RESUMO
UNLABELLED: A fraction of HIV-1 patients are able to generate broadly neutralizing antibodies (bNAbs) after 2 to 4 years of infection. In rare occasions such antibodies are observed close to the first year of HIV-1 infection but never within the first 6 months. In this study, we analyzed the neutralization breadth of sera from 157 antiretroviral-naive individuals who were infected for less than 1 year. A range of neutralizing activities was observed with a previously described panel of six recombinant viruses from five different subtypes (M. Medina-Ramirez et al., J Virol 85:5804-5813, 2011, http://dx.doi.org/10.1128/JVI.02482-10). Some sera were broadly reactive, predominantly targeting envelope epitopes within the V2 glycan-dependent region. The neutralization breadth was positively associated with time postinfection (P = 0.0001), but contrary to what has been reported for chronic infections, no association with the viral load was observed. Notably, five individuals within the first 6 months of infection (two as early as 77 and 96 days postinfection) showed substantial cross-neutralization. This was confirmed with an extended panel of 20 Env pseudoviruses from four different subtypes (two in tier 3, 14 in tier 2, and four in tier 1). Sera from these individuals were capable of neutralizing viruses from four different subtypes with a geometric mean 50% infective dose (ID50) between 100 and 800. These results indicate that induction of cross-neutralizing responses, albeit rare, is achievable even within 6 months of HIV-1 infection. These observations encourage the search for immunogens able to elicit this kind of response in preventive HIV-1 vaccine approaches. IMPORTANCE: There are very few individuals able to mount broadly neutralizing activity (bNA) close to the first year postinfection. It is not known how early in the infection cross-neutralizing responses can be induced. In the present study, we show that bNAbs, despite being rare, can be induced much earlier than previously thought. The identification of HIV-1-infected patients with these activities within the first months of infection and characterization of these responses will help in defining new immunogen designs and neutralization targets for vaccine-mediated induction of bNAbs.
Assuntos
Anticorpos Neutralizantes/sangue , Anticorpos Neutralizantes/imunologia , Epitopos/imunologia , Anticorpos Anti-HIV/sangue , Infecções por HIV/imunologia , HIV-1/imunologia , Adulto , Estudos Transversais , Mapeamento de Epitopos , Epitopos/química , Feminino , Anticorpos Anti-HIV/imunologia , Infecções por HIV/virologia , HIV-1/fisiologia , Humanos , Masculino , Testes de Neutralização , Polissacarídeos/imunologia , Fatores de Tempo , Carga ViralRESUMO
UNLABELLED: The potency and breadth of the recently isolated neutralizing human monoclonal antibodies to HIV-1 have stimulated interest in their use to prevent or to treat HIV-1 infection. Due to the antigenically diverse nature of the HIV-1 envelope (Env), no single antibody is highly active against all viral strains. While the physical combination of two broadly neutralizing antibodies (bNAbs) can improve coverage against the majority of viruses, the clinical-grade manufacturing and testing of two independent antibody products are time and resource intensive. In this study, we constructed bispecific immunoglobulins (IgGs) composed of independent antigen-binding fragments with a common Fc region. We developed four different bispecific IgG variants that included antibodies targeting four major sites of HIV-1 neutralization. We show that these bispecific IgGs display features of both antibody specificities and, in some cases, display improved coverage over the individual parental antibodies. All four bispecific IgGs neutralized 94% to 97% of antigenically diverse viruses in a panel of 206 HIV-1 strains. Among the bispecific IgGs tested, VRC07 × PG9-16 displayed the most favorable neutralization profile. It was superior in breadth to either of the individual antibodies, neutralizing 97% of viruses with a median 50% inhibitory concentration (IC50) of 0.055 µg/ml. This bispecific IgG also demonstrated in vivo pharmacokinetic parameters comparable to those of the parental bNAbs when administered to rhesus macaques. These results suggest that IgG-based bispecific antibodies are promising candidates for the prevention and treatment of HIV-1 infection in humans. IMPORTANCE: To prevent or treat HIV-1 infection, antibodies must potently neutralize nearly all strains of HIV-1. Thus, the physical combination of two or more antibodies may be needed to broaden neutralization coverage and diminish the possibility of viral resistance. A bispecific antibody that has two different antibody binding arms could potentially display neutralization characteristics better than those of any single parental antibody. Here we show that bispecific antibodies contain the binding specificities of the two parental antibodies and that a single bispecific antibody can neutralize 97% of viral strains with a high overall potency. These findings support the use of bispecific antibodies for the prevention or treatment of HIV-1 infection.
