RESUMO
Chimeric Simian-Human Immunodeficiency Viruses (SHIVs) are an important tool for evaluating anti-HIV Env interventions in nonhuman primate (NHP) models. However, most unadapted SHIVs do not replicate well in vivo limiting their utility. Furthermore, adaptation in vivo often negatively impacts fundamental properties of the Env, including neutralization profiles. Transmitted/founder (T/F) viruses are particularly important to study since they represent viruses that initiated primary HIV-1 infections and may have unique attributes. Here we combined in vivo competition and rational design to develop novel subtype C SHIVs containing T/F envelopes. We successfully generated 19 new, infectious subtype C SHIVs, which were tested in multiple combinatorial pools in Indian-origin rhesus macaques. Infected animals attained peak viremia within 5 weeks ranging from 103 to 107 vRNA copies/mL. Sequence analysis during primary infection revealed 7 different SHIVs replicating in 8 productively infected animals with certain clones prominent in each animal. We then generated 5 variants each of 6 SHIV clones (3 that predominated and 3 undetectable after pooled in vivo inoculations), converting a serine at Env375 to methionine, tyrosine, histidine, tryptophan or phenylalanine. Overall, most Env375 mutants replicated better in vitro and in vivo than wild type with both higher and earlier peak viremia. In 4 of these SHIV clones (with and without Env375 mutations) we also created mutations at position 281 to include serine, alanine, valine, or threonine. Some Env281 mutations imparted in vitro replication dynamics similar to mutations at 375; however, clones with both mutations did not exhibit incremental benefit. Therefore, we identified unique subtype C T/F SHIVs that replicate in rhesus macaques with improved acute phase replication kinetics without altering phenotype. In vivo competition and rational design can produce functional SHIVs with globally relevant HIV-1 Envs to add to the growing number of SHIV clones for HIV-1 research in NHPs.
Assuntos
Infecções por HIV/virologia , HIV-1/genética , Mutação , Síndrome de Imunodeficiência Adquirida dos Símios/virologia , Vírus da Imunodeficiência Símia/genética , Produtos do Gene env do Vírus da Imunodeficiência Humana/metabolismo , Animais , Regulação Viral da Expressão Gênica , Humanos , Macaca mulatta , Projetos de Pesquisa , Replicação Viral , Produtos do Gene env do Vírus da Imunodeficiência Humana/genéticaRESUMO
To protect against human immunodeficiency virus (HIV-1) infection, broadly neutralizing antibodies (bnAbs) must be active at the portals of viral entry in the gastrointestinal or cervicovaginal tracts. The localization and persistence of antibodies at these sites is influenced by the neonatal Fc receptor (FcRn), whose role in protecting against infection in vivo has not been defined. Here, we show that a bnAb with enhanced FcRn binding has increased gut mucosal tissue localization, which improves protection against lentiviral infection in non-human primates. A bnAb directed to the CD4-binding site of the HIV-1 envelope (Env) protein (denoted VRC01) was modified by site-directed mutagenesis to increase its binding affinity for FcRn. This enhanced FcRn-binding mutant bnAb, denoted VRC01-LS, displayed increased transcytosis across human FcRn-expressing cellular monolayers in vitro while retaining FcγRIIIa binding and function, including antibody-dependent cell-mediated cytotoxicity (ADCC) activity, at levels similar to VRC01 (the wild type). VRC01-LS had a threefold longer serum half-life than VRC01 in non-human primates and persisted in the rectal mucosa even when it was no longer detectable in the serum. Notably, VRC01-LS mediated protection superior to that afforded by VRC01 against intrarectal infection with simian-human immunodeficiency virus (SHIV). These findings suggest that modification of FcRn binding provides a mechanism not only to increase serum half-life but also to enhance mucosal localization that confers immune protection. Mutations that enhance FcRn function could therefore increase the potency and durability of passive immunization strategies to prevent HIV-1 infection.
