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Human respiratory syncytial virus (RSV) is a common cause of severe respiratory disease among infants, immunocompromised individuals, and the elderly. No licensed vaccine is currently available. In this study, we evaluated two parainfluenza virus 5 (PIV5)-vectored vaccines expressing RSV F (PIV5/F) or G (PIV5/G) protein in the cotton rat and African green monkey models for their replication, immunogenicity, and efficacy of protection against RSV challenge. Following a single intranasal inoculation, both animal species shed the vaccine viruses for a limited time but without noticeable clinical symptoms. In cotton rats, the vaccines elicited RSV F- or G-specific serum antibodies and conferred complete lung protection against RSV challenge at doses as low as 103 PFU. Neither vaccine produced the enhanced lung pathology observed in animals immunized with formalin-inactivated RSV. In African green monkeys, vaccine-induced serum and mucosal antibody responses were readily detected, as well. PIV5/F provided nearly complete protection against RSV infection in the upper and lower respiratory tract at a dose of 106 PFU of vaccine. At the same dose levels, PIV5/G was less efficacious. Both PIV5/F and PIV5/G were also able to boost neutralization titers in RSV-preexposed African green monkeys. Overall, our data indicated that PIV5/F is a promising RSV vaccine candidate.IMPORTANCE A safe and efficacious respiratory syncytial virus (RSV) vaccine remains elusive. We tested the recombinant parainfluenza virus 5 (PIV5) vectors expressing RSV glycoproteins for their immunogenicity and protective efficacy in cotton rats and African green monkeys, which are among the best available animal models to study RSV infection. In both species, a single dose of intranasal immunization with PIV5-vectored vaccines was able to produce systemic and local immunity and to protect animals from RSV challenge. The vaccines could also boost RSV neutralization antibody titers in African green monkeys that had been infected previously. Our data suggest that PIV5-vectored vaccines could potentially protect both the pediatric and elderly populations and support continued development of the vector platform.
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Vírus da Parainfluenza 5/genética , Infecções por Vírus Respiratório Sincicial/prevenção & controle , Vacinas contra Vírus Sincicial Respiratório/imunologia , Vírus Sincicial Respiratório Humano/imunologia , Animais , Anticorpos Antivirais/sangue , Anticorpos Antivirais/imunologia , Chlorocebus aethiops , Modelos Animais de Doenças , Vetores Genéticos , Pulmão/virologia , Ratos , Infecções por Vírus Respiratório Sincicial/imunologia , Infecções por Vírus Respiratório Sincicial/virologia , Vacinas contra Vírus Sincicial Respiratório/administração & dosagem , Vacinas contra Vírus Sincicial Respiratório/genética , Vírus Sincicial Respiratório Humano/genética , Sigmodontinae , Vacinação , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/imunologia , Células Vero , Proteínas do Envelope Viral/genética , Proteínas Virais de Fusão/genéticaRESUMO
The Ebola virus disease outbreak that began in Western Africa in December 2013 was unprecedented in both scope and spread, and the global response was slower and less coherent than was optimal given the scale and pace of the epidemic. Past experience with limited localized outbreaks, lack of licensed medical countermeasures, reluctance by first responders to direct scarce resources to clinical research, community resistance to outside interventions, and lack of local infrastructure were among the factors delaying clinical research during the outbreak. Despite these hurdles, the global health community succeeded in accelerating Ebola virus vaccine development, in a 5-month interval initiating phase I trials in humans in September 2014 and initiating phase II/III trails in February 2015. Each of the three Ebola virus disease-affected countries, Sierra Leone, Guinea, and Liberia, conducted a phase II/III Ebola virus vaccine trial. Only one of these trials evaluating recombinant vesicular stomatitis virus expressing Ebola virus glycoprotein demonstrated vaccine efficacy using an innovative mobile ring vaccination trial design based on a ring vaccination strategy responsible for eradicating smallpox that reached areas of new outbreaks. Thoughtful and intensive community engagement in each country enabled the critical community partnership and acceptance of the phase II/III in each country. Due to the delayed clinical trial initiation, relative to the epidemiologic peak of the outbreak in the three countries, vaccine interventions may or may not have played a major role in bringing the epidemic under control. Having demonstrated that clinical trials can be performed during a large outbreak, the global research community can now build on the experience to implement trials more rapidly and efficiently in future outbreaks. Incorporating clinical research needs into planning for future health emergencies and understanding what kind of trial designs is needed for reliable results in an epidemic of limited duration should improve global response to future infectious disease outbreaks.