Assuntos
Anticorpos Biespecíficos , Anticorpos Neutralizantes , Anticorpos Anti-HIV , Infecções por HIV , HIV-1/imunologia , Imunoglobulina G , Animais , Anticorpos Biespecíficos/imunologia , Anticorpos Biespecíficos/farmacocinética , Anticorpos Biespecíficos/farmacologia , Anticorpos Neutralizantes/imunologia , Anticorpos Neutralizantes/farmacologia , Feminino , Anticorpos Anti-HIV/imunologia , Anticorpos Anti-HIV/farmacologia , Infecções por HIV/tratamento farmacológico , Infecções por HIV/imunologia , Humanos , Imunoglobulina G/imunologia , Imunoglobulina G/farmacologia , Macaca mulatta , MasculinoRESUMO
VRC-HIVMAB060-00-AB (VRC01) is a broadly neutralizing HIV-1 monoclonal antibody (mAb) isolated from the B cells of an HIV-infected patient. It is directed against the HIV-1 CD4 binding site and is capable of potently neutralizing the majority of diverse HIV-1 strains. This Phase I dose-escalation study in healthy adults was conducted at the National Institutes of Health (NIH) Clinical Center (Bethesda, MD, USA). Primary objectives were the safety, tolerability and pharmacokinetics (PK) of VRC01 intravenous (i.v.) infusion at 5, 20 or 40 mg/kg, given either once (20 mg/kg) or twice 28 days apart (all doses), and of subcutaneous (s.c.) delivery at 5 mg/kg compared to s.c. placebo given twice, 28 days apart. Cumulatively, 28 subjects received 43 VRC01 and nine received placebo administrations. There were no serious adverse events or dose-limiting toxicities. Mean 28-day serum trough concentrations after the first infusion were 35 and 57 µg/ml for groups infused with 20 mg/kg (n = 8) and 40 mg/kg (n = 5) doses, respectively. Mean 28-day trough concentrations after the second infusion were 56 and 89 µg/ml for the same two doses. Over the 5-40 mg/kg i.v. dose range (n = 18), the clearance was 0.016 l/h and terminal half-life was 15 days. After infusion VRC01 retained expected neutralizing activity in serum, and anti-VRC01 antibody responses were not detected. The human monoclonal antibody (mAb) VRC01 was well tolerated when delivered i.v. or s.c. The mAb demonstrated expected half-life and pharmacokinetics for a human immunoglobulin G. The safety and PK results support and inform VRC01 dosing schedules for planning HIV-1 prevention efficacy studies.
Assuntos
Anticorpos Monoclonais , Anticorpos Neutralizantes , Anticorpos Anti-HIV , Infecções por HIV , HIV-1 , Adolescente , Adulto , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais/efeitos adversos , Anticorpos Monoclonais/farmacocinética , Anticorpos Neutralizantes/administração & dosagem , Anticorpos Neutralizantes/efeitos adversos , Anticorpos Amplamente Neutralizantes , Relação Dose-Resposta a Droga , Feminino , Anticorpos Anti-HIV/administração & dosagem , Anticorpos Anti-HIV/efeitos adversos , Infecções por HIV/sangue , Infecções por HIV/tratamento farmacológico , Meia-Vida , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
Cellular entry of human immunodeficiency virus type 1 (HIV-1) requires binding to both CD4 (ref, 1, 2) and to one of the chemokine receptors recently discovered to act as coreceptors. Viruses that infect T-cell lines to form syncytia (syncytium-inducing, SI) are frequently found in late-stage HIV disease and utilize the chemokine receptor CXCR-4; macrophage-tropic viruses are non-syncytium-inducing (NSI), found throughout disease and utilize CCR-5 (ref. 3-11). We postulated that CCR-5 gene defects might reduce infection risk in seronegative subjects and prolong AIDS-free survival in seropositive subjects with NSI but not SI virus. Homozygous (delta ccr5/delta ccr5) and heterozygous (CCR5/delta ccr5) CCR-5 deletions (delta ccr5) were found in 7 (2.7%) and 51 (19.5%), respectively, of 261 seronegative subjects from the San Francisco Men's Health Study. CCR-5/delta ccr5 genotype was identified in 33 of 172 (19.2%) nonprogressors and 25 of 234 (10.7%) progressors from the seropositive arm of this cohort. The delta ccr5 allele conferred a significant protective effect against HIV-1 infection (P = 0.001) and a survival advantage against disease progression (P = 0.02). Although both progressing and nonprogressing CCR5/delta ccr5 subjects were identified, a distinct survival advantage was shown for those with NSI virus (P < 0.0001). Thus, the protective effect of delta ccr5 against disease progression is lost when the infecting virus uses CXCR-4 as a coreceptor.