Assuntos
Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Infecções por HIV/imunologia , Infecções por HIV/prevenção & controle , Antígenos de Histocompatibilidade Classe I/imunologia , Receptores Fc/imunologia , Síndrome de Imunodeficiência Adquirida dos Símios/imunologia , Síndrome de Imunodeficiência Adquirida dos Símios/prevenção & controle , Administração Retal , Animais , Anticorpos Neutralizantes/análise , Anticorpos Neutralizantes/sangue , Anticorpos Neutralizantes/genética , Anticorpos Antivirais/análise , Anticorpos Antivirais/sangue , Anticorpos Antivirais/genética , Afinidade de Anticorpos/genética , Afinidade de Anticorpos/imunologia , Citotoxicidade Celular Dependente de Anticorpos/imunologia , Sítios de Ligação/genética , Antígenos CD4/metabolismo , Feminino , HIV/química , HIV/imunologia , Anticorpos Anti-HIV/análise , Anticorpos Anti-HIV/sangue , Anticorpos Anti-HIV/genética , Anticorpos Anti-HIV/imunologia , Proteína gp160 do Envelope de HIV/química , Proteína gp160 do Envelope de HIV/imunologia , Meia-Vida , Imunidade nas Mucosas/imunologia , Imunização Passiva , Mucosa Intestinal/imunologia , Macaca mulatta , Masculino , Camundongos , Mutagênese Sítio-Dirigida , Receptores de IgG/imunologia , Receptores de IgG/metabolismo , Reto/imunologia , Vírus da Imunodeficiência Símia/imunologia , TranscitoseRESUMO
UNLABELLED: Extraordinary antibodies capable of near pan-neutralization of HIV-1 have been identified. One of the broadest is antibody 10E8, which recognizes the membrane-proximal external region (MPER) of the HIV-1 envelope and neutralizes >95% of circulating HIV-1 strains. If delivered passively, 10E8 might serve to prevent or treat HIV-1 infection. Antibody 10E8, however, is markedly less soluble than other antibodies. Here, we describe the use of both structural biology and somatic variation to develop optimized versions of 10E8 with increased solubility. From the structure of 10E8, we identified a prominent hydrophobic patch; reversion of four hydrophobic residues in this patch to their hydrophilic germ line counterparts resulted in an â¼10-fold decrease in turbidity. We also used somatic variants of 10E8, identified previously by next-generation sequencing, to optimize heavy and light chains; this process yielded several improved variants. Of these, variant 10E8v4 with 26 changes versus the parent 10E8 was the most soluble, with a paratope we showed crystallographically to be virtually identical to that of 10E8, a potency on a panel of 200 HIV-1 isolates also similar to that of 10E8, and a half-life in rhesus macaques of â¼10 days. An anomaly in 10E8v4 size exclusion chromatography that appeared to be related to conformational isomerization was resolved by engineering an interchain disulfide. Thus, by combining a structure-based approach with natural variation in potency and solubility from the 10E8 lineage, we successfully created variants of 10E8 which retained the potency and extraordinary neutralization breadth of the parent 10E8 but with substantially increased solubility. IMPORTANCE: Antibody 10E8 could be used to prevent HIV-1 infection, if manufactured and delivered economically. It suffers, however, from issues of solubility, which impede manufacturing. We hypothesized that the physical characteristic of 10E8 could be improved through rational design, without compromising breadth and potency. We used structural biology to identify hydrophobic patches on 10E8, which did not appear to be involved in 10E8 function. Reversion of hydrophobic residues in these patches to their hydrophilic germ line counterparts increased solubility. Next, clues from somatic variants of 10E8, identified by next-generation sequencing, were incorporated. A combination of structure-based design and somatic variant optimization led to 10E8v4, with substantially improved solubility and similar potency compared to the parent 10E8. The cocrystal structure of antibody 10E8v4 with its HIV-1 epitope was highly similar to that with the parent 10E8, despite 26 alterations in sequence and substantially improved solubility. Antibody 10E8v4 may be suitable for manufacturing.
Assuntos
Anticorpos Neutralizantes/química , Anticorpos Neutralizantes/genética , Anticorpos Anti-HIV/química , HIV-1/imunologia , Animais , Anticorpos Neutralizantes/imunologia , Anticorpos Neutralizantes/metabolismo , Técnicas de Química Analítica , Cristalografia por Raios X , Dissulfetos , Anticorpos Anti-HIV/genética , Anticorpos Anti-HIV/imunologia , Anticorpos Anti-HIV/metabolismo , Meia-Vida , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Interações Hidrofóbicas e Hidrofílicas , Macaca mulatta , Modelos Moleculares , SolubilidadeRESUMO
UNLABELLED: Pathogen-specific neutralizing antibodies protect against many viral infections and can potentially prevent human immunodeficiency virus (HIV) transmission in humans. However, neutralizing antibodies have so far only been shown to protect nonhuman primates (NHP) against lentiviral infection when given shortly before challenge. Thus, the clinical utility and feasibility of passive antibody transfer to confer long-term protection against HIV-1 are still debated. Here, we investigate the potential of a broadly neutralizing HIV-1 antibody to provide long-term protection in a NHP model of HIV-1 infection. A human antibody was simianized to avoid immune rejection and used to sustain therapeutic levels for â¼5 months. Two months after the final antibody administration, animals were completely protected against viral challenge. These findings demonstrate the feasibility and potential of long-term passive antibody for protection against HIV-1 in humans and provide a model to test antibody therapies for other diseases in NHP. IMPORTANCE: Antibodies against HIV are potential drugs that may be able to prevent HIV infection in humans. However, the long-term protective capacity of antibodies against HIV has not been assessed. Here, we repetitively administered a macaque version of a human anti-HIV antibody to monkeys, after which the antibody persisted in the blood for >5 months. Moreover, the antibody could be sustained at protective levels for 108 days, conferring protection 52 days after the last dose in a monkey model of HIV infection. Thus, passive antibody transfer can provide durable protection against infection by viruses that cause AIDS in primates.