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Pesquisa Biomédica/estatística & dados numéricos , Vacinas contra Ebola/provisão & distribuição , Doença pelo Vírus Ebola/epidemiologia , Doença pelo Vírus Ebola/prevenção & controle , África Ocidental/epidemiologia , Surtos de Doenças , Doença pelo Vírus Ebola/transmissão , HumanosRESUMO
BACKGROUND: ERVEBO®, a live recombinant vesicular stomatitis virus (VSV) vaccine containing the Zaire ebolavirus glycoprotein (GP) in place of the VSV GP (rVSVΔG-ZEBOV-GP), was advanced through clinical development by Merck & Co., Inc., Rahway, NJ, USA in collaboration with multiple partners to prevent Ebola virus disease (EVD) and has been approved for human use in several countries. METHODS: We evaluated data from three Phase 2/3 clinical trials conducted in Liberia (PREVAIL), Guinea (FLW), and Sierra Leone (STRIVE) during the 2013-2016 West African EVD outbreak to assess immune responses using validated assays. We performed a post hoc analysis of the association of vaccine response with sex, age (18-50 yrs & >50 yrs), and baseline (BL) GP-enzyme-linked immunosorbent assay (ELISA) titer (<200 & ≥200 EU/mL), including individual study (PREVAIL, FLW, or STRIVE) data and pooled data from all 3 studies. The endpoints were total IgG antibody response (EU/mL) measured by the GP-ELISA and neutralizing antibody response measured by the plaque reduction neutralization test (PRNT) to rVSVΔG-ZEBOV-GP at Days 28, 180, and 365 postvaccination. RESULTS: In the overall pooled population, in all subgroups, and in each trial independently, GP-ELISA and PRNT geometric mean titers increased from BL, generally peaking at Day 28 and persisting through Day 365. Immune responses were greater in women and participants with BL GP-ELISA ≥ 200 EU/mL, but did not differ across age groups. CONCLUSION: These data demonstrate that rVSVΔG-ZEBOV-GP elicits a robust and durable immune response through 12 months postvaccination in participants regardless of age, sex, or BL GP-ELISA titer. The higher immune responses observed in women and participants with pre-existing immunity are consistent with those described previously and for other vaccines. Trials were registered as follows: PREVAIL: ClinicalTrials.gov NCT02344407; FLW: Pan African Clinical Trials Registry PACTR201503001057193; STRIVE: ClinicalTrials.gov NCT02378753. Protocols V920-009, 011, and 018.
Assuntos
Vacinas contra Ebola , Ebolavirus , Doença pelo Vírus Ebola , Adolescente , Adulto , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Adulto Jovem , Anticorpos Neutralizantes , Anticorpos Antivirais , Ensaio de Imunoadsorção Enzimática , Glicoproteínas , Doença pelo Vírus Ebola/epidemiologia , Imunogenicidade da Vacina , Imunoglobulina G , Proteínas do Envelope ViralRESUMO
BACKGROUND: The COVID-19 pandemic has increased the need for innovative quantitative decision tools to support rapid development of safe and efficacious vaccines against SARS-CoV-2. To meet that need, we developed and applied a model-based meta-analysis (MBMA) approach integrating non-clinical and clinical immunogenicity and protection data. METHODS: A systematic literature review identified studies of vaccines against SARS-CoV-2 in rhesus macaques (RM) and humans. Summary-level data of 13 RM and 8 clinical trials were used in the analysis. A RM MBMA model was developed to quantify the relationship between serum neutralizing (SN) titres after vaccination and peak viral load (VL) post-challenge in RM. The translation of the RM MBMA model to a clinical protection model was then carried out to predict clinical efficacies based on RM data alone. Subsequently, clinical SN and efficacy data were integrated to develop three predictive models of efficacy - a calibrated RM MBMA, a joint (RM-Clinical) MBMA, and the clinical MBMA model. The three models were leveraged to predict efficacies of vaccine candidates not included in the model and efficacies against newer strains of SARS-CoV-2. FINDINGS: Clinical efficacies predicted based on RM data alone were in reasonable agreement with the reported data. The SN titre predicted to provide 50% efficacy was estimated to be about 21% of the mean human convalescent titre level, and that value was consistent across the three models. Clinical efficacies predicted from the MBMA models agreed with reported efficacies for two vaccine candidates (BBV152 and CoronaVac) not included in the modelling and for efficacies against delta variant. INTERPRETATION: The three MBMA models are predictive of protection against SARS-CoV-2 and provide a translational framework to enable early Go/No-Go and study design decisions using non-clinical and/or limited clinical immunogenicity data in the development of novel SARS-CoV-2 vaccines. FUNDING: This study was funded by Merck Sharp & Dohme LLC, a subsidiary of Merck & Co., Inc., Rahway, NJ, USA.
Assuntos
COVID-19 , Vacinas Virais , Animais , Anticorpos Neutralizantes , Anticorpos Antivirais , COVID-19/prevenção & controle , Vacinas contra COVID-19 , Humanos , Macaca mulatta , Pandemias/prevenção & controle , SARS-CoV-2RESUMO
This review describes key aspects of the development of the rVSVΔG-ZEBOV-GP Ebola vaccine and key activities which are continuing to further expand our knowledge of the product. Extensive partnerships and innovative approaches were used to address the various challenges encountered during this process. The rVSVΔG-ZEBOV-GP Ebola vaccine was initially approved by the European Medicines Agency and prequalified by the World Health Organization in November 2019. It was approved by the United States Food and Drug Administration in December 2019 and approved in five African countries within 90 days of prequalification. The development resulted in the first stockpile of a registered Ebola vaccine that is available to support outbreak response. This also provides insights into how the example of rVSVΔG-ZEBOV-GP can inform the development of vaccines for Sudan ebolavirus, Marburg virus, and other emerging epidemic diseases in terms of the types of approaches and data needed to support product registration, availability, and the use of a filovirus vaccine.
