induced HIV-1-specific T-cell responses in systemic/local tissues in mice. This suggests that prime-boost regimens combining adjuvanted protein and low-seroprevalent chimpanzee adenoviral vectors represent an attractive vaccination strategy for clinical evaluation.
Assuntos
Vetores Genéticos/genética , HIV-1/imunologia , Proteínas Recombinantes de Fusão/imunologia , Linfócitos T/imunologia , Vacinas Virais/imunologia , Adenoviridae , Animais , Anticorpos Antivirais/sangue , Citocinas/imunologia , Ensaio de Imunoadsorção Enzimática , Genes pol/genética , Antígenos HIV/genética , Proteína do Núcleo p24 do HIV/genética , Injeções Intramusculares , Macaca , Camundongos , Pan troglodytes , Proteínas Recombinantes de Fusão/administração & dosagem , Proteínas Recombinantes de Fusão/genética , Vacinas Virais/administração & dosagem , Produtos do Gene gag do Vírus da Imunodeficiência Humana/genética , Produtos do Gene nef do Vírus da Imunodeficiência Humana/genéticaRESUMO
Preclinical studies of viral vector-based HIV-1 vaccine candidates have previously shown partial protection against neutralization-resistant virus challenges in rhesus monkeys. In this study, we evaluated the protective efficacy of adenovirus serotype 26 (Ad26) vector priming followed by purified envelope (Env) glycoprotein boosting. Rhesus monkeys primed with Ad26 vectors expressing SIVsmE543 Env, Gag, and Pol and boosted with AS01B-adjuvanted SIVmac32H Env gp140 demonstrated complete protection in 50% of vaccinated animals against a series of repeated, heterologous, intrarectal SIVmac251 challenges that infected all controls. Protective efficacy correlated with the functionality of Env-specific antibody responses. Comparable protection was also observed with a similar Ad/Env vaccine against repeated, heterologous, intrarectal SHIV-SF162P3 challenges. These data demonstrate robust protection by Ad/Env vaccines against acquisition of neutralization-resistant virus challenges in rhesus monkeys.
Assuntos
Vacinas contra a AIDS/imunologia , Vacinas contra Adenovirus/imunologia , Produtos do Gene env/imunologia , HIV-1/imunologia , Vacinas contra a SAIDS/imunologia , Síndrome de Imunodeficiência Adquirida dos Símios/prevenção & controle , Transferência Adotiva , Animais , Anticorpos Neutralizantes/imunologia , Feminino , Produtos do Gene gag/imunologia , Produtos do Gene pol/imunologia , Vetores Genéticos/imunologia , Antígenos de Histocompatibilidade Classe I/genética , Antígenos de Histocompatibilidade Classe I/imunologia , Imunização Secundária , Macaca mulatta , Masculino , Vírus da Imunodeficiência Símia/imunologiaRESUMO
The HPV-16/18 vaccine (Cervarix) is a prophylactic vaccine for the prevention of cervical cancer and contains recombinant virus-like particles (VLPs) assembled from the L1 major capsid proteins of human papillomavirus (HPV) strains 16 and 18. Although a correlate of protection has yet to be identified, HPV-specific antibodies are thought to prevent virus infection of the genital mucosa. Therefore, antigen-specific antibodies as assessed by ELISA or pseudovirion-based neutralisation assay are frequently measured in clinical trials to substantiate the immune responses induced by the vaccine. Measuring antigen-antibody binding avidities, which reflects the degree of affinity maturation in the B-cells, is another valuable method to assess the quality of the antibody responses. Here we describe the antigen-specific antibody avidities in samples taken from a clinical trial examining the feasibility of adopting a two-dose (Months 0 and 6) schedule for 9-14 year olds instead of the three-dose schedule (Months 0, 1 and 6). Antibody avidity (i.e. avidity index [AI]) was determined in the ELISA by the ratio of antibody concentrations in serum samples treated or not with the chaotropic agent NaSCN. Importantly, in the comparison between the groups of two-dose and three-dose recipients, no differences in AIs were observed at Months 7, 24 and 48. The results suggest that from Month 7 to 48, the quality of the antibody response in terms of avidity was similar in the two-dose recipients to that in the three-dose recipients. Hence these results support the adoption of a two-dose schedule in 9-14 year-old girls.
