RESUMO
As part of the continuing effort to develop an effective HIV vaccine, we generated a poxviral vaccine vector (previously described) designed to improve on the results of the RV144 phase III clinical trial. The construct, NYVAC-KC, is a replication-competent, attenuated recombinant of the vaccinia virus strain NYVAC. NYVAC is a vector that has been used in many previous clinical studies but is replication deficient. Here, we report a side-by-side comparison of replication-restricted NYVAC and replication-competent NYVAC-KC in a nonhuman primate study, which utilized a prime-boost regimen similar to that of RV144. NYVAC-C and NYVAC-C-KC express the HIV-1 antigens gp140, and Gag/Gag-Pol-Nef-derived virus-like particles (VLPs) from clade C and were used as the prime, with recombinant virus plus envelope protein used as the boost. In nearly every T and B cell immune assay against HIV-1, including neutralization and antibody binding, NYVAC-C-KC induced a greater immune response than NYVAC-C, indicating that replication competence in a poxvirus may improve upon the modestly successful regimen used in the RV144 clinical trial.IMPORTANCE Though the RV144 phase III clinical trial showed promise that an effective vaccine against HIV-1 is possible, a successful vaccine will require improvement over the vaccine candidate (ALVAC) used in the RV144 study. With that goal in mind, we have tested in nonhuman primates an attenuated but replication-competent vector, NYVAC-KC, in direct comparison to its parental vector, NYVAC, which is replication restricted in human cells, similar to the ALVAC vector used in RV144. We have utilized a prime-boost regimen for administration of the vaccine candidate that is similar to the one used in the RV144 study. The results of this study indicate that a replication-competent poxvirus vector may improve upon the effectiveness of the RV144 clinical trial vaccine candidate.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Antígenos HIV/imunologia , Infecções por HIV/imunologia , HIV-1/imunologia , Vacinas Virais/administração & dosagem , Replicação Viral , Produtos do Gene env do Vírus da Imunodeficiência Humana/imunologia , Animais , Anticorpos Neutralizantes/sangue , Anticorpos Anti-HIV/sangue , Infecções por HIV/prevenção & controle , Infecções por HIV/virologia , Humanos , Macaca mulatta , Masculino , Vacinação , Vaccinia virus/imunologia , Vacinas Virais/imunologiaRESUMO
The use of heterologous immunization regimens and improved vector systems has led to increases in immunogenicity of HIV-1 vaccine candidates in nonhuman primates. In order to resolve interrelations between different delivery modalities, three different poxvirus boost regimens were compared. Three groups of rhesus macaques were each primed with the same DNA vaccine encoding Gag, Pol, Nef, and gp140. The groups were then boosted with either the vaccinia virus strain NYVAC or a variant with improved replication competence in human cells, termed NYVAC-KC. The latter was administered either by scarification or intramuscularly. Finally, macaques were boosted with adjuvanted gp120 protein to enhance humoral responses. The regimen elicited very potent CD4+ and CD8+ T cell responses in a well-balanced manner, peaking 2 weeks after the boost. T cells were broadly reactive and polyfunctional. All animals exhibited antigen-specific humoral responses already after the poxvirus boost, which further increased following protein administration. Polyclonal reactivity of IgG antibodies was highest against HIV-1 clade C Env proteins, with considerable cross-reactivity to other clades. Substantial effector functional activities (antibody-dependent cell-mediated cytotoxicity and antibody-dependent cell-mediated virus inhibition) were observed in serum obtained after the last protein boost. Notably, major differences between the groups were absent, indicating that the potent priming induced by the DNA vaccine initially framed the immune responses in such a way that the subsequent boosts with NYVAC and protein led only to an increase in the response magnitudes without skewing the quality. This study highlights the importance of selecting the best combination of vector systems in heterologous prime-boost vaccination regimens.IMPORTANCE The evaluation of HIV vaccine efficacy trials indicates that protection would most likely correlate with a polyfunctional immune response involving several effector functions from all arms of the immune system. Heterologous prime-boost regimens have been shown to elicit vigorous T cell and antibody responses in nonhuman primates that, however, qualitatively and quantitatively differ depending on the respective vector systems used. The present study evaluated a DNA prime and poxvirus and protein boost regimen and compared how two poxvirus vectors with various degrees of replication capacity and two different delivery modalities-conventional intramuscular delivery and percutaneous delivery by scarification-impact several immune effectors. It was found that despite the different poxvirus boosts, the overall immune responses in the three groups were similar, suggesting the potent DNA priming as the major determining factor of immune responses. These findings emphasize the importance of selecting optimal priming agents in heterologous prime-boost vaccination settings.