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Single-stranded oligoribonucleotides (ORNs) stimulate innate immune responses through TLR7 and TLR8. Specific linkages and chemical modifications incorporated into synthetic ORN can greatly enhance nuclease stability, selectivity, and potency. In the present study, we have synthesized 15 ORN containing different sequence compositions and chemical modifications and studied their TLR7- and TLR8-mediated immune response profiles in HEK293 cells expressing human TLR7 or TLR8, human PBMCs, mDCs and pDCs, non-human primate (NHP) PBMCs, and in vivo in mice and NHPs. Based on the results obtained, eight of the ORNs containing specific chemical modifications induced immune responses through both TLR7 and TLR8, including activation of NF-κB in TLR7- and TLR8-transfected cell lines; induction of IFN-α, IL-6, TNF-α, IL-12, and IP-10 in human PBMCs; IFN-α induction in human pDCs; CD80 upregulation in human pDCs and mDCs; IL-12 induction following acute administration in mice; IFN-α, IP-10, IL-6, and IL-12 induction in NHP PBMCs; and IFN-α, IP-10, and IL-6 induction following acute administration in NHPs. Seven of the ORNs show selectivity for TLR8-induced responses; they specifically activate only TLR8-transfected cell lines, induce cytokines other than IFN-α in human and NHP PBMCs, activate mDCs more than pDCs, and do not induce IL-12 acutely in mice, consistent with the lack of functional TLR8 in mice. The novel TLR8-selective ORNs also induce cytokines other than IFN-α acutely in NHPs. In conclusion, we have designed and synthesized novel ORNs with varying sequence compositions and chemical modifications, which selectively act as agonists of TLR8 or dual agonists of TLR7 and TLR8.
Assuntos
Oligorribonucleotídeos/farmacologia , Receptor 7 Toll-Like/agonistas , Receptor 8 Toll-Like/agonistas , Animais , Sequência de Bases , Linhagem Celular , Citocinas/biossíntese , Citocinas/sangue , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/imunologia , Desenho de Fármacos , Feminino , Células HEK293 , Humanos , Imunidade Inata , Interleucina-12/biossíntese , Macaca mulatta , Camundongos , Camundongos Endogâmicos C57BL , Oligorribonucleotídeos/síntese química , Oligorribonucleotídeos/genética , Oligorribonucleotídeos/imunologia , Proteínas Recombinantes/agonistas , Proteínas Recombinantes/genética , Receptor 7 Toll-Like/genética , Receptor 8 Toll-Like/genética , TransfecçãoRESUMO
BACKGROUND: We report composite results from the Merck phase I program of near-consensus clade B human immunodeficiency virus (HIV) type 1 gag vaccines. METHODS: Healthy HIV-uninfected adults were enrolled in 6 blinded placebo-controlled studies evaluating the immunogenicity of (1) a 4-dose regimen of a DNA vaccine, (2) a 3-dose priming regimen of the DNA vaccine with a booster dose of an adenovirus type 5 (Ad5)-vectored vaccine, or (3) a 3-dose regimen of the Ad5 vaccine. The DNA plasmid was provided with or without an aluminum phosphate or CRL1005 adjuvant. The primary end point was the unfractionated HIV-1 gag-specific interferon gamma enzyme-linked immunospot (ELISpot) response 4 weeks after the final dose. RESULTS: Overall, 254 (83%) of 307 subjects randomized to the vaccine groups were evaluable. Adjuvants did not enhance immunogenicity of the DNA vaccine. Postboost ELISpot responder frequencies were higher for Ad5-containing regimens than for the DNA/DNA regimen (33%) but were similar for DNA/Ad5 (55%) and Ad5/Ad5 (50%). DNA/DNA elicited mainly a CD4 response, whereas Ad5/Ad5 elicited mainly a CD8 response; DNA/Ad5 generated CD4 and CD8 responses comparable to those of DNA/DNA and Ad5/Ad5, respectively. CONCLUSIONS: The DNA vaccine alone or as a priming regimen for the Ad5 vaccine did not increase unfractionated ELISpot responses compared with the Ad5 vaccine alone. Qualitative T cell responses to different vaccine regimens deserve further study.