Assuntos
Afinidade de Anticorpos , Esquemas de Imunização , Vacinas contra Papillomavirus/imunologia , Adolescente , Adulto , Anticorpos Antivirais/sangue , Criança , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Pessoa de Meia-Idade , Infecções por Papillomavirus/prevenção & controle , Ensaios Clínicos Controlados Aleatórios como Assunto , Neoplasias do Colo do Útero/prevenção & controle , Vacinas de Partículas Semelhantes a Vírus/imunologia , Adulto JovemRESUMO
Intracellular cytokine staining (ICS) assay is increasingly used in vaccine clinical trials to measure antigen-specific T-cell mediated immune (CMI) responses in cryopreserved peripheral blood mononuclear cells (PBMCs) and whole blood. However, recent observations indicate that several parameters involved in blood processing can impact PBMC viability and CMI responses, especially in antiretroviral therapy (ART)-naïve HIV-1-infected individuals. In this phase I study (NCT01610427), we collected blood samples from 22 ART-naïve HIV-1-infected adults. PBMCs were isolated and processed for ICS assay. The individual and combined effects of the following parameters were investigated: time between blood collection and PBMC processing (time-to-process: 2, 7 or 24 h); time between PBMC thawing and initiation of in vitro stimulation with HIV-1 antigens (resting-time: 0, 2, 6 and 18 h); and duration of antigen-stimulation in PBMC cultures (stimulation-time: 6h or overnight). The cell recovery after thawing, cell viability after ICS and magnitude of HIV-specific CD8(+) T-cell responses were considered to determine the optimal combination of process conditions. The impact of time-to-process (2 or 4 h) on HIV-specific CD8(+) T-cell responses was also assessed in a whole blood ICS assay. A higher quality of cells in terms of recovery and viability (up to 81% and >80% respectively) was obtained with shorter time-to-process (less than 7 h) and resting-time (less than 2 h) intervals. Longer (overnight) rather than shorter (6 h) stimulation-time intervals increased the frequency of CD8(+)-specific T-cell responses using ICS in PBMCs without change of the functionality. The CD8(+) specific T-cell responses detected using fresh whole blood showed a good correlation with the responses detected using frozen PBMCs. Our results support the need of standardized procedures for the evaluation of CMI responses, especially in HIV-1-infected, ART-naïve patients.
Assuntos
Vacinas contra a AIDS/imunologia , Antirretrovirais/uso terapêutico , Coleta de Amostras Sanguíneas/normas , Linfócitos T CD8-Positivos/imunologia , Infecções por HIV/terapia , Vacinas contra a AIDS/uso terapêutico , Adolescente , Adulto , Reações Antígeno-Anticorpo , Linfócitos T CD4-Positivos/imunologia , Sobrevivência Celular , Criopreservação , Feminino , Citometria de Fluxo/métodos , Infecções por HIV/imunologia , Soropositividade para HIV/imunologia , HIV-1/imunologia , Testes Hematológicos/métodos , Humanos , Imunidade Celular , Técnicas In Vitro , Masculino , Pessoa de Meia-Idade , Coloração e Rotulagem/métodos , Fatores de Tempo , Carga Viral , Adulto JovemRESUMO
The HIV epidemic is greatest in Sub-Saharan Africa and India where HIV-1 subtype C is predominant. To control the spread of HIV in these parts of the world a preventive HIV-1 subtype C vaccine is urgently required. Here we report the immunogenicity of a candidate HIV-1 subtype C vaccine delivered by a recombinant measles vector carrying an insert encoding HIV-1 subtype C Gag, RT and Nef (MV1-F4), in MHC-typed non-human primates. HIV-1 specific cytokine secreting CD4+ and CD8+ T cell responses were detected in 15 out of 16 vaccinees. These HIV-specific T cell responses persisted in lymphoid tissues. Anti-HIV-1 antibody responses were detected in 15 out of 16 vaccinees and titres were boosted by a second immunisation carried out 84 days later. These findings support further exploration of the MV1-F4 vector as a candidate HIV-1 subtype C vaccine or as part of a wider vaccine strategy.