Assuntos
Linfócitos T CD4-Positivos/virologia , Infecções por HIV/virologia , HIV-1/genética , Receptores de Citocinas/genética , Receptores de HIV/genética , Linhagem Celular , Infecções por HIV/transmissão , Homozigoto , Humanos , Mutação , Receptores CCR5RESUMO
The development of the human immunodeficiency virus-1 (HIV-1)/simian immunodeficiency virus (SIV) chimeric virus macaque model (SHIV) permits the in vivo evaluation of anti-HIV-1 envelope glycoprotein immune responses. Using this model, others, and we have shown that passively infused antibody can protect against an intravenous challenge. However, HIV-1 is most often transmitted across mucosal surfaces and the intravenous challenge model may not accurately predict the role of antibody in protection against mucosal exposure. After controlling the macaque estrous cycle with progesterone, anti-HIV-1 neutralizing monoclonal antibodies 2F5 and 2G12, and HIV immune globulin were tested. Whereas all five control monkeys displayed high plasma viremia and rapid CD4 cell decline, 14 antibody-treated macaques were either completely protected against infection or against pathogenic manifestations of SHIV-infection. Infusion of all three antibodies together provided the greatest amount of protection, but a single monoclonal antibody, with modest virus neutralizing activity, was also protective. Compared with our previous intravenous challenge study with the same virus and antibodies, the data indicated that greater protection was achieved after vaginal challenge. This study demonstrates that antibodies can affect transmission and subsequent disease course after vaginal SHIV-challenge; the data begin to define the type of antibody response that could play a role in protection against mucosal transmission of HIV-1.
Assuntos
Anticorpos Monoclonais/administração & dosagem , HIV-1/imunologia , Síndrome de Imunodeficiência Adquirida dos Símios/prevenção & controle , Vírus da Imunodeficiência Símia/imunologia , Vagina/imunologia , Animais , Quimera , Feminino , Anticorpos Anti-HIV/análise , Anticorpos Anti-HIV/sangue , HIV-1/genética , Imunidade nas Mucosas , Imunização Passiva , Macaca mulatta , Testes de Neutralização , Síndrome de Imunodeficiência Adquirida dos Símios/imunologia , Síndrome de Imunodeficiência Adquirida dos Símios/transmissão , Vírus da Imunodeficiência Símia/genéticaRESUMO
Dengue virus (DV), an arthropod-borne flavivirus, causes a febrile illness for which there is no antiviral treatment and no vaccine. Macrophages are important in dengue pathogenesis; however, the initial target cell for DV infection remains unknown. As DV is introduced into human skin by mosquitoes of the genus Aedes, we undertook experiments to determine whether human dendritic cells (DCs) were permissive for the growth of DV. Initial experiments demonstrated that blood-derived DCs were 10-fold more permissive for DV infection than were monocytes or macrophages. We confirmed this with human skin DCs (Langerhans cells and dermal/interstitial DCs). Using cadaveric human skin explants, we exposed skin DCs to DV ex vivo. Of the human leukocyte antigen DR-positive DCs that migrated from the skin, emigrants from both dermis and epidermis, 60-80% expressed DV antigens. These observations were supported by histologic findings from the skin rash of a human subject who received an attenuated tetravalent dengue vaccine. Immunohistochemistry of the skin showed CD1a-positive DCs double-labeled with an antibody against DV envelope glycoprotein. These data demonstrate that human skin DCs are permissive for DV infection, and provide a potential mechanism for the transmission of DV into human skin.