Assuntos
Antígenos HIV/imunologia , Infecções por HIV/imunologia , HIV-1/imunologia , Linfócitos T/imunologia , Vacinas de DNA/administração & dosagem , Vacinas Virais/imunologia , Replicação Viral , Animais , Anticorpos Neutralizantes/sangue , Anticorpos Anti-HIV/sangue , Infecções por HIV/prevenção & controle , Infecções por HIV/virologia , Humanos , Macaca mulatta , Masculino , Poxviridae , Vacinação , Vacinas de DNA/imunologia , Vaccinia virus/imunologiaRESUMO
The goal of the STAR Sexually Transmitted Infection Clinical Trial Group (STI CTG) Programmatic meeting on Sexually Transmitted Infections (STIs) in Pregnancy and Reproductive Health in April 2018 was to review the latest research and develop recommendations to improve prevention and management of STIs during pregnancy. Experts from academia, government, nonprofit, and industry discussed the burden of STIs during pregnancy; the impact of STIs on adverse pregnancy and birth outcomes; interventions that work to reduce STIs in pregnancy, and the evidence, policy, and technology needed to improve STI care during pregnancy. Key points of the meeting are as follows: (i) alternative treatments and therapies for use during pregnancy are needed; (ii) further research into the relationship between the vaginal microbiome and STIs during pregnancy should be supported; (iii) more research to determine whether STI tests function equally well in pregnant as nonpregnant women is needed; (iv) development of new lower cost, rapid point-of-care testing assays could allow for expanded STI screening globally; (v) policies should be implemented that create standard screening and treatment practices globally; (vi) federal funding should be increased for STI testing and treatment initiatives supported by the Centers for Disease Control and Prevention (CDC), the Centers of Excellence in STI Treatment, public STD clinics, and the President's Emergency Plan for AIDS Relief (PEPFAR).
Assuntos
Ensaios Clínicos como Assunto , Saúde Reprodutiva , Infecções Sexualmente Transmissíveis/prevenção & controle , Congressos como Assunto , Feminino , Infecções por HIV/prevenção & controle , Humanos , Testes Imediatos , Gravidez , Complicações Infecciosas na Gravidez/prevenção & controleRESUMO
The goal of the Sexually Transmitted Infection Clinical Trial Group's Antimicrobial Resistance (AMR) in Neisseria gonorrhoeae (NG) meeting was to assemble experts from academia, government, nonprofit and industry to discuss the current state of research, gaps and challenges in research and technology and priorities and new directions to address the continued emergence of multidrug-resistant NG infections. Topics discussed at the meeting, which will be the focus of this article, include AMR NG global surveillance initiatives, the use of whole genome sequencing and bioinformatics to understand mutations associated with AMR, mechanisms of AMR, and novel antibiotics, vaccines and other methods to treat AMR NG. Key points highlighted during the meeting include: (i) US and International surveillance programs to understand AMR in NG; (ii) the US National Strategy for combating antimicrobial-resistant bacteria; (iii) surveillance needs, challenges, and novel technologies; (iv) plasmid-mediated and chromosomally mediated mechanisms of AMR in NG; (v) novel therapeutic (eg, sialic acid analogs, factor H [FH]/Fc fusion molecule, monoclonal antibodies, topoisomerase inhibitors, fluoroketolides, LpxC inhibitors) and preventative (eg, peptide mimic) strategies to combat infection. The way forward will require renewed political will, new funding initiatives, and collaborations across academic and commercial research and public health programs.
Assuntos
Farmacorresistência Bacteriana Múltipla , Gonorreia/tratamento farmacológico , Processos Grupais , Neisseria gonorrhoeae/efeitos dos fármacos , Infecções Sexualmente Transmissíveis/tratamento farmacológico , Antibacterianos/efeitos adversos , Antibacterianos/uso terapêutico , Gestão de Antimicrobianos/métodos , Vacinas Bacterianas/uso terapêutico , Barbitúricos/uso terapêutico , Monitoramento Epidemiológico , Humanos , Isoxazóis , Macrolídeos/uso terapêutico , Testes de Sensibilidade Microbiana , Morfolinas , Mutação , Neisseria gonorrhoeae/genética , Neisseria gonorrhoeae/imunologia , Neisseria gonorrhoeae/isolamento & purificação , Oxazolidinonas , Saúde Pública/métodos , Compostos de Espiro/uso terapêutico , Inibidores da Topoisomerase/uso terapêutico , Triazóis/uso terapêutico , Organização Mundial da SaúdeRESUMO
The nonreplicating attenuated poxvirus vector NYVAC expressing clade C(CN54) HIV-1 Env(gp120) and Gag-Pol-Nef antigens (NYVAC-C) showed limited immunogenicity in phase I clinical trials. To enhance the capacity of the NYVAC vector to trigger broad humoral responses and a more balanced activation of CD4+ and CD8+ T cells, here we compared the HIV-1-specific immunogenicity elicited in nonhuman primates immunized with two replicating NYVAC vectors that have been modified by the insertion of the K1L and C7L vaccinia virus host range genes and express the clade C(ZM96) trimeric HIV-1 gp140 protein or a Gag(ZM96)-Pol-Nef(CN54) polyprotein as Gag-derived virus-like particles (termed NYVAC-C-KC). Additionally, one NYVAC-C-KC vector was generated by deleting the viral gene B19R, an inhibitor of the type I interferon response (NYVAC-C-KC-ΔB19R). An immunization protocol mimicking that of the RV144 phase III clinical trial was used. Two groups of macaques received two doses of the corresponding NYVAC-C-KC vectors (weeks 0 and 4) and booster doses with NYVAC-C-KC vectors plus the clade C HIV-1 gp120 protein (weeks 12 and 24). The two replicating NYVAC-C-KC vectors induced enhanced and similar HIV-1-specific CD4+ and CD8+ T cell responses, similar levels of binding IgG antibodies, low levels of IgA antibodies, and high levels of antibody-dependent cellular cytotoxicity responses and HIV-1-neutralizing antibodies. Small differences within the NYVAC-C-KC-ΔB19R group were seen in the magnitude of CD4+ and CD8+ T cells, the induction of some cytokines, and the neutralization of some HIV-1 isolates. Thus, replication-competent NYVAC-C-KC vectors acquired relevant immunological properties as vaccine candidates against HIV/AIDS, and the viral B19 molecule exerts some control of immune functions.IMPORTANCE It is of special importance to find a safe and effective HIV/AIDS vaccine that can induce strong and broad T cell and humoral immune responses correlating with HIV-1 protection. Here we developed novel replicating poxvirus NYVAC-based HIV/AIDS vaccine candidates expressing clade C HIV-1 antigens, with one of them lacking the vaccinia virus B19 protein, an inhibitor of the type I interferon response. Immunization of nonhuman primates with these novel NYVAC-C-KC vectors and the protein component gp120 elicited high levels of T cell and humoral immune responses, with the vector containing a deletion in B19R inducing a trend toward a higher magnitude of CD4+ and CD8+ T cell responses and neutralization of some HIV-1 strains. These poxvirus vectors could be considered HIV/AIDS vaccine candidates based on their activation of potential immune correlates of protection.