Assuntos
Vacinas contra a AIDS/imunologia , Portadores de Fármacos , Vírus do Sarampo/genética , Vacinas contra a AIDS/administração & dosagem , Vacinas contra a AIDS/genética , Animais , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Citocinas/metabolismo , Vetores Genéticos , Anticorpos Anti-HIV/sangue , Transcriptase Reversa do HIV/genética , Transcriptase Reversa do HIV/imunologia , HIV-1/genética , HIV-1/imunologia , Macaca fascicularis , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologia , Produtos do Gene gag do Vírus da Imunodeficiência Humana/genética , Produtos do Gene gag do Vírus da Imunodeficiência Humana/imunologia , Produtos do Gene nef do Vírus da Imunodeficiência Humana/genética , Produtos do Gene nef do Vírus da Imunodeficiência Humana/imunologiaRESUMO
Live attenuated measles virus is one of the most efficient and safest vaccines available, making it an attractive candidate vector for a HIV/AIDS vaccine aimed at eliciting cell-mediated immune responses (CMI). Here we have characterized the potency of CMI responses generated in mice and non-human primates after intramuscular immunisation with a candidate recombinant measles vaccine carrying an HIV-1 insert encoding Clade B Gag, RT and Nef (MV1-F4). Eight Mauritian derived, MHC-typed cynomolgus macaques were immunised with 10(5) TCID(50) of MV1-F4, four of which were boosted 28 days later with the same vaccine. F4 and measles virus (MV)-specific cytokine producing T cell responses were detected in 6 and 7 out of 8 vaccinees, respectively. Vaccinees with either M6 or recombinant MHC haplotypes demonstrated the strongest cytokine responses to F4 peptides. Polyfunctional analysis revealed a pattern of TNFα and IL-2 responses by CD4+ T cells and TNFα and IFNγ responses by CD8+ T cells to F4 peptides. HIV-specific CD4+ and CD8+ T cells expressing cytokines waned in peripheral blood lymphocytes by day 84, but CD8+ T cell responses to F4 peptides could still be detected in lymphoid tissues more than 3 months after vaccination. Anti-F4 and anti-MV antibody responses were detected in 6 and 8 out of 8 vaccinees, respectively. Titres of anti-F4 and MV antibodies were boosted in vaccinees that received a second immunisation. MV1-F4 carrying HIV-1 Clade B inserts induces robust boostable immunity in non-human primates. These results support further exploration of the MV1-F4 vector modality in vaccination strategies that may limit HIV-1 infectivity.
Assuntos
Vacinas contra a AIDS/imunologia , Síndrome da Imunodeficiência Adquirida/prevenção & controle , Anticorpos Antivirais/biossíntese , HIV-1/imunologia , Imunização Secundária , Vacina contra Sarampo/genética , Vacinas contra a AIDS/administração & dosagem , Vacinas contra a AIDS/genética , Síndrome da Imunodeficiência Adquirida/imunologia , Síndrome da Imunodeficiência Adquirida/virologia , Animais , Anticorpos Antivirais/imunologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/virologia , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/virologia , Produtos do Gene gag/genética , Produtos do Gene gag/imunologia , Humanos , Imunidade Celular , Memória Imunológica , Injeções Intramusculares , Interferon gama/biossíntese , Interferon gama/imunologia , Interleucina-2/biossíntese , Interleucina-2/imunologia , Macaca fascicularis , Masculino , Vacina contra Sarampo/administração & dosagem , Vacina contra Sarampo/imunologia , Camundongos , DNA Polimerase Dirigida por RNA/genética , DNA Polimerase Dirigida por RNA/imunologia , Fator de Necrose Tumoral alfa/biossíntese , Fator de Necrose Tumoral alfa/imunologia , Vacinas Sintéticas , Produtos do Gene nef do Vírus da Imunodeficiência Humana/genética , Produtos do Gene nef do Vírus da Imunodeficiência Humana/imunologiaRESUMO
As a new human immunodeficiency virus type 1 (HIV-1) vaccine approach, the live-attenuated measles virus (MV) Schwarz vaccine strain was genetically engineered to express the F4 antigen (MV1-F4). F4 is a fusion protein comprising HIV-1 antigens p17 and p24, reverse transcriptase and Nef. This study assessed the toxicity, biodistribution and shedding profiles of MV1-F4. Cynomolgus macaques were intramuscularly immunized one or three times with the highest dose of MV1-F4 intended for clinical use, the reference (Schwarz) measles vaccine or saline, and monitored clinically for 11 or 85 days. Toxicological parameters included local and systemic clinical signs, organ weights, haematology, clinical and gross pathology and histopathology. Both vaccines were well tolerated, with no morbidity, clinical signs or gross pathological findings observed. Mean spleen weights were increased after three doses of either vaccine, which corresponded with increased numbers and/or sizes of germinal centers. This was likely a result of the immune response to the vaccines. Either vaccine virus replicated preferentially in secondary lymphoid organs and to a lesser extent in epithelium-rich tissues (e.g., intestine, urinary bladder and trachea) and the liver. At the expected peak of viremia, viral RNA was detected in some biological fluid samples from few animals immunized with either vaccine, but none of these samples contained infectious virus. In conclusion, no shedding of infectious viral particles was identified in cynomolgus monkeys after injection of MV1-F4 or Schwarz measles vaccines. Furthermore, no toxic effect in relation to the MV vaccination was found with these vaccines in this study.