Assuntos
Anticorpos Neutralizantes/administração & dosagem , Anticorpos Antivirais/administração & dosagem , HIV-1/imunologia , Síndrome de Imunodeficiência Adquirida dos Símios/imunologia , Síndrome de Imunodeficiência Adquirida dos Símios/prevenção & controle , Vírus da Imunodeficiência Símia/imunologia , Animais , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Modelos Animais de Doenças , Imunização Passiva , Macaca , Síndrome de Imunodeficiência Adquirida dos Símios/virologia , Resultado do TratamentoRESUMO
Broadly neutralizing antibodies (bnAbs) can prevent lentiviral infection in nonhuman primates and may slow the spread of human immunodeficiency virus type 1 (HIV-1). Although protection by passive transfer of human bnAbs has been demonstrated in monkeys, durable expression is essential for its broader use in humans. Gene-based expression of bnAbs provides a potential solution to this problem, although immune responses to the viral vector or to the antibody may limit its durability and efficacy. Here, we delivered an adeno-associated viral vector encoding a simianized form of a CD4bs bnAb, VRC07, and evaluated its immunogenicity and protective efficacy. The expressed antibody circulated in macaques for 16 weeks at levels up to 66 g/ml, although immune suppression with cyclosporine (CsA) was needed to sustain expression. Gene-delivered simian VRC07 protected against simian-human immunodeficiency virus (SHIV) infection in monkeys 5.5 weeks after treatment. Gene transfer of an anti-HIV antibody can therefore protect against infection by viruses that cause AIDS in primates when the host immune responses are controlled.
Assuntos
Anticorpos Neutralizantes/imunologia , Anticorpos Anti-HIV/imunologia , Infecções por HIV/prevenção & controle , HIV-1/imunologia , Imunoglobulina G/genética , Modelos Moleculares , Sequência de Aminoácidos , Animais , Anticorpos Neutralizantes/genética , Ciclosporina/farmacologia , Primers do DNA/genética , Ensaio de Imunoadsorção Enzimática , Técnicas de Transferência de Genes , Terapia Genética/métodos , Vetores Genéticos/genética , Anticorpos Anti-HIV/genética , Humanos , Imunossupressores/farmacologia , Macaca mulatta , Dados de Sequência Molecular , Testes de Neutralização , Reação em Cadeia da Polimerase em Tempo RealRESUMO
UNLABELLED: Over the past 5 years, a new generation of highly potent and broadly neutralizing HIV-1 antibodies has been identified. These antibodies can protect against lentiviral infection in nonhuman primates (NHPs), suggesting that passive antibody transfer would prevent HIV-1 transmission in humans. To increase the protective efficacy of such monoclonal antibodies, we employed next-generation sequencing, computational bioinformatics, and structure-guided design to enhance the neutralization potency and breadth of VRC01, an antibody that targets the CD4 binding site of the HIV-1 envelope. One variant, VRC07-523, was 5- to 8-fold more potent than VRC01, neutralized 96% of viruses tested, and displayed minimal autoreactivity. To compare its protective efficacy to that of VRC01 in vivo, we performed a series of simian-human immunodeficiency virus (SHIV) challenge experiments in nonhuman primates and calculated the doses of VRC07-523 and VRC01 that provide 50% protection (EC50). VRC07-523 prevented infection in NHPs at a 5-fold lower concentration than VRC01. These results suggest that increased neutralization potency in vitro correlates with improved protection against infection in vivo, documenting the improved functional efficacy of VRC07-523 and its potential clinical relevance for protecting against HIV-1 infection in humans. IMPORTANCE: In the absence of an effective HIV-1 vaccine, alternative strategies are needed to block HIV-1 transmission. Direct administration of HIV-1-neutralizing antibodies may be able to prevent HIV-1 infections in humans. This approach could be especially useful in individuals at high risk for contracting HIV-1 and could be used together with antiretroviral drugs to prevent infection. To optimize the chance of success, such antibodies can be modified to improve their potency, breadth, and in vivo half-life. Here, knowledge of the structure of a potent neutralizing antibody, VRC01, that targets the CD4-binding site of the HIV-1 envelope protein was used to engineer a next-generation antibody with 5- to 8-fold increased potency in vitro. When administered to nonhuman primates, this antibody conferred protection at a 5-fold lower concentration than the original antibody. Our studies demonstrate an important correlation between in vitro assays used to evaluate the therapeutic potential of antibodies and their in vivo effectiveness.