Assuntos
Vacinas contra a AIDS/imunologia , Anticorpos Neutralizantes/sangue , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Anticorpos Anti-HIV/sangue , Proteína gp120 do Envelope de HIV/imunologia , Vacinas de Partículas Semelhantes a Vírus/imunologia , Produtos do Gene env do Vírus da Imunodeficiência Humana/metabolismo , Vacinas contra a AIDS/genética , Animais , Anticorpos Neutralizantes/imunologia , Citotoxicidade Celular Dependente de Anticorpos/imunologia , Anticorpos Anti-HIV/imunologia , Antígenos HIV/imunologia , Infecções por HIV/prevenção & controle , Interferon Tipo I/genética , Macaca mulatta , Masculino , Receptores de Interferon/genética , Receptores de Interferon/imunologia , Vacinação , Vaccinia virus/genética , Produtos do Gene env do Vírus da Imunodeficiência Humana/genéticaRESUMO
We compared the HIV-1-specific immune responses generated by targeting HIV-1 envelope protein (Env gp140) to either CD40 or LOX-1, two endocytic receptors on dendritic cells (DCs), in rhesus macaques primed with a poxvirus vector (NYVAC-KC) expressing Env gp140. The DC-targeting vaccines, humanized recombinant monoclonal antibodies fused to Env gp140, were administered as a boost with poly-ICLC adjuvant either alone or coadministered with the NYVAC-KC vector. All the DC-targeting vaccine administrations with poly-ICLC increased the low-level serum anti-Env IgG responses elicited by NYVAC-KC priming significantly more (up to a P value of 0.01) than in a group without poly-ICLC. The responses were robust and cross-reactive and contained antibodies specific to multiple epitopes within gp140, including the C1, C2, V1, V2, and V3, C4, C5, and gp41 immunodominant regions. The DC-targeting vaccines also elicited modest serum Env-specific IgA responses. All groups gave serum neutralization activity limited to tier 1 viruses and antibody-dependent cytotoxicity responses (ADCC) after DC-targeting boosts. Furthermore, CD4+ and CD8+ T cell responses specific to multiple Env epitopes were strongly boosted by the DC-targeting vaccines plus poly-ICLC. Together, these results indicate that prime-boost immunization via NYVAC-KC and either anti-CD40.Env gp140/poly-ICLC or anti-LOX-1.Env gp140/poly-ICLC induced balanced antibody and T cell responses against HIV-1 Env. Coadministration of NYVAC-KC with the DC-targeting vaccines increased T cell responses but had minimal effects on antibody responses except for suppressing serum IgA responses. Overall, targeting Env to CD40 gave more robust T cell and serum antibody responses with broader epitope representation and greater durability than with LOX-1.IMPORTANCE An effective vaccine to prevent HIV-1 infection does not yet exist. An approach to elicit strong protective antibody development is to direct virus protein antigens specifically to dendritic cells, which are now known to be the key cell type for controlling immunity. In this study, we have tested in nonhuman primates two prototype vaccines engineered to direct the HIV-1 coat protein Env to dendritic cells. These vaccines bind to either CD40 or LOX-1, two dendritic cell surface receptors with different functions and tissue distributions. We tested the vaccines described above in combination with attenuated virus vectors that express Env. Both vaccines, but especially that delivered via CD40, raised robust immunity against HIV-1 as measured by monitoring potentially protective antibody and T cell responses in the blood. The safety and efficacy of the CD40-targeted vaccine justify further development for future human clinical trials.