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Vacinas contra a AIDS/imunologia , DNA Viral/imunologia , Genes gag/imunologia , HIV-1/imunologia , Imunização Secundária/métodos , Adenoviridae/imunologia , Adjuvantes Imunológicos , Adulto , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Placebos , Adulto JovemRESUMO
BACKGROUND: Establishment of immune correlates of protection can provide a measurable criterion for assessing protection against infection or disease. For some vaccines, such as the measles vaccine, antibodies serve as the correlate of protection, but for others, such as human papillomavirus, the correlate of protection remains unknown. Merck & Co, Kenilworth, NJ, USA, in collaboration with multiple partners, developed a live recombinant vesicular stomatitis virus vaccine (rVSVΔG-ZEBOV-GP [ERVEBO]) containing the Zaire ebolavirus glycoprotein (GP) in place of the recombinant vesicular stomatitis virus GP to prevent Ebola virus disease. Seroresponse, defined as post-vaccination GP-ELISA of 200 ELISA units (EU) per mL or higher and two-times or more above baseline, was proposed; however, correlates of protection have not been determined. The objective of this post-hoc analysis was to infer possible correlates of protection for rVSVΔG-ZEBOV-GP. METHODS: In this post-hoc analysis we included vaccinated participants with serology data from three phase 2/3 immunogenicity trials in Guinea, Sierra Leone, and Liberia (n=2199). Two of the trials were open-label, single-arm trials (one randomised [STRIVE], one non-randomised [FLW]); and one trial was randomised, placebo-controlled with two vaccine comparators (PREVAIL). Endpoints were total IgG antibody response (EU per mL) to rVSVΔG-ZEBOV-GP measured by GP-ELISA and neutralising antibody response to rVSVΔG-ZEBOV-GP measured by plaque reduction neutralisation test at days 14, 28, 180, and 365 after vaccination. Reverse cumulative distribution curves of the antibody concentrations were used to estimate statistical correlates of protection between 70% and 100% that might be applied to vaccine efficacy and effectiveness estimates. FINDINGS: Although GP-ELISA and plaque reduction neutralisation tests showed similar response patterns, GP-ELISA provided a wider range of measurable titres and better differentiation for estimating correlates of protection compared with the plaque reduction neutralisation test. At day 14 after vaccination in the FLW trial, 1060 (100%) of 1060 participants had GP-ELISA levels at or above 68 EU per mL and 742 (70%) of 1060 had levels at or above 313 EU per mL. At day 28 after vaccination in the pooled population, 1953 (100%) of 1953 participants had levels at or above 73 EU per mL and 1368 (70%) of 1953 participants had levels at or above 735 EU per mL. GP-ELISA seroresponse 200 EU per mL or higher and two-times or more increase in antibody level from baseline occurred in 80% or higher of participants at each assessment and in 94% or higher of participants at any time after vaccination. INTERPRETATION: Our results are consistent with previous work suggesting that seroresponse defined as GP-ELISA of 200 EU per mL or higher and two-times or more from baseline associated with vaccination might be the most appropriate dichotomous correlate of protection and falls within the seroprotective threshold range described herein. FUNDING: Merck Sharp & Dohme, Biomedical Advanced Research and Development Authority, Office of the Assistant Secretary for Preparedness and Response, US Department of Health and Human Services.
Assuntos
Vacinas contra Ebola , Ebolavirus , Estomatite Vesicular , Anticorpos Antivirais , Ensaios Clínicos Fase II como Assunto , Ensaios Clínicos Fase III como Assunto , República Democrática do Congo , Glicoproteínas , HumanosRESUMO
Novel agonists of TLR9 with two 5'-ends and synthetic immune stimulatory motifs, referred to as immune modulatory oligonucleotides (IMOs) are potent agonists of TLR9. In the present study, we have designed and synthesized 15 novel IMOs by incorporating specific chemical modifications and studied their immune response profiles both in vitro and in vivo. Analysis of the immunostimulatory profiles of these IMOs in human and NHP cell-based assays suggest that changes in the number of synthetic immunostimulatory motifs gave only a subtle change in immune stimulation of pDCs as indicated by IFN-alpha production and pDC maturation while the addition of self-complementary sequences produced more dramatic changes in both pDC and B cell stimulation. All IMOs induced cytokine production in vivo immediately after administration in mice. Representative compounds were also compared for the ability to stimulate cytokine production in vivo (IFN-alpha and IP-10) in rhesus macaques after intra-muscular administration.
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Linfócitos B/efeitos dos fármacos , Linfócitos B/metabolismo , Células Dendríticas/efeitos dos fármacos , Oligonucleotídeos/farmacologia , Receptor Toll-Like 9/agonistas , Animais , Linfócitos B/imunologia , Linfócitos B/patologia , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Células Dendríticas/patologia , Feminino , Humanos , Interferon-alfa/metabolismo , Ativação Linfocitária/efeitos dos fármacos , Macaca mulatta , Camundongos , Camundongos Endogâmicos C57BL , Oligonucleotídeos/síntese química , Oligonucleotídeos/química , Oligonucleotídeos/imunologia , Pirimidinonas/metabolismo , Tiazóis/metabolismoRESUMO
rVSVΔG-ZEBOV-GP is a live, attenuated, recombinant vesicular stomatitis virus (rVSV)-based vaccine for the prevention of Ebola virus disease caused by Zaire ebolavirus. As a replication-competent genetically modified organism, rVSVΔG-ZEBOV-GP underwent various environmental evaluations prior to approval, the most in-depth being the environmental risk assessment (ERA) required by the European Medicines Agency. This ERA, as well as the underlying methodology used to arrive at a sound conclusion about the environmental risks of rVSVΔG-ZEBOV-GP, are described in this review. Clinical data from vaccinated adults demonstrated only infrequent, low-level shedding and transient, low-level viremia, indicating a low person-to-person infection risk. Animal data suggest that it is highly unlikely that vaccinated individuals would infect animals with recombinant virus vaccine or that rVSVΔG-ZEBOV-GP would spread within animal populations. Preclinical studies in various hematophagous insect vectors showed that these species were unable to transmit rVSVΔG-ZEBOV-GP. Pathogenicity risk in humans and animals was found to be low, based on clinical and preclinical data. The overall risk for non-vaccinated individuals and the environment is thus negligible and can be minimized further through defined mitigation strategies. This ERA and the experience gained are relevant to developing other rVSV-based vaccines, including candidates under investigation for prevention of COVID-19.