Assuntos
Vacinas contra a AIDS/imunologia , Antígenos HIV/imunologia , Vacina contra Sarampo/imunologia , Vírus do Sarampo/imunologia , Vacinas contra a AIDS/farmacocinética , Vacinas contra a AIDS/toxicidade , Animais , Feminino , Engenharia Genética/métodos , HIV-1/imunologia , Injeções Intramusculares , Macaca fascicularis , Masculino , Vacina contra Sarampo/farmacocinética , Vacina contra Sarampo/toxicidade , Tamanho do Órgão/imunologia , RNA Viral/metabolismo , Fatores de Tempo , Distribuição Tecidual , Replicação Viral , Eliminação de Partículas ViraisRESUMO
The Schwarz strain of measles virus (MV), a live attenuated RNA virus, is one of the safest and most effective human vaccines available. Immunization with MV vaccine expressing heterologous antigen is an attractive strategy to prevent emerging viral diseases. West Nile virus (WNV), which recently emerged in North America, is an important mosquito-borne flavivirus that causes numerous cases of human encephalitis, thus urging the development of a vaccine. To evaluate the efficacy of recombinant MV for the prevention of WNV encephalitis, we constructed a live attenuated Schwarz MV (MVSchw-sE(WNV)) expressing the secreted form of the envelope glycoprotein from the virulent IS-98-ST1 strain of WNV. Inoculation of MV-susceptible mice with MVSchw-sE(WNV) induced both high levels of specific anti-WNV neutralizing antibodies and protection from a lethal challenge with WNV. Passive administration with antisera to MVSchw-sE(WNV) prevented WNV encephalitis in BALB/c mice challenged with a high dose of WNV. The present study is the first to report that a recombinant live attenuated vector based on an approved and widely used MV vaccine can protect against a heterologous, medically important pathogen.
Assuntos
Encefalite/prevenção & controle , Vacina contra Sarampo/imunologia , Proteínas do Envelope Viral/imunologia , Vacinas Virais/uso terapêutico , Vírus do Nilo Ocidental/imunologia , Animais , Anticorpos Antivirais/imunologia , Chlorocebus aethiops , Clonagem Molecular , Encefalite/virologia , Vacina contra Sarampo/administração & dosagem , Vacina contra Sarampo/genética , Camundongos , Camundongos Endogâmicos BALB C , Células Vero , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/metabolismo , Febre do Nilo Ocidental/prevenção & controle , Vírus do Nilo Ocidental/genética , Vírus do Nilo Ocidental/patogenicidadeRESUMO
Live attenuated measles vaccine (MV) could provide a safe and efficient pediatric vaccination vector to immunize children simultaneously against measles and human immunodeficiency virus type 1 (HIV-1). To evaluate the capacity of a vector derived from the certified Schwarz measles vaccine (MVSchw) to prime effective cytotoxic T cells (CTL) and broad neutralizing antibodies against HIV-1 conserved epitopes, we generated recombinant MVSchw viruses expressing HIV-1 antigens. We demonstrate that a recombinant MVSchw virus expressing an HIV-1-derived CTL polyepitope primes effective HLA-A0201-restricted CTLs against multiple conserved HIV-1 epitopes in mice susceptible to measles and humanized for the major histocompatibility complex (MHC) class-I molecule HLA-A0201. Additionally, we show that a recombinant MVSchw virus expressing an HIV-1(89.6) gp140 glycoprotein whose hyper variable V1, V2 and V3 loops were deleted (DeltaV1V2V3gp140), induces broadly neutralizing antibodies against HIV-1 primary isolates. These results show that the MVSchw pediatric vaccination vector induces efficient cellular and humoral HIV-specific immune responses.