Assuntos
Vacinas contra a AIDS/imunologia , Linfócitos T CD4-Positivos/imunologia , Antígenos CD40/imunologia , Linfócitos T CD8-Positivos/imunologia , Anticorpos Anti-HIV/imunologia , HIV-1/imunologia , Receptores Depuradores Classe E/imunologia , Produtos do Gene env do Vírus da Imunodeficiência Humana/imunologia , Vacinas contra a AIDS/genética , Animais , Anticorpos Neutralizantes/imunologia , Células CHO , Carboximetilcelulose Sódica/análogos & derivados , Cricetulus , Células Dendríticas/imunologia , Imunoglobulina A/imunologia , Imunoglobulina G/imunologia , Macaca mulatta , Masculino , Poli I-C/imunologia , Polilisina/análogos & derivados , Polilisina/imunologia , VacinaçãoRESUMO
UNLABELLED: In a follow-up to the modest efficacy observed in the RV144 trial, researchers in the HIV vaccine field seek to substantiate and extend the results by evaluating other poxvirus vectors and combinations with DNA and protein vaccines. Earlier clinical trials (EuroVacc trials 01 to 03) evaluated the immunogenicity of HIV-1 clade C GagPolNef and gp120 antigens delivered via the poxviral vector NYVAC. These showed that a vaccination regimen including DNA-C priming prior to a NYVAC-C boost considerably enhanced vaccine-elicited immune responses compared to those with NYVAC-C alone. Moreover, responses were improved by using three as opposed to two DNA-C primes. In the present study, we assessed in nonhuman primates whether such vaccination regimens can be streamlined further by using fewer and accelerated immunizations and employing a novel generation of improved DNA-C and NYVAC-C vaccine candidates designed for higher expression levels and more balanced immune responses. Three different DNA-C prime/NYVAC-C+ protein boost vaccination regimens were tested in rhesus macaques. All regimens elicited vigorous and well-balanced CD8(+)and CD4(+)T cell responses that were broad and polyfunctional. Very high IgG binding titers, substantial antibody-dependent cellular cytotoxicity (ADCC), and modest antibody-dependent cell-mediated virus inhibition (ADCVI), but very low neutralization activity, were measured after the final immunizations. Overall, immune responses elicited in all three groups were very similar and of greater magnitude, breadth, and quality than those of earlier EuroVacc vaccines. In conclusion, these findings indicate that vaccination schemes can be simplified by using improved antigens and regimens. This may offer a more practical and affordable means to elicit potentially protective immune responses upon vaccination, especially in resource-constrained settings. IMPORTANCE: Within the EuroVacc clinical trials, we previously assessed the immunogenicity of HIV clade C antigens delivered in a DNA prime/NYVAC boost regimen. The trials showed that the DNA prime crucially improved the responses, and three DNA primes with a NYVAC boost appeared to be optimal. Nevertheless, T cell responses were primarily directed toward Env, and humoral responses were modest. The aim of this study was to assess improved antigens for the capacity to elicit more potent and balanced responses in rhesus macaques, even with various simpler immunization regimens. Our results showed that the novel antigens in fact elicited larger numbers of T cells with a polyfunctional profile and a good Env-GagPolNef balance, as well as high-titer and Fc-functional antibody responses. Finally, comparison of the different schedules indicates that a simpler regimen of only two DNA primes and one NYVAC boost in combination with protein may be very efficient, thus showing that the novel antigens allow for easier immunization protocols.
Assuntos
Vacinas contra a AIDS/imunologia , Primers do DNA , HIV-1/imunologia , Vacinas de DNA/imunologia , Vacinas contra a AIDS/genética , Animais , Anticorpos Neutralizantes/imunologia , Citotoxicidade Celular Dependente de Anticorpos , Anticorpos Anti-HIV/imunologia , Antígenos HIV/imunologia , Interferon gama/biossíntese , Masculino , Linfócitos T/imunologia , Vacinação/métodos , Vacinas de DNA/genética , Produtos do Gene gag do Vírus da Imunodeficiência Humana/genética , Produtos do Gene gag do Vírus da Imunodeficiência Humana/imunologiaRESUMO
The goal of the point-of-care (POC) sexually transmitted infection (STI) Diagnostics meeting was to review the state-of-the-art research and develop recommendations for the use of POC STI diagnostics. Experts from academia, government, nonprofit, and industry discussed POC diagnostics for STIs such as Chlamydia trachomatis, human papillomavirus, Neisseria gonorrhoeae, Trichomonas vaginalis, and Treponema pallidum. Key objectives included a review of current and emerging technologies, clinical and public health benefits, POC STI diagnostics in developing countries, regulatory considerations, and future areas of development. Key points of the meeting are as follows: (i) although some rapid point-of-care tests are affordable, sensitive, specific, easy to perform, and deliverable to those who need them for select sexually transmitted infections, implementation barriers exist at the device, patient, provider, and health system levels; (ii) further investment in research and development of point-of-care tests for sexually transmitted infections is needed, and new technologies can be used to improve diagnostic testing, test uptake, and treatment; (iii) efficient deployment of self-testing in supervised (ie, pharmacies, clinics, and so on) and/or unsupervised (ie, home, offices, and so on) settings could facilitate more screening and diagnosis that will reduce the burden of sexually transmitted infections; (iv) development of novel diagnostic technologies has outpaced the generation of guidance tools and documents issued by regulatory agencies; and (v) questions regarding quality management are emerging including the mechanism by which poor-performing diagnostics are removed from the market and quality assurance of self-testing is ensured.