Assuntos
Anticorpos Neutralizantes/imunologia , Anticorpos Anti-HIV/imunologia , HIV-1/imunologia , Imunização Passiva/métodos , Síndrome de Imunodeficiência Adquirida dos Símios/prevenção & controle , Animais , Anticorpos Neutralizantes/administração & dosagem , Anticorpos Neutralizantes/genética , Anticorpos Anti-HIV/administração & dosagem , Anticorpos Anti-HIV/genética , HIV-1/genética , Macaca mulatta , Masculino , Dados de Sequência Molecular , Análise de Sequência de DNARESUMO
Antibody (Ab)-dependent cellular cytotoxicity (ADCC) is thought to potentially play a role in vaccine-induced protection from HIV-1. The characteristics of such antibodies remain incompletely understood. Furthermore, correlates between ADCC and HIV-1 immune status are not clearly defined. We screened the sera of 20 HIV-1-positive (HIV-1(+)) patients for ADCC. Normal human peripheral blood mononuclear cells were used to derive HIV-infected CD4(+) T cell targets and autologous, freshly isolated, natural killer (NK) cells in a novel assay that measures granzyme B (GrB) and HIV-1-infected CD4(+) T cell elimination (ICE) by flow cytometry. We observed that complex sera mediated greater levels of ADCC than anti-HIV-1 envelope glycoprotein (Env)-specific monoclonal antibodies and serum-mediated ADCC correlated with the amount of IgG and IgG1 bound to HIV-1-infected CD4(+) T cells. No correlation between ADCC and viral load, CD4(+) T cell count, or neutralization of HIV-1(SF162) or other primary viral isolates was detected. Sera pooled from clade B HIV-1(+) individuals exhibited breadth in killing targets infected with HIV-1 from clades A/E, B, and C. Taken together, these data suggest that the total amount of IgG bound to an HIV-1-infected cell is an important determinant of ADCC and that polyvalent antigen-specific Abs are required for a robust ADCC response. In addition, Abs elicited by a vaccine formulated with immunogens from a single clade may generate a protective ADCC response in vivo against a variety of HIV-1 species. Increased understanding of the parameters that dictate ADCC against HIV-1-infected cells will inform efforts to stimulate ADCC activity and improve its potency in vaccinees.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/virologia , Citotoxicidade Imunológica , Anticorpos Anti-HIV/imunologia , HIV-1/imunologia , Imunoglobulina G/imunologia , Anticorpos Neutralizantes/sangue , Contagem de Linfócito CD4 , Sobrevivência Celular , Citometria de Fluxo , Anticorpos Anti-HIV/sangue , Infecções por HIV/imunologia , Humanos , Carga ViralRESUMO
The RV144 trial demonstrated that an experimental AIDS vaccine can prevent human immunodeficiency virus type 1 (HIV-1) infection in humans. Because of its limited efficacy, further understanding of the mechanisms of preventive AIDS vaccines remains a priority, and nonhuman primate (NHP) models of lentiviral infection provide an opportunity to define immunogens, vectors, and correlates of immunity. In this study, we show that prime-boost vaccination with a mismatched SIV envelope (Env) gene, derived from simian immunodeficiency virus SIVmac239, prevents infection by SIVsmE660 intrarectally. Analysis of different gene-based prime-boost immunization regimens revealed that recombinant adenovirus type 5 (rAd5) prime followed by replication-defective lymphocytic choriomeningitis virus (rLCMV) boost elicited robust CD4 and CD8 T-cell and humoral immune responses. This vaccine protected against infection after repetitive mucosal challenge with efficacies of 82% per exposure and 62% cumulatively. No effect was seen on viremia in infected vaccinated monkeys compared to controls. Protection correlated with the presence of neutralizing antibodies to the challenge viruses tested in peripheral blood mononuclear cells. These data indicate that a vaccine expressing a mismatched Env gene alone can prevent SIV infection in NHPs and identifies an immune correlate that may guide immunogen selection and immune monitoring for clinical efficacy trials.
Assuntos
Adenoviridae , Produtos do Gene env/imunologia , Vírus da Coriomeningite Linfocítica , Vacinas contra a SAIDS/imunologia , Síndrome de Imunodeficiência Adquirida dos Símios/imunologia , Vírus da Imunodeficiência Símia/imunologia , Vacinas de DNA/imunologia , Vacinas contra a AIDS/genética , Vacinas contra a AIDS/imunologia , Animais , Feminino , Produtos do Gene env/genética , HIV-1/genética , HIV-1/imunologia , Imunização Secundária/métodos , Macaca mulatta , Masculino , Camundongos , Vacinas contra a SAIDS/genética , Síndrome de Imunodeficiência Adquirida dos Símios/genética , Síndrome de Imunodeficiência Adquirida dos Símios/prevenção & controle , Transdução Genética , Vacinas de DNA/genéticaRESUMO
We assessed whether the sublingual (s.l.) route would be an effective means of delivering vaccines against influenza virus in mice by using either formalin-inactivated or live influenza A/PR/8 virus (H1N1). Sublingual administration of inactivated influenza virus given on two occasions induced both systemic and mucosal antibody responses and conferred protection against a lethal intranasal (i.n.) challenge with influenza virus. Coadministration of a mucosal adjuvant (mCTA-LTB) enhanced these responses and resulted in complete protection against respiratory viral challenge. In addition, s.l. administration of formalin-inactivated A/PR/8 plus mCTA-LTB induced systemic expansion of IFN-gamma-secreting T cells and virus-specific cytotoxic T lymphocyte responses. Importantly, a single s.l. administration of live A/PR/8 virus was not pathogenic and induced protection mediated by both acquired and innate immunity. Moreover, s.l. administration of live A/PR/8 virus conferred heterosubtypic protection against respiratory challenge with H3N2 virus. Unlike the i.n. route, the A/PR/8 virus, whether live or inactivated, did not migrate to or replicate in the CNS after s.l. administration. Based on these promising findings, we propose that the s.l. mucosal route offers an attractive alternative to mucosal routes for administering influenza vaccines.