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Results from Merck's phase II adenovirus type 5 (Ad5) gag/pol/nef test-of-concept trial showed that the vaccine lacked efficacy against human immunodeficiency virus (HIV) infection in a high-risk population. Among the many questions to be explored following this outcome are whether (i) the Ad5 vaccine induced the quality of T-cell responses necessary for efficacy and (ii) the lack of efficacy in the Ad5 vaccine can be generalized to other vector approaches intended to induce HIV type 1 (HIV-1)-specific T-cell responses. Here we present a comprehensive evaluation of the T-cell response profiles from cohorts of clinical trial subjects who received the HIV CAM-1 gag insert delivered by either a regimen with DNA priming followed by Ad5 boosting (n = 50) or a homologous Ad5/Ad5 prime-boost regimen (n = 70). The samples were tested using a statistically qualified nine-color intracellular cytokine staining assay measuring interleukin-2 (IL-2), tumor necrosis factor alpha, macrophage inflammatory protein 1beta, and gamma interferon production and expression of CD107a. Both vaccine regimens induced CD4(+) and CD8(+) HIV gag-specific T-cell responses which variably expressed several intracellular markers. Several trends were observed in which the frequencies of HIV-1-specific CD4(+) T cells and IL-2 production from antigen-specific CD8(+) T cells in the DNA/Ad5 cohort were more pronounced than in the Ad5/Ad5 cohort. Implications of these results for future vaccine development will be discussed.
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Adenoviridae/metabolismo , Genes gag/genética , Linfócitos T/virologia , Vacinas contra a AIDS/metabolismo , Linfócitos T CD4-Positivos/virologia , Linfócitos T CD8-Positivos/virologia , Quimiocina CCL4/biossíntese , Estudos de Coortes , DNA/metabolismo , Humanos , Interleucina-2/biossíntese , Proteína 1 de Membrana Associada ao Lisossomo/biossíntese , Modelos Biológicos , Fenótipo , Linfócitos T/metabolismo , Fator de Necrose Tumoral alfa/biossínteseRESUMO
BACKGROUND: The safety and immunogenicity of the MRK adenovirus type 5 human immunodeficiency virus type 1 clade B gag/pol/nef vaccine, a replication-incompetent adenovirus type 5-vectored vaccine designed to elicit cell-mediated immunity against conserved human immunodeficiency virus proteins, was assessed in a phase 1 trial. METHODS: Healthy adults not infected with human immunodeficiency virus were enrolled in a multicenter, dose-escalating, blind, placebo-controlled study to evaluate a 3-dose homologous prime-boost regimen of the trivalent MRK adenovirus type 5 human immunodeficiency virus type 1 vaccine containing from 3 x 10(6) to 1 x 10(11) viral particles per 1-mL dose administered on day 1, during week 4 and during week 26. Adverse events were recorded for 29 days after each intradeltoid injection. The primary immunogenicity end point was the proportion of study participants with a positive unfractionated Gag-, Pol-, or Nef-specific interferon-gamma enzyme-linked immunosorbent spot response measured 4 weeks after administration of the last dose. RESULTS: Of 259 randomized individuals, 257 (99%) received > or = 1 dose of vaccine or placebo and were included in the safety analyses. Enzyme-linked immunosorbent spot results were available for 217 study participants (84%) at week 30. No serious vaccine-related adverse events occurred. No study participant discontinued participation because of vaccine-related adverse events. The frequency of injection-site reactions was dose dependent. Vaccine doses of > or = 3 x 10(9) viral particles elicited positive enzyme-linked immunosorbent spot responses to > or = 1 vaccine component in > 60% of recipients. High baseline antibody titers against adenovirus type 5 diminished enzyme-linked immunosorbent spot responses at all doses except the 3 x 10(10) viral particle dose. CONCLUSIONS: The vaccine was generally well tolerated and induced cell-mediated immune responses against human immunodeficiency virus type 1 peptides in most healthy adults. Despite these findings, vaccination in a proof-of-concept trial with use of this vaccine was discontinued because of lack of efficacy.