Assuntos
Testes Imediatos/tendências , Infecções Sexualmente Transmissíveis/diagnóstico , Congressos como Assunto , Humanos , Saúde Pública/métodosRESUMO
UNLABELLED: We compared the HIV-1-specific cellular and humoral immune responses elicited in rhesus macaques immunized with two poxvirus vectors (NYVAC and ALVAC) expressing the same HIV-1 antigens from clade C, Env gp140 as a trimeric cell-released protein and a Gag-Pol-Nef polyprotein as Gag-induced virus-like particles (VLPs) (referred to as NYVAC-C and ALVAC-C). The immunization protocol consisted of two doses of the corresponding poxvirus vector plus two doses of a combination of the poxvirus vector and a purified HIV-1 gp120 protein from clade C. This immunogenicity profile was also compared to that elicited by vaccine regimens consisting of two doses of the ALVAC vector expressing HIV-1 antigens from clades B/E (ALVAC-vCP1521) plus two doses of a combination of ALVAC-vCP1521 and HIV-1 gp120 protein from clades B/E (similar to the RV144 trial regimen) or clade C. The results showed that immunization of macaques with NYVAC-C stimulated at different times more potent HIV-1-specific CD4(+) T-cell responses and induced a trend toward higher-magnitude HIV-1-specific CD8(+) T-cell immune responses than did ALVAC-C. Furthermore, NYVAC-C induced a trend toward higher levels of binding IgG antibodies against clade C HIV-1 gp140, gp120, or murine leukemia virus (MuLV) gp70-scaffolded V1/V2 and toward best cross-clade-binding IgG responses against HIV-1 gp140 from clades A, B, and group M consensus, than did ALVAC-C. Of the linear binding IgG responses, most were directed against the V3 loop in all immunization groups. Additionally, NYVAC-C and ALVAC-C also induced similar levels of HIV-1-neutralizing antibodies and antibody-dependent cellular cytotoxicity (ADCC) responses. Interestingly, binding IgA antibody levels against HIV-1 gp120 or MuLV gp70-scaffolded V1/V2 were absent or very low in all immunization groups. Overall, these results provide a comprehensive survey of the immunogenicity of NYVAC versus ALVAC expressing HIV-1 antigens in nonhuman primates and indicate that NYVAC may represent an alternative candidate to ALVAC in the development of a future HIV-1 vaccine. IMPORTANCE: The finding of a safe and effective HIV/AIDS vaccine immunogen is one of the main research priorities. Here, we generated two poxvirus-based HIV vaccine candidates (NYVAC and ALVAC vectors) expressing the same clade C HIV-1 antigens in separate vectors, and we analyzed in nonhuman primates their immunogenicity profiles. The results showed that immunization with NYVAC-C induced a trend toward higher HIV-1-specific cellular and humoral immune responses than did ALVAC-C, indicating that this new NYVAC vector could be a novel optimized HIV/AIDS vaccine candidate for human clinical trials.
Assuntos
Vacinas contra a AIDS/imunologia , Produtos do Gene env/metabolismo , Vetores Genéticos/imunologia , Infecções por HIV/prevenção & controle , Vacinas Sintéticas/imunologia , Vacinas Virais/imunologia , Animais , Anticorpos Neutralizantes , Embrião de Galinha , Anticorpos Anti-HIV , Antígenos HIV/metabolismo , Macaca mulatta , Poxviridae/genética , Regiões Promotoras Genéticas/genética , Ensaio de Placa ViralRESUMO
To control HIV infection there is a need for vaccines to induce broad, potent and long-term B and T cell immune responses. With the objective to accelerate and maintain the induction of substantial levels of HIV-1 Env-specific antibodies and, at the same time, to enhance balanced CD4 and CD8 T cell responses, we evaluated the effect of concurrent administration of MF59-adjuvanted Env protein together with DNA or NYVAC vectors at priming to establish if early administration of Env leads to early induction of antibody responses. The primary goal was to assess the immunogenicity endpoint at week 26. Secondary endpoints were (i) to determine the quality of responses with regard to RV144 correlates of protection and (ii) to explore a potential impact of two late boosts. In this study, five different prime/boost vaccination regimens were tested in rhesus macaques. Animals received priming immunizations with either NYVAC or DNA alone or in combination with Env protein, followed by NYVAC + protein or DNA + protein boosts. All regimens induced broad, polyfunctional and well-balanced CD4 and CD8 T cell responses, with DNA-primed regimens eliciting higher response rates and magnitudes than NYVAC-primed regimens. Very high plasma binding IgG titers including V1/V2 specific antibodies, modest antibody-dependent cellular cytotoxicity (ADCC) and moderate neutralization activity were observed. Of note, early administration of the MF59-adjuvanted Env protein in parallel with DNA priming leads to more rapid elicitation of humoral responses, without negatively affecting the cellular responses, while responses were rapidly boosted after repeated immunizations, indicating the induction of a robust memory response. In conclusion, our findings support the use of the Env protein component during priming in the context of an heterologous immunization regimen with a DNA and/or NYVAC vector as an optimized immunization protocol against HIV infection.
Assuntos
Vacinas contra a AIDS , Infecções por HIV , Soropositividade para HIV , HIV-1 , Animais , Anticorpos Neutralizantes , DNA , Produtos do Gene env , Anticorpos Anti-HIV , Infecções por HIV/prevenção & controle , Macaca mulattaRESUMO
As sexual transmission of human immunodeficiency virus-1 (HIV-1) occurs via the mucosa, an ideal HIV-1 vaccine should induce both mucosal and systemic immunity. We therefore sought to evaluate the induction of mucosal responses using a DNA env prime-gp120 protein boost approach in which sequential nasal and parenteral protein administration was performed with two novel carbohydrate-based adjuvants. These adjuvants, Advax-M and Advax-P, were specifically designed for mucosal and systemic immune enhancement, respectively. Murine intranasal immunization with gp120/Advax-M adjuvant elicited gp120-specific IgA in serum and mucosal secretions that was markedly enhanced by DNA priming. Boosting of DNA-primed mice with gp120/Advax-M and gp120/Advax-P by sequential intranasal and intramuscular immunization, or vice versa, elicited persistent mucosal gp120-specific IgA, systemic IgG and memory T- and B-cell responses. Induction of homologous, but not heterologous, neutralizing activity was noted in the sera of all immunized groups. While confirmation of efficacy is required in challenge studies using non-human primates, these results suggest that the combination of DNA priming with sequential nasal and parenteral protein boosting, with appropriate mucosal and systemic adjuvants, could generate strong mucosal and systemic immunity and may block HIV-1 mucosal transmission and infection.