Assuntos
Vírus da Influenza A Subtipo H1N1/imunologia , Vacinas contra Influenza/administração & dosagem , Infecções por Orthomyxoviridae/prevenção & controle , Vacinas de Produtos Inativados/administração & dosagem , Administração Sublingual , Animais , Anticorpos Antivirais/sangue , Antígenos Virais/análise , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Sistema Nervoso Central/imunologia , Imunidade Inata , Imunidade nas Mucosas , Interferon gama/metabolismo , Camundongos , Infecções por Orthomyxoviridae/imunologia , VacinaçãoRESUMO
An effective AIDS vaccine must control highly diverse circulating strains of human immunodeficiency virus type 1 (HIV-1). Among HIV-1 gene products, the envelope (Env) protein contains variable as well as conserved regions. In this report, an informatic approach to the design of T-cell vaccines directed to HIV-1 Env M group global sequences was tested. Synthetic Env antigens were designed to express mosaics that maximize the inclusion of common potential T-cell epitope (PTE) 9-mers and minimize the inclusion of rare epitopes likely to elicit strain-specific responses. DNA vaccines were evaluated using intracellular cytokine staining in inbred mice with a standardized panel of highly conserved 15-mer PTE peptides. One-, two-, and three-mosaic sets that increased theoretical epitope coverage were developed. The breadth and magnitude of T-cell immunity stimulated by these vaccines were compared to those for natural strain Envs; additional comparisons were performed on mutant Envs, including gp160 or gp145 with or without V regions and gp41 deletions. Among them, the two- or three-mosaic Env sets elicited the optimal CD4 and CD8 responses. These responses were most evident in CD8 T cells; the three-mosaic set elicited responses to an average of eight peptide pools, compared to two pools for a set of three natural Envs. Synthetic mosaic HIV-1 antigens can therefore induce T-cell responses with expanded breadth and may facilitate the development of effective T-cell-based HIV-1 vaccines.
Assuntos
Vacinas contra a AIDS/imunologia , HIV-1/imunologia , Linfócitos T/imunologia , Vacinas de DNA/imunologia , Produtos do Gene env do Vírus da Imunodeficiência Humana/imunologia , Animais , Epitopos de Linfócito T/imunologia , Feminino , Imunidade Celular , Camundongos , Modelos Estatísticos , Linfócitos T/virologiaRESUMO
Human immunodeficiency virus type 1 (HIV-1) infection is characterized by the rapid onset of intestinal T-cell depletion that initiates the progression to AIDS. The induction of protective immunity in the intestinal mucosa therefore represents a potentially desirable feature of a preventive AIDS vaccine. In this study, we have evaluated the ability of an enteric adenovirus, recombinant adenovirus 41 (rAd41), to elicit intestinal and systemic immune responses by different immunization routes, alone or in combination with rAd5. rAd41 expressing HIV envelope (Env) protein induced cellular immune responses comparable to those of rAd5-based vectors after either a single intramuscular injection or a DNA prime/rAd boost. Oral priming with rAd41-Env followed by intramuscular boosting with rAd5-Env stimulated a more potent CD8(+) T-cell response in the small intestine than the other immunization regimens. Furthermore, the direct injection of rAd41-Env into ileum together with intramuscular rAd5-Env boosting increased Env-specific cellular immunity markedly in mucosal as well as systemic compartments. These data demonstrate that heterologous rAd41 oral or ileal priming with rAd5 intramuscular boosting elicits enhanced intestinal mucosal cellular immunity and that oral or ileal vector delivery for primary immunization facilitates the generation of mucosal immunity.
Assuntos
Vacinas contra a AIDS/administração & dosagem , Vacinas contra a AIDS/imunologia , Adenoviridae/imunologia , Vetores Genéticos/imunologia , Administração Oral , Animais , Linfócitos T CD8-Positivos/imunologia , Feminino , Imunidade nas Mucosas , Imunização/métodos , Imunização Secundária , Injeções Intramusculares , Intestino Delgado/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/imunologia , Produtos do Gene env do Vírus da Imunodeficiência Humana/genética , Produtos do Gene env do Vírus da Imunodeficiência Humana/imunologiaRESUMO
Effective vaccines for human immunodeficiency virus type 1 (HIV-1) will likely need to stimulate protective immunity in the intestinal mucosa, where HIV-1 infection causes severe CD4(+) T-cell depletion. While replication-competent recombinant adenovirus (rAd) vectors can stimulate adenovirus-specific mucosal immunity after replication, oral delivery of replication-defective rAd vectors encoding specific immunogens has proven challenging. In this study, we have systematically identified barriers to effective gut delivery of rAd vectors and identified sites and strategies to induce potent cellular and humoral immunity. Vector-mediated gene transfer by rAd5 was susceptible to low-pH buffer, gastric and pancreatic proteases, and extracellular mucins. Using ex vivo organ explants, we found that transduction with rAd5 was highest in the ileum and colon among all intestinal segments. Transgene expression was 100-fold higher after direct surgical introduction into the ileum than after oral gavage, with rAd5 showing greater potency than the rAd35 or the rAd41 vector. A single immunization of rAd5 encoding HIV-1 gp140B to the ileum stimulated potent CD8(+) T-cell responses in the intestinal and systemic compartments, and these responses were further enhanced by intramuscular rAd5 boosting. These studies suggest that induction of primary immune responses by rAd5 gut immunization and subsequent systemic boosting elicits potent antigen-specific gut mucosal responses.