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Vacinas contra a AIDS/efeitos adversos , Vacinas contra a AIDS/imunologia , Infecções por HIV/prevenção & controle , HIV-1/imunologia , Vacinas contra a AIDS/administração & dosagem , Adenoviridae , Adulto , Feminino , Proteínas de Fusão gag-pol/imunologia , Genes gag , Genes pol , Anticorpos Anti-HIV/biossíntese , Infecções por HIV/imunologia , HIV-1/genética , Humanos , Masculino , Segurança , Vacinas de DNA/administração & dosagem , Vacinas de DNA/efeitos adversos , Vacinas de DNA/imunologiaRESUMO
The importance of host cellular immune responses, particularly CD8(+) cytotoxic T-lymphocyte (CTL) responses, in control of human immunodeficiency virus type 1 (HIV-1) infection has been demonstrated in many clinical studies. These studies, along with vaccination challenge studies in rhesus macaques, indicate the importance of cellular immune responses against HIV-1. Toward this end, we evaluated anti-HIV-1 cellular immune responses in a cohort of 54 subjects who were chronically infected with HIV-1. By validation of IFN-gamma ELISpot assay, we established a dual cut-off criterion for scoring a positive response. The magnitude and frequency of cellular immune responses were measured against HIV-1 antigens (Gag, Pol, Nef, Rev, and Tat), using synthetic peptides as antigens in ELISpot assay. Here we showed that HIV-1 Gag, Pol, and Nef were frequent targets of T cell responses in these subjects, whereas Tat and Rev were less frequently recognized. We further evaluated the possible association between host cellular immune responses and corresponding plasma viral loads in this cohort. By performing ranking correlation analysis, we demonstrated a positive correlation between host viral loads and ELISpot responses of HIV Gag and Pol in untreated subjects. For the subjects under antiviral regimens, however, we did not find any significant association. Our findings suggest that the high levels of ELISpot responses in chronically infected subjects were reflective of their persistent viral infection.
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Linfócitos T CD8-Positivos/imunologia , Infecções por HIV/imunologia , HIV-1/imunologia , Linfócitos T Citotóxicos/imunologia , Adulto , Negro ou Afro-Americano/estatística & dados numéricos , Contagem de Linfócito CD4 , Doença Crônica , Estudos de Coortes , Estudos Transversais , Ensaio de Imunoadsorção Enzimática , Feminino , Citometria de Fluxo , Produtos do Gene gag/imunologia , Produtos do Gene nef/imunologia , Produtos do Gene pol/imunologia , Produtos do Gene tat/imunologia , Infecções por HIV/virologia , Hispânico ou Latino/estatística & dados numéricos , Humanos , Imunidade Celular , Interferon gama/imunologia , Masculino , RNA Viral/sangue , Carga Viral , População Branca/estatística & dados numéricos , Produtos do Gene nef do Vírus da Imunodeficiência Humana , Produtos do Gene tat do Vírus da Imunodeficiência HumanaRESUMO
Currently, there are numerous candidate HIV vaccines aimed at inducing T-cell mediated immune responses against HIV. To assess the immunogenicity of such vaccines, a reliable T cell assay must be utilized and typically one of the following assays is chosen for this purpose: bulk culture CTL, MHC I tetramer staining, IFN-gamma ELISPOT, or IFN-gamma intracellular cytokine staining. In this paper we report a comparison of the T cell responses detected by each assay in a large cohort of healthy normal volunteers vaccinated with adenovirus serotype 5 expressing HIV gag. Using stringently validated formats of each of these assays and pools of overlapping HIV gag peptides, we demonstrate that there is a high degree of correlation between all four of the common T cell assays, but inherent differences in the sensitivity of each assay to detect responders. In this study, the ELISPOT assay is shown to have the greatest sensitivity in detecting vaccine responses, while the ICS assay, although less sensitive, has the advantage of providing additional information on the phenotype of the responding cells.
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Vacinas contra a AIDS/imunologia , Produtos do Gene gag/imunologia , Infecções por HIV/prevenção & controle , Linfócitos T/fisiologia , Adenoviridae , Adolescente , Adulto , Feminino , Antígenos HIV/imunologia , HIV-1 , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
Sub-unit vaccines are primarily designed to include antigens required to elicit protective immune responses and to be safer than whole-inactivated or live-attenuated vaccines. But their purity and inability to self-adjuvant often result in weaker immunogenicity. Emerging evidence suggests that bio-engineered nanoparticles can be used as immunomodulatory adjuvants. Therefore, in this study we explored the potential of novel Merck-proprietary lipid nanoparticle (LNP) formulations to enhance immune responses to sub-unit viral antigens. Immunization of BALB/c and C57BL/6 mice revealed that LNPs alone or in combination with a synthetic TLR9 agonist, immune-modulatory oligonucleotides, IMO-2125 (IMO), significantly enhanced immune responses to hepatitis B virus surface antigen (HBsAg) and ovalbumin (OVA). LNPs enhanced total B-cell responses to both antigens tested, to levels comparable to known vaccine adjuvants including aluminum based adjuvant, IMO alone and a TLR4 agonist, 3-O-deactytaled monophosphoryl lipid A (MPL). Investigation of the quality of B-cell responses demonstrated that the combination of LNP with IMO agonist elicited a stronger Th1-type response (based on the IgG2a:IgG1 ratio) than levels achieved with IMO alone. Furthermore, the LNP adjuvant significantly enhanced antigen specific cell-mediated immune responses. In ELISPOT assays, depletion of specific subsets of T cells revealed that the LNPs elicited potent antigen-specific CD4(+) and CD8(+)T cell responses. Intracellular FACS analyses revealed that LNP and LNP+IMO formulated antigens led to higher frequency of antigen-specific IFNγ(+)TNFα(+)IL-2(+), multi-functional CD8(+)T cell responses, than unadjuvanted vaccine or vaccine with IMO only. Overall, our results demonstrate that lipid nanoparticles can serve as future sub-unit vaccine adjuvants to boost both B-cell and T-cell responses in vivo, and that addition of IMO can be used to manipulate the quality of immune responses.