Assuntos
Vírus da Dengue/crescimento & desenvolvimento , Células de Langerhans/virologia , Pele/virologia , Células Sanguíneas/virologia , Derme/virologia , Exantema , Humanos , Macrófagos/virologia , Monócitos/virologia , Pele/citologia , Proteínas Virais/isolamento & purificação , Vacinas Virais/efeitos adversosRESUMO
Clinical investigation in humans and experimental lentivirus infection in nonhuman primates have advanced our understanding of immune responses that control HIV-1 disease. Recently, immunization approaches in macaques have shown that the immune response can control viremia and improve clinical outcome. When such vaccine strategies are formulated to be similarly immunogenic in humans, they could form the basis for the development of candidate AIDS vaccines that would prevent infection, suppress progression to disease or reduce HIV-1 transmission in humans.
Assuntos
Vacinas contra a AIDS/farmacologia , Infecções por HIV/prevenção & controle , Animais , Vetores Genéticos , Anticorpos Anti-HIV/biossíntese , Antígenos HIV/administração & dosagem , Infecções por HIV/imunologia , HIV-1/imunologia , Humanos , Imunidade Celular , Macaca , Vacinas de DNA/farmacologia , Vacinas Sintéticas/farmacologiaRESUMO
OBJECTIVE: While transmission of drug-resistant HIV-1 has been reported, estimates of prevalence of resistance in drug-naïve populations are incomplete. We investigated the prevalence of genotypic mutations and phenotypic antiretroviral resistance in a cohort of HIV-1 infected U.S. military personnel prior to the institution of antiretroviral therapy. DESIGN: Cross-sectional cohort study. METHODS: Plasma was obtained from 114 recently HIV-1 infected subjects enrolled in an epidemiological study. Genotypic resistance was determined by consensus sequencing of a PCR product from the HIV-1 pol gene. Sequences were interpreted by a phenotypic-genotypic correlative database. Resistance phenotypes were determined by a recombinant virus cell culture assay. RESULTS: Genotypic mutations and phenotypic resistance were found at a higher than expected frequency. Resistance to non-nucleoside reverse transcriptase inhibitors was most common, with a prevalence of 15% of 95 subjects by genotype and 26% of 91 subjects by phenotype. Genotypic and phenotypic resistance respectively were found in 4% and 8% of subjects for nucleoside reverse transcriptase inhibitors and in 10% and 1% for protease inhibitors. One subject harbored virus with resistance to all three drug classes. CONCLUSIONS: A substantial frequency of resistance to antiretroviral drugs was identified in a therapy-naïve U.S. cohort. In most cases, the genotypic and phenotypic assays yielded similar results, although the genotypic assay could detect some protease inhibitor resistance-associated mutations in the absence of phenotypic resistance. These data suggest the need for optimization of treatment guidelines based on current estimates of the prevalence of drug resistance in HIV-1 seroconverters.
Assuntos
Fármacos Anti-HIV/farmacologia , Infecções por HIV/virologia , HIV-1/efeitos dos fármacos , Militares , Inibidores da Transcriptase Reversa/farmacologia , Adulto , Estudos de Coortes , Estudos Transversais , Resistência Microbiana a Medicamentos/genética , Resistência a Múltiplos Medicamentos/genética , Feminino , Genes pol , Genótipo , Infecções por HIV/tratamento farmacológico , Infecções por HIV/epidemiologia , HIV-1/classificação , HIV-1/genética , Humanos , Masculino , Testes de Sensibilidade Microbiana/métodos , Pessoa de Meia-Idade , Mutação , Fenótipo , RNA Viral/análise , Recombinação Genética , Estados UnidosRESUMO
Concern that ADE of HIV infection could occur in vivo, as a result of HIV immunization, has arisen for several reasons. Immune-mediated disease enhancement occurs in several human and animal viral diseases, including lentiviral diseases. Tropism for host M/M cells is a common characteristic in these diseases. Sera from naturally infected, and possibly HIV-immunized, individuals have been shown to contain infection enhancing antibodies in vitro. Finally, there is considerable genetic, and potentially antigenic, diversity among HIV-1 isolates. This workshop was convened to evaluate these concerns regarding ADE of HIV infection in human HIV vaccine trials and to propose studies that would address this potential risk. Although there is currently no evidence that immune-mediated enhancement of disease occurs in HIV, there is clearly a need for carefully designed experiments to further evaluate this issue. As there are several notable diseases for which in vitro ADE does not correlate with ADE in vivo, in vitro data are insufficient to deter development of current HIV-1 vaccine candidates. In vivo correlates of protection/enhancement are necessary to evaluate the ADE risk accurately. The development of an HIV animal model that would allow testing of vaccine candidates is of primary importance.