Assuntos
Vacinas contra a AIDS/imunologia , Anticorpos Anti-HIV/sangue , Imunidade nas Mucosas , Imunização Secundária/métodos , Linfócitos T/imunologia , Vacinação/métodos , Adjuvantes Imunológicos/administração & dosagem , Administração Intranasal , Animais , Feminino , Anticorpos Anti-HIV/análise , Proteína gp120 do Envelope de HIV/imunologia , HIV-1/imunologia , Imunoglobulina A/análise , Imunoglobulina A/sangue , Imunoglobulina G/sangue , Injeções Intramusculares , Camundongos , Camundongos Endogâmicos BALB C , Testes de Neutralização , Vacinas de DNA/imunologia , Vacinas de Subunidades Antigênicas/imunologiaRESUMO
Intramuscular needle injection of HIV-1 DNA vaccines typically elicits weak immune responses in immunized individuals. To improve such responses, the immunogenicity of a vaccine consisting of electroporated DNA followed by intramuscular protein boost was evaluated in rabbits and macaques. In macaques, electroporation of low dose DNA encoding HIV-1 env followed by gp120 protein elicited Th1 cytokines and functional CTL that persisted for over 1 year. In both macaques and rabbits, robust anti-envelope antibodies, elicited by electroporated DNA, were augmented by gp120 protein and such responses neutralized sensitive SHIV isolates. These findings highlight efficient priming of immune responses by electroporated DNA that in conjunction with protein boost may give rise to long-term immunity in immunized hosts.
Assuntos
Vacinas contra a AIDS/administração & dosagem , Vacinas contra a AIDS/imunologia , Eletroporação/métodos , HIV-1/imunologia , Linfócitos T/imunologia , Vacinas de DNA/administração & dosagem , Vacinas de DNA/imunologia , Animais , Anticorpos/imunologia , HIV-1/genética , Macaca fascicularis , Coelhos , Transfecção/métodosRESUMO
Selection of potent yet low reactogenic adjuvants for protein immunization is important for HIV-1 vaccine development. Immunogenicity of electroporated DNA (HIV env) and recombinant gp120, administered with either QS-21 or the orally administered immunomodulator, Talabostat, was evaluated in BALB/c mice. Electroporation of low dose DNA elicited Th1 cytokines and anti-envelope antibodies. Immunization with gp120 protein alone with or without Talabostat elicited lower Th1 and Th2 cytokine levels but comparable anti-gp120 antibodies to QS-21-formulated protein. Boosting of DNA-primed mice with gp120/Talabostat induced similar anti-gp120 antibody titers and slightly higher levels of Th1 and Th2 cytokines relative to QS-21-formulated protein. Induction of CD8(+) and CD4(+) T cells and functional CTL activity was noted. These results highlight the potential use of orally administered Talabostat for efficient protein boosting of antibody and T-cell responses primed by DNA.
Assuntos
Vacinas contra a AIDS/imunologia , Ácidos Borônicos/administração & dosagem , Dipeptídeos/administração & dosagem , Proteína gp120 do Envelope de HIV/imunologia , HIV-1/imunologia , Vacinas de DNA/imunologia , Animais , Formação de Anticorpos , Citocinas/metabolismo , DNA Viral/genética , Eletroporação , Feminino , Proteína gp120 do Envelope de HIV/administração & dosagem , Proteína gp120 do Envelope de HIV/genética , Camundongos , Camundongos Endogâmicos BALB C , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Células Th1/efeitos dos fármacos , Células Th1/imunologia , Células Th2/efeitos dos fármacos , Células Th2/imunologiaRESUMO
The enormous genetic variability reported in HIV-1 has posed problems in the treatment of infected individuals. This is evident in the form of HIV-1 resistant to antiviral agents, neutralizing antibodies and cytotoxic T lymphocytes (CTLs) involving multiple viral gene products. Based on this, it has been suggested that a comprehensive analysis of the polymorphisms in HIV proteins is of value for understanding the virus transmission and pathogenesis as well as for the efforts towards developing anti-viral therapeutics and vaccines. This study, for the first time, describes an in-depth analysis of genetic variation in Vpr using information from global HIV-1 isolates involving a total of 976 Vpr sequences. The polymorphisms at the individual amino acid level were analyzed. The residues 9, 33, 39, and 47 showed a single variant amino acid compared to other residues. There are several amino acids which are highly polymorphic. The residues that show ten or more variant amino acids are 15, 16, 28, 36, 37, 48, 55, 58, 59, 77, 84, 86, 89, and 93. Further, the variant amino acids noted at residues 60, 61, 34, 71 and 72 are identical. Interestingly, the frequency of the variant amino acids was found to be low for most residues. Vpr is known to contain multiple CTL epitopes like protease, reverse transcriptase, Env, and Gag proteins of HIV-1. Based on this, we have also extended our analysis of the amino acid polymorphisms to the experimentally defined and predicted CTL epitopes. The results suggest that amino acid polymorphisms may contribute to the immune escape of the virus. The available data on naturally occurring polymorphisms will be useful to assess their potential effect on the structural and functional constraints of Vpr and also on the fitness of HIV-1 for replication.