Assuntos
Vacinas contra a AIDS/imunologia , Adenoviridae/imunologia , Vetores Genéticos/imunologia , Infecções por HIV/imunologia , HIV-1/imunologia , Mucosa Intestinal/imunologia , Vacinas contra a AIDS/administração & dosagem , Adenoviridae/genética , Animais , Células Cultivadas , Feminino , Vetores Genéticos/genética , Infecções por HIV/virologia , Humanos , Imunidade Celular , Imunidade nas Mucosas , Mucosa Intestinal/virologia , Camundongos , Camundongos Endogâmicos BALB C , Linfócitos T/imunologia , Produtos do Gene env do Vírus da Imunodeficiência Humana/administração & dosagem , Produtos do Gene env do Vírus da Imunodeficiência Humana/imunologiaRESUMO
Zika virus (ZIKV) is the cause of a pandemic associated with microcephaly in newborns and Guillain-Barre syndrome in adults. Currently, there are no available treatments or vaccines for ZIKV, and the development of a safe and effective vaccine is a high priority for many global health organizations. We describe the development of ZIKV vaccine candidates using the self-amplifying messenger RNA (SAM) platform technology delivered by cationic nanoemulsion (CNE) that allows bedside mixing and is particularly useful for rapid responses to pandemic outbreaks. Two immunizations of either of the two lead SAM (CNE) vaccine candidates elicited potent neutralizing antibody responses to ZIKV in mice and nonhuman primates. Both SAM (CNE) vaccines protected these animals from ZIKV challenge, with one candidate providing complete protection against ZIKV infection in nonhuman primates. The data provide a preclinical proof of concept that a SAM (CNE) vaccine candidate can rapidly elicit protective immunity against ZIKV.
Assuntos
Vacinas Virais , Infecção por Zika virus , Zika virus , Animais , Anticorpos Antivirais , Camundongos , RNA Mensageiro/genética , Zika virus/genética , Infecção por Zika virus/prevenção & controleRESUMO
The emergence of Zika virus (ZIKV) in the Americas stimulated the development of multiple ZIKV vaccine candidates. We previously developed two related DNA vaccine candidates encoding ZIKV structural proteins that were immunogenic in animal models and humans. We sought to identify neutralizing antibody (NAb) properties induced by each vaccine that correlated with protection in nonhuman primates (NHPs). Despite eliciting equivalent NAb titers in NHPs, these vaccines were not equally protective. The transfer of equivalent titers of vaccine-elicited NAb into AG129 mice also revealed nonequivalent protection, indicating qualitative differences among antibodies (Abs) elicited by these vaccines. Both vaccines elicited Abs with similar binding titers against envelope protein monomers and those incorporated into virus-like particles, as well as a comparable capacity to orchestrate phagocytosis. Functional analysis of vaccine-elicited NAbs from NHPs and humans revealed a capacity to neutralize the structurally mature form of the ZIKV virion that varied in magnitude among vaccine candidates. Conversely, sensitivity to the virion maturation state was not a characteristic of NAbs induced by natural or experimental infection. Passive transfer experiments in mice revealed that neutralization of mature ZIKV virions more accurately predicts protection from ZIKV infection. These findings demonstrate that NAb correlates of protection may differ among vaccine antigens when assayed using standard neutralization platforms and suggest that measurements of Ab quality, including the capacity to neutralize mature virions, will be critical for defining correlates of ZIKV vaccine-induced immunity.
Assuntos
Vacinas Virais , Infecção por Zika virus , Zika virus , Animais , Anticorpos Neutralizantes , Anticorpos Antivirais , Camundongos , Infecção por Zika virus/prevenção & controleRESUMO
Her-2/neu is a well-characterized tumor-associated antigen overexpressed in human carcinomas such as breast cancer. Because Her-2/neu is a self-antigen with poor immunogenicity due to immunologic tolerance, active immunotherapy targeting Her-2/neu should incorporate methods to overcome immunologic tolerance to self-proteins. In this study, we developed a tolerogenic tumor model in mice using mouse Her-2/neu as self-antigen and investigated whether genetic vaccination with DNA plasmid and/or adenoviral vector expressing the extracellular and transmembrane domain of syngeneic mouse Her-2/neu or xenogenic human Her-2/neu could induce mouse Her-2/neu-specific CTL responses. Interestingly, adenoviral vectors expressing xenogenic human Her-2/neu (AdhHM) proved capable of breaking immune tolerance and of thereby inducing self-reactive CTL and antibodies, but not to the degree required to induce therapeutic antitumor immunity. In attempting to generate therapeutic antitumor immunity against established tumors, we adopted several approaches. Treatment with agonistic anti-glucocorticoid-induced TNFR family-related receptor (GITR) antibody plus AdhHM immunization significantly increased self-reactive CTL responses, and alpha-galactosylceramide (alphaGalCer)-loaded dendritic cells (DC) transduced with AdhHM were shown to break self-tolerance in a tolerogenic murine tumor model. Furthermore, gemcitabine treatment together with either AdhHM plus agonistic anti-GITR antibody administration or alphaGalCer-loaded DC transduced with AdhHM showed potent therapeutic antitumor immunity and perfect protection against preexisting tumors. Gemcitabine treatment attenuated the tumor-suppressive environment by eliminating CD11b(+)/Gr-1(+) myeloid-derived suppressor cells. When combined with immunotherapies, gemcitabine offers a promising strategy for the Ag-specific treatment of human cancer.