Assuntos
Vacinas contra a AIDS/efeitos adversos , Animais , Ensaios Clínicos como Assunto/métodos , Modelos Animais de Doenças , Variação Genética , HIV/genética , HIV/fisiologia , Anticorpos Anti-HIV/sangue , Infecções por HIV/etiologia , Infecções por HIV/prevenção & controle , Humanos , Técnicas In Vitro , Fatores de Risco , Viroses/etiologia , Replicação Viral/imunologiaRESUMO
To ascertain the antigenic relationship between HIV-1 viruses belonging to various genetically defined subgroups (clades), shared epitopes need to be defined. Human monoclonal antibodies (MAbs) are particularly useful for this purpose because they can detect complex regions of viral proteins that may be missed by sequence analysis and because, by definition, they react with epitopes that stimulate the human immune system. Monoclonal antibodies derived from the cells of HIV-1 clade B-infected subjects have been used extensively for this purpose. Here we describe the first human MAb derived from a clade E-infected individual; the MAb is specific for the V3 loop, recognizing a core epitope represented by the amino acids TRTSVR on the N-terminal side of the crown of the V3 loop. The IgG1(kappa) MAb, designated 1324E, binds to the clade E consensus V3 loop, to rgp120 proteins from clade E and to peripheral blood mononuclear cells infected in vitro with the virus that infected the subject from whose cells the MAb-producing heterohybridoma was derived. Strong cross-reactivity of the MAb to the V3 peptides, rgp120 proteins, and native monomeric gp120s representing clades A and C, as well as to cells infected with a clade C primary isolate, revealed a shared V3 epitope between these clades. When tested for its neutralizing ability, MAb 1324E neutralized a clade E isolate that had been adapted for growth in H9 cells but failed to neutralize five clade E primary isolates.
Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Anti-HIV/imunologia , Proteína gp120 do Envelope de HIV/imunologia , HIV-1/imunologia , Fragmentos de Peptídeos/imunologia , Sequência de Aminoácidos , Afinidade de Anticorpos , Especificidade de Anticorpos , Sequência Consenso , Reações Cruzadas , Mapeamento de Epitopos , Citometria de Fluxo , Proteína gp120 do Envelope de HIV/química , Infecções por HIV/imunologia , Infecções por HIV/virologia , HIV-1/classificação , HIV-1/isolamento & purificação , Humanos , Dados de Sequência Molecular , Testes de Neutralização , Fragmentos de Peptídeos/química , Filogenia , Proteínas Recombinantes/imunologiaRESUMO
Because the immune response to HIV depends on viral gene expression, we examined the HIV-specific immune responses in persons whose viral load after highly active antiretroviral therapy (HAART) was <400 on at least 3 occasions over a 12-month interval. Eleven patients were identified. While there was little change in mean HIV-binding antibody (Ab) titers in this group, two persons mounted increases in HIV envelope-specific binding antibody. Neutralizing antibody (NAb) titers against a panel of HIV-1 primary isolates (BZ167, US1, and CM237) increased post-HAART (80% neutralization titer against US1, p = 0.06; against CM237, p = 0.04). The two persons with large increases in binding antibody also had increases in primary isolate NAb. Roughly half of HAART recipients had significant increases in neutralizing antibody to the primary isolates US1 and CM237. Compared with CD4-matched, non-HAART controls, there were significant increases in NAb against the subtype B primary isolate US1 (p < 0.0009); no increases were seen against more easily neutralized primary isolate BZ167. There were no differences after HAART in antibody-directed cellular cytotoxicity (ADCC). HAART resulted in a partial restoration of lymphoproliferative responses to recall antigens (tetanus and diphtheria). New responses developed to HIV Gag p24. No patient responded to HIV Env gp160 or gp120 either before or after HAART. The data underscore the lack of functional reconstitution of HIV-specific, CD4-mediated responses despite durable suppression of viral replication. In the setting of stable anti-HIV Ab levels, the development of increased NAb in certain individuals suggests that control of the virus by HAART may assist in immune control of HIV.