Assuntos
Epitopos de Linfócito T/imunologia , Produtos do Gene vpr/química , Genes vpr , Infecções por HIV/imunologia , HIV-1/genética , Polimorfismo Genético , Linfócitos T Citotóxicos/imunologia , Sequência de Aminoácidos , Produtos do Gene vpr/genética , Produtos do Gene vpr/imunologia , Infecções por HIV/virologia , HIV-1/imunologia , Humanos , Dados de Sequência Molecular , Alinhamento de SequênciaRESUMO
HIV-1 infection can be controlled by anti-retroviral drug therapy, but this is a lifetime treatment and the virus remains latent and rapidly rebounds if therapy is stopped. HIV-1-infected individuals under this drug regimen have increased rates of cancers, cardiovascular diseases, and autoimmunity due to compromised immunity. A therapeutic vaccine boosting cellular immunity against HIV-1 is therefore desirable and, possibly combined with other immune modulating agents, could obviate the need for long-term drug therapies. An approach to elicit strong T cell-based immunity is to direct virus protein antigens specifically to dendritic cells (DCs), which are the key cell type for controlling immune responses. For eliciting therapeutic cellular immunity in HIV-1-infected individuals, we developed vaccines comprised of five T cell epitope-rich regions of HIV-1 Gag, Nef, and Pol (HIV5pep) fused to monoclonal antibodies that bind either, the antigen presenting cell activating receptor CD40, or the endocytic dendritic cell immunoreceptor DCIR. The study aimed to demonstrate vaccine safety, establish efficacy for broad T cell responses in both primed and naïve settings, and identify one candidate vaccine for human therapeutic development. The vaccines were administered to Rhesus macaques by intradermal injection with poly-ICLC adjuvant. The animals were either i) naïve or, ii) previously primed with modified vaccinia Ankara vector (MVA) encoding HIV-1 Gag, Pol, and Nef (MVA GagPolNef). In the MVA-primed groups, both DC-targeting vaccinations boosted HIV5pep-specific blood CD4+ T cells producing multiple cytokines, but did not affect the MVA-elicited CD8+ T cell responses. In the naive groups, both DC-targeting vaccines elicited antigen-specific polyfunctional CD4+ and CD8+ T cell responses to multiple epitopes and these responses were unchanged by a subsequent MVA GagPolNef boost. In both settings, the T cell responses elicited via the CD40-targeting vaccine were more robust and were detectable in all the animals, favoring further development of the CD40-targeting vaccine for therapeutic vaccination of HIV-1-infected individuals.
Assuntos
Vacinas contra a AIDS/imunologia , Antígenos CD40/imunologia , Células Dendríticas/imunologia , Epitopos de Linfócito T/imunologia , Anticorpos Anti-HIV/imunologia , HIV-1/imunologia , Receptores Imunológicos/imunologia , Animais , Macaca mulatta , Masculino , Terapia de Alvo MolecularRESUMO
OBJECTIVE: To determine if immunization with non-myristoylated nef would elicit enhanced cellular immune responses resulting from improved presentation of Nef peptides by MHC-I on the cell surface, and enhanced T-cell help. DESIGN: The myristoylation site of HIV and SIV Nef is required for several Nef functions that modulate the immune response in an infected host, including downregulation of MHC-I, MHC-II, and CD4, and increased expression of the invariant chain on the cell surface. We constructed replication-competent Ad5- and Ad7-HIV recombinants encoding wild-type nef (nefWT) or a nef mutant (nefNM) lacking 19 amino-terminal amino acids, including the myristoylation site, and sequentially immunized chimpanzees mucosally, first with Ad5-HIVnef recombinants and subsequently with Ad7-HIVnef recombinants. METHODS: Peripheral blood lymphocytes were evaluated over the immunization course for Nef-specific cellular immune responses by interferon (IFN)-gamma ELISPOT and T-cell proliferation assays. Nef-specific CD4 and CD8 memory T cells that produced intracellular IFN-gamma, interleukin-2, and tumor necrosis factor (TNF)-alpha were assessed by flow cytometry. RESULTS: In comparison to immunization with Ad-HIVnefWT, Ad-HIVnefNM elicited statistically significant increases in numbers of IFN-gamma-secreting cells after the Ad7-HIVnefNM immunization and increased T-cell proliferative responses following both Ad5- and Ad7-HIVnefNM immunizations. Nef-specific CD4 and CD8 memory T-cell populations secreting TNF-alpha were also significantly increased in the Ad-HIVnefNM immunization group. CONCLUSIONS: The results support the hypothesis that immunization with Ad-recombinants encoding HIVnefNM rather than HIVnefWT elicits enhanced cellular immunity resulting from improved antigen presentation and greater T-cell help.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Produtos do Gene nef/imunologia , Infecções por HIV/imunologia , Vacinas contra a AIDS , Adenoviridae , Animais , Proliferação de Células , Regulação para Baixo , Feminino , Imunidade Celular , Memória Imunológica , Masculino , Pan troglodytes , Fator de Necrose Tumoral alfa/imunologia , Produtos do Gene nef do Vírus da Imunodeficiência HumanaRESUMO
A promising concept for human immunodeficiency virus (HIV) vaccines focuses immunity on the highly conserved transition state structures and epitopes that appear when the HIV glycoprotein gp120 binds to its receptor, CD4. We are developing chimeric antigens (full-length single chain, or FLSC) in which gp120 and CD4 sequences are flexibly linked to allow stable intrachain complex formation between the two moieties (A. DeVico et al., Proc Natl Acad Sci U S A 104:17477-17482, 2007, doi:10.1073/pnas.0707399104; T. R. Fouts et al., J Virol 74:11427-11436, 2000, doi:10.1128/JVI.74.24.11427-11436.2000). Proof of concept studies with nonhuman primates show that FLSC elicited heterologous protection against simian-human immunodeficiency virus (SHIV)/simian immunodeficiency virus (SIV) (T. R. Fouts et al., Proc Natl Acad Sci U S A 112:E992-E999, 2016, doi:10.1073/pnas.1423669112), which correlated with antibodies against transition state gp120 epitopes. Nevertheless, advancement of any vaccine that comprises gp120-CD4 complexes must consider whether the CD4 component breaks tolerance and becomes immunogenic in the autologous host. To address this, we performed an immunotoxicology study with cynomolgus macaques vaccinated with either FLSC or a rhesus variant of FLSC containing macaque CD4 sequences (rhFLSC). Enzyme-linked immunosorbent assay (ELISA) binding titers, primary CD3(+) T cell staining, and temporal trends in T cell subset frequencies served to assess whether anti-CD4 autoantibody responses were elicited by vaccination. We find that immunization with multiple high doses of rhFLSC did not elicit detectable antibody titers despite robust responses to rhFLSC. In accordance with these findings, immunized animals had no changes in circulating CD4(+) T cell counts or evidence of autoantibody reactivity with cell surface CD4 on primary naive macaque T cells. Collectively, these studies show that antigens using CD4 sequences to stabilize transition state gp120 structures are unlikely to elicit autoimmune antibody responses, supporting the advancement of gp120-CD4 complex-based antigens, such as FLSC, into clinical testing.