Assuntos
Adjuvantes Imunológicos/administração & dosagem , Linfócitos B/imunologia , Lipossomos/administração & dosagem , Nanopartículas/administração & dosagem , Linfócitos T/imunologia , Vacinas de Subunidades Antigênicas/imunologia , Animais , Feminino , Antígenos de Superfície da Hepatite B/imunologia , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Ovalbumina/imunologia , Resultado do Tratamento , Vacinas de Subunidades Antigênicas/administração & dosagemRESUMO
Monitoring antigen-specific memory B cells and the antibodies they encode is important for understanding the specificity, breadth and duration of immune response to an infection or vaccination. The antibodies isolated could further help design vaccine antigens for raising relevant protective immune responses. However, developing assays to measure and isolate antigen-specific memory B cells is technically challenging due to the low frequencies of these cells that exist in the circulating blood. Here, we describe a flow cytometry method to identify and isolate dengue envelope-specific memory B cells using a labeled dengue envelope protein. We enumerated dengue-envelope specific memory B cells from a cohort of dengue seropositive donors using this direct flow cytometry assay. A more established and conventional assay, the cultured B ELISPOT, was used as a benchmark comparator. Furthermore, we were able to confirm the single-sorted memory B-cell specificity by culturing B cells and differentiating them into plasma cells using cell lines expressing CD40L. The culture supernatants were assayed for antigen binding and the ability of the antibodies to neutralize the cognate dengue virus. Moreover, we successfully isolated the heavy and light Ig sequences and expressed them as full-length recombinant antibodies to reproduce the activity seen in culture supernatants. Mapping of these antibodies revealed a novel epitope for dengue 2 virus serotype. In conclusion, we established a reproducible methodology to enumerate antigen-specific memory B cells and assay their encoded antibodies for functional characterization.
Assuntos
Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Linfócitos B/imunologia , Vírus da Dengue/imunologia , Memória Imunológica , Proteínas do Envelope Viral/imunologia , Linfócitos B/citologia , Técnicas de Cultura de Células , Feminino , Citometria de Fluxo , Humanos , MasculinoRESUMO
Congenital human cytomegalovirus (HCMV) infection occurs in ~0.64% of infants born each year in the United States and is the leading nongenetic cause of childhood neurodevelopmental disabilities. No licensed HCMV vaccine is currently available. Natural immunity to HCMV in women before pregnancy is associated with a reduced risk of fetal infection, suggesting that a vaccine is feasible if it can reproduce immune responses elicited by natural infection. On the basis of this premise, we developed a whole-virus vaccine candidate from the live attenuated AD169 strain, with genetic modifications to improve its immunogenicity and attenuation. We first restored the expression of the pentameric gH/gL/pUL128-131 protein complex, a major target for neutralizing antibodies in natural immunity. We then incorporated a chemically controlled protein stabilization switch in the virus, enabling us to regulate viral replication with a synthetic compound named Shield-1. The virus replicated as efficiently as its parental virus in the presence of Shield-1 but failed to produce progeny upon removal of the compound. The vaccine was immunogenic in multiple animal species and induced durable neutralizing antibodies, as well as CD4+ and CD8+ T cells, to multiple viral antigens in nonhuman primates.
Assuntos
Infecções por Citomegalovirus/congênito , Infecções por Citomegalovirus/prevenção & controle , Vacinas contra Citomegalovirus/uso terapêutico , Transmissão Vertical de Doenças Infecciosas/prevenção & controle , Animais , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD8-Positivos/citologia , Citomegalovirus , Feminino , Humanos , Imunogenicidade da Vacina , Interferon gama/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Gravidez , Coelhos , Vacinas Atenuadas/uso terapêutico , Proteínas Virais/imunologiaRESUMO
ZOSTAVAX(®) is a live attenuated varicella-zoster virus (VZV) vaccine that is licensed for the protection of individuals ≥50 years against shingles and its most common complication, postherpetic neuralgia. While IFNγ responses increase upon vaccination, the quality of the T cell response has not been elucidated. By using polychromatic flow cytometry, we characterized the breadth, magnitude, and quality of ex vivo CD4(+) and CD8(+) T cell responses induced 3-4 weeks after ZOSTAVAX vaccination of healthy adults. We show, for the first time that the highest frequencies of VZV-specific CD4(+) T cells were poly-functional CD154(+)IFNγ(+)IL-2(+)TNFα(+) cells, which were boosted upon vaccination. The CD4(+) T cells were broadly reactive to several VZV proteins, with immediate early (IE) 63 ranking the highest among them in the fold rise of poly-functional cells, followed by IE62, gB, open reading frame (ORF) 9, and gE. We identified a novel poly-functional ORF9-specific CD8(+) T cell population in 62% of the subjects, and these were boosted upon vaccination. Poly-functional CD4(+) and CD8(+) T cells produced significantly higher levels of IFNγ, IL-2, and TNFα compared to mono-functional cells. After vaccination, a boost in the expression of IFNγ by poly-functional IE63- and ORF9-specific CD4(+) T cells and IFNγ, IL-2, and TNFα by ORF9-specific poly-functional CD8(+) T cells was observed. Responding poly-functional T cells exhibited both effector (CCR7(-)CD45RA(-)CD45RO(+)), and central (CCR7(+)CD45RA(-)CD45RO(+)) memory phenotypes, which expressed comparable levels of cytokines. Altogether, our studies demonstrate that a boost in memory poly-functional CD4(+) T cells and ORF9-specific CD8(+) T cells may contribute toward ZOSTAVAX efficacy.