Assuntos
Fármacos Anti-HIV/uso terapêutico , Terapia Antirretroviral de Alta Atividade , Anticorpos Anti-HIV/biossíntese , Infecções por HIV/imunologia , Imunidade Celular , Contagem de Linfócito CD4 , Anticorpos Anti-HIV/imunologia , Proteína do Núcleo p24 do HIV/imunologia , Infecções por HIV/tratamento farmacológico , Infecções por HIV/virologia , HIV-1/imunologia , HIV-1/isolamento & purificação , Humanos , Testes de Neutralização , RNA Viral/sangue , Carga ViralRESUMO
Classification of human immunodeficiency virus type 1 (HIV-1) by neutralization serotype may be important for the design of active and passive immunization strategies. Neutralizing antibody serotyping is hindered by the lack of standard reagents and assay format, and by the weak activity of many individual sera. To facilitate cross-clade neutralization analysis, we used an infectivity reduction assay (IRA) and selected clade-specific serum (or plasma) pools from subjects infected with clade B and E HIV-1, respectively. Several serum pools were utilized; some were selected for strong neutralizing activity against intraclade viruses and others were derived from conveniently available samples. Against a panel of 51 clade B and E viruses, serum pools displayed strong neutralization of most intraclade viruses and significantly diminished cross-clade neutralization. Results were confirmed against a blinded panel of 20 viruses. The data indicate that the phylogenetic classification of virus subtypes B and E corresponds to two distinct neutralization serotypes. This approach to neutralizing antibody serotyping may be useful in defining the antigenic relationship among viruses from other clades.
Assuntos
Soropositividade para HIV/diagnóstico , Sorotipagem/métodos , Ensaio de Imunoadsorção Enzimática , Proteína gp120 do Envelope de HIV/metabolismo , Proteína gp160 do Envelope de HIV/metabolismo , Humanos , Testes de Neutralização , Fragmentos de Peptídeos/metabolismoRESUMO
Dendritic cells (DCs) infected with recombinant avipox vectors express the introduced genes and activate antigen-specific T cells. DCs exhibit distinct differentiation-dependent immune functions. Moreover, immature DCs are readily infected by canarypox vectors, but undergo tumor necrosis factor (TNF)-alpha-dependent death, while fewer mature DCs get infected and resist dying. A pilot study was performed using the rhesus macaque system to explore whether immature and mature DCs infected with SIV-recombinant canarypox (vCP180) ex vivo could induce primary virus-specific immune responses in vivo. After subcutaneous (sc) reinjection, functional monocyte-derived DCs migrated to lymph nodes (LNs) within 1-2 days and primed T cells in vivo. This was observed by monitoring dye-labeled DCs in the draining LNs and tetanus toxoid (TT)-specific T cell responses after injection of TT-loaded DCs. DCs from simian immunodeficiency virus (SIV)-naïve rhesus macaques were infected with vCP180 (SIVmac142 gag, pol, and env genes), and sc reinjected into donor animals. Low-level SIV-specific T cell proliferation, but little if any interferon (IFN)-gamma production was detected. DCs pulsed with vCP180 in combination with TT and keyhole limpet hemocyanin (KLH) (to activate additional T cells and provide "helper" cytokines) induced SIV-, TT-, and KLH-specific T cell responses, including IFN-gamma responses not seen when vCP180-carrying DCs were used alone. Interleukin (IL)-10 and low-level antibody responses were also observed. This pilot study provides the proof of principle that sc injected ex vivo SIV-recombinant canarypox-infected DCs safely induce low-level SIV-specific immune responses in vivo.