Assuntos
Autoanticorpos/sangue , Antígenos CD4/imunologia , Anticorpos Anti-HIV/sangue , Proteína gp120 do Envelope de HIV/imunologia , Proteínas Recombinantes/imunologia , Animais , Antígenos CD4/genética , Contagem de Linfócito CD4 , Ensaio de Imunoadsorção Enzimática , Feminino , Proteína gp120 do Envelope de HIV/genética , Macaca fascicularis , Masculino , Proteínas Recombinantes/genética , Subpopulações de Linfócitos T/imunologiaRESUMO
Improved antigenicity against HIV-1 envelope (Env) protein is needed to elicit vaccine-induced protective immunity in humans. Here we describe the first tests in non-human primates (NHPs) of Env gp140 protein fused to a humanized anti-LOX-1 recombinant antibody for delivering Env directly to LOX-1-bearing antigen presenting cells, especially dendritic cells (DC). LOX-1, or 1ectin-like oxidized low-density lipoprotein (LDL) receptor-1, is expressed on various antigen presenting cells and endothelial cells, and is involved in promoting humoral immune responses. The anti-LOX-1 Env gp140 fusion protein was tested for priming immune responses and boosting responses in animals primed with replication competent NYVAC-KC Env gp140 vaccinia virus. Anti-LOX-1 Env gp140 vaccination elicited robust cellular and humoral responses when used for either priming or boosting immunity. Co-administration with Poly ICLC, a TLR3 agonist, was superior to GLA, a TLR4 agonist. Both CD4+ and CD8+ Env-specific T cell responses were elicited by anti-LOX-1 Env gp140, but in particular the CD4+ T cells were multifunctional and directed to multiple epitopes. Serum IgG and IgA antibody responses induced by anti-LOX-1 Env gp140 against various gp140 domains were cross-reactive across HIV-1 clades; however, the sera neutralized only HIV-1 bearing sequences most similar to the clade C 96ZM651 Env gp140 carried by the anti-LOX-1 vehicle. These data, as well as the safety of this protein vaccine, justify further exploration of this DC-targeting vaccine approach for protective immunity against HIV-1.
Assuntos
HIV-1/imunologia , Receptores Depuradores Classe E/imunologia , Produtos do Gene env do Vírus da Imunodeficiência Humana/imunologia , Vacinas contra a AIDS/imunologia , Animais , Anticorpos Monoclonais Humanizados/imunologia , Anticorpos Antivirais/imunologia , Especificidade de Anticorpos , Humanos , Macaca mulatta , Masculino , Camundongos , Proteínas Recombinantes de Fusão/imunologia , Linfócitos T/imunologiaRESUMO
The chemokine and HIV receptor CXCR4 has been shown to play a role in chemotaxis and HIV-1 entry into T cells. Dibutyryl cAMP (DcAMP), an analog of cAMP, has been shown to increase CXCR4 cell surface expression and HIV-1 infectivity, but the molecular mechanism(s) responsible is unknown. Here we show that DcAMP treatment of purified human T lymphocytes increased transcription of CXCR4 mRNA as well as cell surface and intracellular CXCR4 protein expression. DcAMP-mediated stimulation of human PBL increased T-trophic HIV-1 (X4) fusion and viral replication as measured by syncytia formation and p24 levels, respectively. To determine the region(s) of the CXCR4 promoter required for cAMP responsiveness, truncations and point mutations of the CXCR4 promoter (nucleotides -1098 to +59) fused to luciferase were constructed and transiently transfected into human PBL. Deletional analysis demonstrated that the -1098 to -93 region of the CXCR4 promoter construct could be eliminated; the residual (-93 to +59) promoter retained cAMP responsiveness. Site-directed mutagenesis of a putative cAMP-responsive element (CRE) in the 5' UTR (+41 to +49) significantly and specifically attenuated the ability of DcAMP to drive the minimal CXCR4 promoter. Electrophoretic mobility shift assays demonstrated the formation of a complex between the CREB transcription factor and the putative CXCR4 CRE site. Our findings demonstrate a CRE element within the CXCR4 promoter that regulates CXCR4 transcription in response to changes in cAMP signaling. The cAMP-dependent up-regulation of CXCR4 mRNA results in increased CXCR4 intracellular and cell surface protein expression as well as increased HIV infectivity.