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Neoplasias da Mama/prevenção & controle , Neoplasias do Colo/prevenção & controle , Imunoterapia , Fragmentos de Peptídeos/imunologia , Receptor ErbB-2/imunologia , Linfócitos T Citotóxicos/imunologia , Adenoviridae/genética , Animais , Antígenos CD/genética , Antígenos CD/imunologia , Antígenos CD/metabolismo , Antígenos de Diferenciação/genética , Antígenos de Diferenciação/imunologia , Antígenos de Diferenciação/metabolismo , Neoplasias da Mama/genética , Neoplasias da Mama/imunologia , Linfócitos T CD8-Positivos/imunologia , Antígeno CTLA-4 , Linhagem Celular Tumoral , Proliferação de Células , Neoplasias do Colo/genética , Neoplasias do Colo/imunologia , Terapia Combinada , Ciclofosfamida/administração & dosagem , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Desoxicitidina/administração & dosagem , Desoxicitidina/análogos & derivados , Docetaxel , Doxorrubicina/administração & dosagem , Feminino , Citometria de Fluxo , Galactosilceramidas/imunologia , Vetores Genéticos/administração & dosagem , Proteína Relacionada a TNFR Induzida por Glucocorticoide , Humanos , Imunização , Camundongos , Células Mieloides/citologia , Células Mieloides/imunologia , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/farmacologia , Receptor ErbB-2/genética , Receptores de Fator de Crescimento Neural/genética , Receptores de Fator de Crescimento Neural/metabolismo , Receptores do Fator de Necrose Tumoral/genética , Receptores do Fator de Necrose Tumoral/metabolismo , Linfócitos T Reguladores , Taxoides/administração & dosagem , Transfecção , Vacinas de DNA/administração & dosagem , GencitabinaRESUMO
Western, Eastern, and Venezuelan equine encephalitis viruses (WEEV, EEEV, and VEEV, respectively) are important mosquito-borne agents that pose public health and bioterrorism threats. Despite considerable advances in understanding alphavirus replication, there are currently no available effective vaccines or antiviral treatments against these highly lethal pathogens. To develop a potential countermeasure for viral encephalitis, we generated a trivalent, or three-component, EEV vaccine composed of virus-like particles (VLPs). Monovalent VLPs elicited neutralizing antibody responses and protected mice and nonhuman primates (NHPs) against homologous challenges, but they were not cross-protective. In contrast, NHPs immunized with trivalent VLPs were completely protected against aerosol challenge by each of these three EEVs. Passive transfer of IgG from immunized NHPs protected mice against aerosolized EEV challenge, demonstrating that the mechanism of protection was humoral. Because they are replication incompetent, these trivalent VLPs represent a potentially safe and effective vaccine that can protect against diverse encephalitis viruses.
Assuntos
Vírus da Encefalite/imunologia , Encefalite por Arbovirus/imunologia , Encefalite por Arbovirus/prevenção & controle , Vacinas de Partículas Semelhantes a Vírus/imunologia , Animais , Anticorpos Neutralizantes/imunologia , Encefalite por Arbovirus/patologia , Encefalite por Arbovirus/virologia , Imunização , Imunoglobulina G/imunologia , Macaca fascicularis , Camundongos Endogâmicos BALB C , Vacinas de Partículas Semelhantes a Vírus/ultraestruturaRESUMO
BACKGROUND: Human monoclonal antibodies that potently and broadly neutralise HIV-1 are under development to prevent and treat HIV-1 infection. In this phase 1 clinical trial we aimed to determine the safety, tolerability, and pharmacokinetic profile of the broadly neutralising monoclonal antibody VRC07-523LS, an engineered variant of VRC01 that targets the CD4 binding site of the HIV-1 envelope protein. METHODS: This phase 1, open-label, dose-escalation clinical trial was done at the National Institutes of Health Clinical Center in Bethesda, MD, USA. Individuals were recruited from the greater Washington, DC, area by IRB-approved written and electronic media. We enrolled healthy, HIV-1-negative adults aged 18-50 years. Inclusion criteria were good general health, measured through clinical laboratory tests, medical history, and physical examination. Participants self-selected into one of seven open groups during enrolment without randomisation. Four groups received a single intravenous dose of 1, 5, 20, or 40 mg/kg of VRC07-523LS, and one group received a single 5 mg/kg subcutaneous dose. Two groups received three doses of either 20 mg/kg intravenous VRC07-523LS, or 5 mg/kg subcutaneous VRC07-523LS at 12-week intervals. The primary outcome was the safety and tolerability of VRC07-523LS, assessed by dose, route, and number of administrations. This study is registered with ClinicalTrials.gov, NCT03015181. FINDINGS: Between Feb 21, 2017, and September 13, 2017, we enrolled 26 participants, including 11 (42%) men and 15 (58%) women. Two (8%) participants withdrew from the study early: one participant in group 1 enrolled in the study but never received VRC07-523LS, and one participant in group 6 chose to withdraw after a single administration. One (4%) participant in group 7 received only one of the three scheduled administrations. 17 participants received intravenous administrations and 8 participants received subcutaneous administrations. VRC07-523LS was safe and well tolerated, we observed no serious adverse events or dose-limiting toxic effects. All reported local and systemic reactogenicity was mild to moderate in severity. The most commonly reported symptoms following intravenous administration were malaise or myalgia in three (18%) participants and headache or chills in two (12%) participants. The most commonly reported symptoms following subcutaneous administration were pain and tenderness in four participants (50%) and malaise or headache in three (38%) participants. INTERPRETATION: Safe and well tolerated, VRC07-523LS is a strong and practical candidate for inclusion in HIV-1 prevention and therapeutic strategies. The results from this trial also indicate that an HIV-1 broadly neutralising monoclonal antibody engineered for improved pharmacokinetic and neutralisation properties can be safe for clinical use. FUNDING: National Institutes of Health.