Assuntos
Vírus da Varíola dos Canários/imunologia , Células Dendríticas/virologia , Vírus da Imunodeficiência Símia/imunologia , Animais , Vírus da Varíola dos Canários/genética , Vírus da Varíola dos Canários/fisiologia , Células Dendríticas/imunologia , Vetores Genéticos , Macaca mulatta , Vírus da Imunodeficiência Símia/genética , Vírus da Imunodeficiência Símia/fisiologia , Linfócitos T Citotóxicos/imunologia , Linfócitos T Citotóxicos/fisiologia , Vacinas SintéticasRESUMO
Antibodies elicited by passive or active immunization protect against numerous virus diseases. This in vivo protective immunity is often associated with in vitro detection of neutralizing antibodies (NAb) (e.g., polio, measles, influenza, respiratory syncytial virus, yellow fever, dengue, rabies, varicella, hepatitis A, hepatitis B) (1-7). Since human protection from HIV-1-associated disease has not yet been achieved, the role of NAb in protective immunity is not defined. Nonetheless, based on experience with other viruses, it is reasonable to assume that NAb will play an important role in protection against HIV-1.
RESUMO
The serum titration neutralization assay described in Chapter 33 utilizes a constant amount of infectious virus and indirectly estimates the antibody-mediated reduction in infectious virus by measuring p24 antigen expressed by human peripheral blood mononuclear cells (PBMC). The assay assumes a direct relationship between expressed p24 antigen and the number of target PBMC infected. Therefore, the optimal time of p24 antigen measurement may vary due to different growth kinetics among viruses. An infectivity reduction assay (IRA) directly measures the effect of antibody on virus endpoint infectious titer. The IRA is more labor-intensive and requires more serum than the antibody titration assay, and it is generally done with a single dilution of antibody. However, it has the important advantage of directly measuring the effect of antibody on virus median tissue culture infectious dose (TCID(50)). In addition, the IRA is unaffected by variation in virus growth kinetics and therefore is theoretically more suitable for comparing neutralization among diverse virus isolates. The assay described below calculates virus TCID(50) by serially diluting virus stock in quadruplicate wells with phytohemagglutinin (PHA)-stimulated PBMC. Wells are scored positive or negative for HIV-1 infection by measuring expressed p24 antigen after eight days in culture. Results are expressed as the antibody-mediated reduction in virus TCID(50) (i.e., ratio of virus TCID(50) in control sera to TCID(50) in test sera).
RESUMO
HIV-1 is routinely isolated by cocultivation of patient PBMC with mitogen-stimulated HIV-uninfected donor PBMC (see Chapter 1 ). In this culture system, HIV-1 primarily replicates in CD4(+) T-lymphocytes, and such viruses are termed clinical or primary isolates. As early as 1986, the in vitro replicative capacity and cell tropism of primary HIV-1 isolates were shown to be important in the pathophysiology of HIV-1 infection (1). High replication capacity in PBMC and virus growth and syncytium formation in neoplastic T-cell lines were found to correlate with severity of HIV-1-disease (2-5). Compared to syncytium-inducing (SI) isolates, nonsyncytium-inducing (NSI) strains did not replicate in neoplastic T-cell lines and showed preferential replication in cells of the monocyte-macrophage lineage (6,7). Thus, NSI viruses have often been termed macrophage tropic, whereas SI strains are termed T-cell line tropic.
RESUMO
CD4 was identified in 1984 as the receptor for HIV-1 (1,2). However, it was soon apparent that a second receptor was necessary for HIV-1 infection of CD4(+) cells. This coreceptor was first identified by Berger and colleagues who showed that fusion and entry of T-cell line-adapted strains of HIV-1 into CD4(+) cells were mediated by a member of the seven transmembrane chemokine receptor family (3). This protein was initially termed "fusin" and later found to be the α-chemokine receptor CXCR4. Subsequent reports by several laboratories rapidly identified a ß-chemokine receptor, CCR5, that mediated entry of macrophage tropic HIV-1 isolates into CD4(+) cells (4-8). Since these initial reports, our understanding of HIV-1 cell entry has continued to evolve. It now seems clear that primary HIV-1 strains with a nonsyncytium-inducing pheno-type (in MT2 cells) utilize the CCR5 coreceptor and are designated "R5," whereas syncytium-inducing viruses preferentially utilize CXCR4, but may be dual tropic and are designated either "X4" or X4R5, respectively (9,10). Some viruses also appear to be able to utilize chemokine receptors CCR2B and CCR3 (7-10). In addition, the role of chemokine coreceptors in the pathophysiology of HIV-1 infection and disease progression is just beginning to be understood. Individuals who are homozygous for a 32-bp deletion in the CCR5 gene are only rarely found to be HIV-1-infected, and the heterozygous CCR5/Δccr5 genotype has been associated with a survival advantage against HIV-1 disease progression (11-14).