RESUMO
This review focuses on a dengue virus (DENV) vaccine candidate based on a recombinant subunit approach which targets the DENV envelope glycoprotein (E). Truncated versions of E consisting of the N-terminal portion of E (DEN-80E) have been expressed recombinantly in the Drosophila S2 expression system and shown to have native-like conformation. Preclinical studies demonstrate that formulations containing tetravalent DEN-80E adjuvanted with ISCOMATRIX™ adjuvant induce high titer virus neutralizing antibodies and IFN-γ producing T cells in flavivirus-naïve non-human primates. The preclinical data further suggest that administration of such formulations on a 0, 1, 6 month schedule may result in higher maximum virus neutralizing antibody titers and better durability of those titers compared to administration on a 0, 1, 2 month schedule. In addition, the virus neutralizing antibody titers induced by adjuvanted tetravalent DEN-80E compare favorably to the titers induced by a tetravalent live virus comparator. Furthermore, DEN-80E was demonstrated to be able to boost virus neutralizing antibody titers in macaques that have had a prior DENV exposure. A monovalent version of the vaccine candidate, DEN1-80E, was formulated with Alhydrogel™ and studied in a proof-of-principle Phase I clinical trial by Hawaii Biotech, Inc. (NCT00936429). The clinical trial results demonstrate that both the 10 µg and 50 µg formulations of DEN1-80E with 1.25 mg of elemental aluminum were immunogenic when administered in a 3-injection series (0, 1, 2 months) to healthy, flavivirus-naïve adults. The vaccine formulations induced DENV-1 neutralizing antibodies in the majority of subjects, although the titers in most subjects were modest and waned over time. Both the 10 µg DEN1-80E and the 50 µg DEN1-80E formulations with Alhydrogel™ were generally well tolerated.
Assuntos
Ensaios Clínicos Fase I como Assunto , Vacinas contra Dengue/administração & dosagem , Vacinas contra Dengue/imunologia , Dengue/prevenção & controle , Avaliação Pré-Clínica de Medicamentos , Adjuvantes Imunológicos/administração & dosagem , Hidróxido de Alumínio/administração & dosagem , Animais , Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Colesterol/administração & dosagem , Dengue/epidemiologia , Vacinas contra Dengue/genética , Vacinas contra Dengue/isolamento & purificação , Combinação de Medicamentos , Humanos , Esquemas de Imunização , Interferon gama/metabolismo , Macaca , Fosfolipídeos/administração & dosagem , Saponinas/administração & dosagem , Linfócitos T/imunologia , Vacinas de Subunidades Antigênicas/administração & dosagem , Vacinas de Subunidades Antigênicas/genética , Vacinas de Subunidades Antigênicas/imunologia , Vacinas de Subunidades Antigênicas/isolamento & purificação , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologia , Vacinas Sintéticas/isolamento & purificaçãoRESUMO
UNLABELLED: T cell directed HIV vaccines are based upon the induction of CD8+ T cell memory responses that would be effective in inhibiting infection and subsequent replication of an infecting HIV-1 strain, a process that requires a match or near-match between the epitope induced by vaccination and the infecting viral strain. We compared the frequency and specificity of the CTL epitope responses elicited by the replication-defective Ad5 gag/pol/nef vaccine used in the Step trial with the likelihood of encountering those epitopes among recently sequenced Clade B isolates of HIV-1. Among vaccinees with detectable 15-mer peptide pool ELISpot responses, there was a median of four (one Gag, one Nef and two Pol) CD8 epitopes per vaccinee detected by 9-mer peptide ELISpot assay. Importantly, frequency analysis of the mapped epitopes indicated that there was a significant skewing of the T cell response; variable epitopes were detected more frequently than would be expected from an unbiased sampling of the vaccine sequences. Correspondingly, the most highly conserved epitopes in Gag, Pol, and Nef (defined by presence in >80% of sequences currently in the Los Alamos database www.hiv.lanl.gov) were detected at a lower frequency than unbiased sampling, similar to the frequency reported for responses to natural infection, suggesting potential epitope masking of these responses. This may be a generic mechanism used by the virus in both contexts to escape effective T cell immune surveillance. The disappointing results of the Step trial raise the bar for future HIV vaccine candidates. This report highlights the bias towards less-conserved epitopes present in the same vaccine used in the Step trial. Development of vaccine strategies that can elicit a greater breadth of responses, and towards conserved regions of the genome in particular, are critical requirements for effective T-cell based vaccines against HIV-1. TRIAL REGISTRATION: ClinicalTrials.gov NCT00849680, A Study of Safety, Tolerability, and Immunogenicity of the MRKAd5 Gag/Pol/Nef Vaccine in Healthy Adults.