Assuntos
Anticorpos Monoclonais/farmacocinética , Infecções por HIV/tratamento farmacológico , Administração Cutânea , Administração Intravenosa , Adulto , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais/efeitos adversos , Anticorpos Neutralizantes/administração & dosagem , Anticorpos Neutralizantes/efeitos adversos , Feminino , Voluntários Saudáveis , Humanos , Masculino , Pessoa de Meia-Idade , Adulto JovemRESUMO
Although resting B cells are known for being poorly immunogenic and for inducing T-cell tolerance, we have here attempted to test whether their immunogenicity could be enhanced by CD1d-restricted invariant T cells (iNKT) to a point where they could be used in cellular vaccines. We found that the addition of the iNKT ligand alpha-galactosylceramide (alphaGalCer) to peptide-loaded B cells overcame peptide-specific T-cell unresponsiveness and allowed for the generation of peptide-specific memory CTL immunity. This CTL was induced independently of CD4 T and natural killer cells but required iNKT and CD8 T cells. B cells directly primed CTL, and the alphaGalCer and the peptide must be presented on the same cell. Importantly, our B-cell-based vaccine is comparable in efficiency with dendritic cell-based vaccines, inducing similar CTL responses as well as providing an effective regimen for preventing and suppressing s.c. and metastatic tumors. Therefore, with the help of iNKT, peptide-pulsed B cells can establish long-lasting antitumor immunity and so show promise as the basis for an alternative cell-based vaccine.
Assuntos
Antígenos CD1/imunologia , Linfócitos B/imunologia , Vacinas Anticâncer/imunologia , Neoplasias do Colo/prevenção & controle , Imunoterapia Adotiva/métodos , Melanoma Experimental/prevenção & controle , Linfócitos T Citotóxicos/imunologia , Animais , Antígenos CD1d , Vacinas Anticâncer/uso terapêutico , Neoplasias do Colo/imunologia , Feminino , Galactosilceramidas/imunologia , Memória Imunológica/imunologia , Melanoma Experimental/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BLRESUMO
alpha-GalCer is the first defined and most potent agonistic antigen of the T cell receptor of natural killer T cells. We have prepared a series of 1,2,3-triazole-containing alpha-GalCer analogues in which the lipid chain lengths have been incrementally varied. We found that this isosteric replacement of alpha-GalCer's amide moiety with triazole increases the IL-4 versus IFN-gamma bias of released cytokines. The stimulatory effect was influenced by the length of the attached chain. In particular, the long-chained triazole analogues have a comparable stimulatory effect on cytokine production as alpha-GalCer and exhibit a stronger Th2 cytokine response.
Assuntos
Adjuvantes Imunológicos/síntese química , Galactosilceramidas/síntese química , Triazóis/síntese química , Adjuvantes Imunológicos/química , Adjuvantes Imunológicos/farmacologia , Animais , Antígenos CD1/química , Antígenos CD1d , Linhagem Celular Tumoral , Técnicas de Cocultura , Galactosilceramidas/química , Galactosilceramidas/farmacologia , Humanos , Hibridomas , Interferon gama/metabolismo , Interleucina-2/metabolismo , Interleucina-4/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Modelos Moleculares , Ratos , Receptores de Antígenos de Linfócitos T/agonistas , Baço/citologia , Relação Estrutura-Atividade , Células Th2/imunologia , Triazóis/química , Triazóis/farmacologiaRESUMO
Zika virus (ZIKV) was identified as a cause of congenital disease during the explosive outbreak in the Americas and Caribbean that began in 2015. Because of the ongoing fetal risk from endemic disease and travel-related exposures, a vaccine to prevent viremia in women of childbearing age and their partners is imperative. We found that vaccination with DNA expressing the premembrane and envelope proteins of ZIKV was immunogenic in mice and nonhuman primates, and protection against viremia after ZIKV challenge correlated with serum neutralizing activity. These data not only indicate that DNA vaccination could be a successful approach to protect against ZIKV infection, but also suggest a protective threshold of vaccine-induced neutralizing activity that prevents viremia after acute infection.