Assuntos
Vacinas contra a AIDS/imunologia , Anticorpos Anti-HIV/sangue , HIV-1/imunologia , Antígenos HLA-A/imunologia , Vacina contra Sarampo/imunologia , Linfócitos T Citotóxicos/imunologia , Vacinas Sintéticas/imunologia , Animais , Antígenos CD/análise , Contagem de Linfócito CD4 , Epitopos , Produtos do Gene env/imunologia , Vetores Genéticos , Imunização , Interferon-alfa/análise , Vacina contra Sarampo/genética , Proteína Cofatora de Membrana , Glicoproteínas de Membrana/análise , Camundongos , Vacinas Atenuadas/imunologia , Produtos do Gene env do Vírus da Imunodeficiência HumanaRESUMO
Most of HIV-1 infections are acquired through sexual contact. In the absence of a preventive vaccine, the development of topical microbicides that can block infection at the mucosal tissues is needed. Dermaseptin S4 (DS4) is an antimicrobial peptide derived from amphibian skin, which displays a broad spectrum of activity against bacteria, yeast, filamentous fungi, and herpes simplex virus type 1. We show here that DS4 inhibits cell-free and cell-associated HIV-1 infection of P4-CCR5 indicator cells and human primary T lymphocytes. The peptide is effective against R5 and X4 primary isolates and laboratory-adapted strains of HIV-1. Its activity is directed against HIV-1 particles by disrupting the virion integrity. Increasing the number of DS4-positive charges reduced cytotoxicity without affecting the antiviral activity. The modified DS4 inhibited HIV-1 capture by dendritic cells and subsequent transmission to CD4(+) T cells, as well as HIV-1 binding on HEC-1 endometrial cells and transcytosis through a tight epithelial monolayer.
Assuntos
Proteínas de Anfíbios/farmacologia , Peptídeos Catiônicos Antimicrobianos/farmacologia , Infecções por HIV/virologia , HIV-1/efeitos dos fármacos , HIV-1/patogenicidade , Proteínas de Anfíbios/síntese química , Proteínas de Anfíbios/toxicidade , Animais , Peptídeos Catiônicos Antimicrobianos/síntese química , Peptídeos Catiônicos Antimicrobianos/toxicidade , Linhagem Celular , Células Cultivadas , Células Dendríticas/virologia , Células Epiteliais/virologia , Humanos , Testes de Sensibilidade Microbiana/métodos , Monócitos/virologia , Receptores CCR5/metabolismo , Linfócitos T/virologia , Vírion/efeitos dos fármacos , Vírion/metabolismoRESUMO
The anchored and secreted forms of the human immunodeficiency virus type 1 (HIV-1) 89.6 envelope glycoprotein, either complete or after deletion of the V3 loop, were expressed in a cloned attenuated measles virus (MV) vector. The recombinant viruses grew as efficiently as the parental virus and expressed high levels of the HIV protein. Expression was stable during serial passages. The immunogenicity of these recombinant vectors was tested in mice susceptible to MV and in macaques. High titers of antibodies to both MV and HIV-Env were obtained after a single injection in susceptible mice. These antibodies neutralized homologous SHIV89.6p virus, as well as several heterologous HIV-1 primary isolates. A gp160 mutant in which the V3 loop was deleted induced antibodies that neutralized heterologous viruses more efficiently than antibodies induced by the native envelope protein. A high level of CD8+ and CD4+ cells specific for HIV gp120 was also detected in MV-susceptible mice. Furthermore, recombinant MV was able to raise immune responses against HIV in mice and macaques with a preexisting anti-MV immunity. Therefore, recombinant MV vaccines inducing anti-HIV neutralizing antibodies and specific T lymphocytes responses deserve to be tested as a candidate AIDS vaccine.