RESUMO
Avian influenza virus causes outbreaks in domestic and wild birds around the world, and sporadic human infections have been reported. A DNA vaccine encoding hemagglutinin (HA) protein from the A/Indonesia/5/05 (H5N1) strain was initially tested in two randomized phase I clinical studies. Vaccine Research Center study 304 (VRC 304) was a double-blinded study with 45 subjects randomized to placebo, 1 mg of vaccine, or 4 mg of vaccine treatment groups (n = 15/group) by intramuscular (i.m.) Biojector injection. VRC 305 was an open-label study to evaluate route, with 44 subjects randomized to intradermal (i.d.) injections of 0.5 mg by needle/syringe or by Biojector or 1 mg delivered as two 0.5-mg Biojector injections in the same deltoid or as 0.5 mg in each deltoid (n = 11/group). Injections were administered at weeks 0, 4, and 8 in both studies. Antibody responses to H5 were assessed by hemagglutination inhibition (HAI) assay, enzyme-linked immunosorbent assay (ELISA), and neutralization assay, and the H5 T cell responses were assessed by enzyme-linked immunospot and intracellular cytokine staining assays. There were no vaccine-related serious adverse events, and the vaccine was well tolerated in all groups. At 1 mg, i.d. vaccination compared to i.m. vaccination induced a greater frequency and magnitude of response by ELISA, but there were no significant differences in the frequency or magnitude of response between the i.d. and i.m. routes in the HAI or neutralization assays. T cell responses were more common in subjects who received the 1- or 4-mg dose i.m. These studies demonstrated that the DNA vaccine encoding H5 is safe and immunogenic and served to define the proper dose and route for further studies. The i.d. injection route did not offer a significant advantage over the i.m. route, and no difference was detected by delivery to one site versus splitting the dose between two sites for i.d. vaccine administration. The 4-mg dose (i.m) was further investigated in prime-boost regimens.
Assuntos
Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Vacinas contra Influenza/administração & dosagem , Vacinas contra Influenza/imunologia , Influenza Humana/prevenção & controle , Vacinação/métodos , Vacinas de DNA/administração & dosagem , Vacinas de DNA/imunologia , Adulto , Anticorpos Antivirais/sangue , Citocinas/metabolismo , Método Duplo-Cego , Ensaio de Imunoadsorção Enzimática , ELISPOT , Feminino , Testes de Inibição da Hemaglutinação , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Humanos , Vacinas contra Influenza/genética , Injeções Intradérmicas , Injeções Intramusculares , Masculino , Pessoa de Meia-Idade , Testes de Neutralização , Placebos/administração & dosagem , Linfócitos T/imunologia , Vacinas de DNA/genética , Adulto JovemRESUMO
Ebola virus causes irregular outbreaks of severe hemorrhagic fever in equatorial Africa. Case mortality remains high; there is no effective treatment and outbreaks are sporadic and unpredictable. Studies of Ebola virus vaccine platforms in non-human primates have established that the induction of protective immunity is possible and safety and human immunogenicity has been demonstrated in a previous Phase I clinical trial of a 1st generation Ebola DNA vaccine. We now report the safety and immunogenicity of a recombinant adenovirus serotype 5 (rAd5) vaccine encoding the envelope glycoprotein (GP) from the Zaire and Sudan Ebola virus species, in a randomized, placebo-controlled, double-blinded, dose escalation, Phase I human study. Thirty-one healthy adults received vaccine at 2×10(9) (n=12), or 2×10(10) (n=11) viral particles or placebo (n=8) as an intramuscular injection. Antibody responses were assessed by ELISA and neutralizing assays; and T cell responses were assessed by ELISpot and intracellular cytokine staining assays. This recombinant Ebola virus vaccine was safe and subjects developed antigen specific humoral and cellular immune responses.