RESUMO
Vaccine-induced B cells differentiate along two pathways. The follicular pathway gives rise to germinal centers (GCs) that can take weeks to fully develop. The extrafollicular pathway gives rise to short-lived plasma cells (PCs) that can rapidly secrete protective antibodies within days of vaccination. Rabies virus (RABV) postexposure prophylaxis (PEP) requires rapid vaccine-induced humoral immunity for protection. Therefore, we hypothesized that targeting extrafollicular B cell responses for activation would improve the speed and magnitude of RABV PEP. To test this hypothesis, we constructed, recovered, and characterized a recombinant RABV-based vaccine expressing murine B cell activating factor (BAFF) (rRABV-mBAFF). BAFF is an ideal molecule to improve early pathways of B cell activation, as it links innate and adaptive immunity, promoting potent B cell responses. Indeed, rRABV-mBAFF induced a faster, higher antibody response in mice and enhanced survivorship in PEP settings compared to rRABV. Interestingly, rRABV-mBAFF and rRABV induced equivalent numbers of GC B cells, suggesting that rRABV-mBAFF augmented the extrafollicular B cell pathway. To confirm that rRABV-mBAFF modulated the extrafollicular pathway, we used a signaling lymphocytic activation molecule (SLAM)-associated protein (SAP)-deficient mouse model. In response to antigen, SAP-deficient mice form extrafollicular B cell responses but do not generate GCs. rRABV-mBAFF induced similar anti-RABV antibody responses in SAP-deficient and wild-type mice, demonstrating that BAFF modulated immunity through the extrafollicular and not the GC B cell pathway. Collectively, strategies that manipulate pathways of B cell activation may facilitate the development of a single-dose RABV vaccine that replaces current complicated and costly RABV PEP.IMPORTANCE Effective RABV PEP is currently resource- and cost-prohibitive in regions of the world where RABV is most prevalent. In order to diminish the requirements for rabies immunoglobulin (RIG) and multiple vaccinations for effective prevention of clinical rabies, a more rapidly protective vaccine is needed. This work presents a successful approach to rapidly generate antibody-secreting PCs in response to vaccination by targeting the extrafollicular B cell pathway. We demonstrate that the improved early antibody responses induced by rRABV-mBAFF confer improved protection against RABV in a PEP model. Significantly, activation of the early extrafollicular B cell pathway, such as that demonstrated here, could improve the efficacy of vaccines targeting other pathogens against which rapid protection would decrease morbidity and mortality.
Assuntos
Linfócitos B/fisiologia , Diferenciação Celular , Profilaxia Pós-Exposição/métodos , Vacina Antirrábica/imunologia , Raiva/prevenção & controle , Animais , Anticorpos Antivirais/sangue , Fator Ativador de Células B/genética , Fator Ativador de Células B/metabolismo , Linfócitos B/imunologia , Camundongos , Vacina Antirrábica/administração & dosagem , Vacina Antirrábica/genética , Análise de Sobrevida , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologiaRESUMO
UNLABELLED: Replication-deficient rabies virus (RABV)-based vaccines induce rapid and potent antibody responses via T cell-independent and T cell-dependent mechanisms. To further investigate early events in vaccine-induced antibody responses against RABV infections, we studied the role of macrophages as mediators of RABV-based vaccine immunogenicity. In this report, we show that a recombinant matrix gene-deleted RABV-based vaccine (rRABV-ΔM) infects and activates primary murine macrophages in vitro. Immunization of mice with live RABV-based vaccines results in accumulation of macrophages at the site of immunization, which suggests that macrophages in tissues support the development of effective anti-RABV B cell responses. However, we show that draining lymph node macrophages, but not macrophages at the site of immunization, are essential for the generation of germinal center B cells, follicular T helper cells, and RABV-specific antibodies. Our findings have implications for the design of new RABV-based vaccines for which early immunological events are important for the protection against RABV in postexposure settings. IMPORTANCE: More than two-thirds of the world's population live in regions where rabies is endemic. Postexposure prophylaxis is the primary means of treating humans. Identifying immunological principles that guide the development of rapid and potent antibody responses against rabies infections will greatly increase our ability to produce more-effective rabies vaccines. Here we report that macrophages in the draining lymph node, but not in the tissue at the site of immunization are important for vaccine-induced antibody responses to rabies. Information gleaned from this study may help guide the development of a single-dose vaccine against rabies infections.
Assuntos
Formação de Anticorpos , Linfócitos B/imunologia , Centro Germinativo/imunologia , Linfonodos/imunologia , Macrófagos/imunologia , Vacina Antirrábica/imunologia , Animais , Anticorpos Antivirais/sangue , Feminino , Injeções Intradérmicas , Camundongos Endogâmicos C57BL , Vacina Antirrábica/administração & dosagem , Linfócitos T Auxiliares-Indutores/imunologiaRESUMO
B cells secreting IgG antibodies, but not IgM, are thought to be solely responsible for vaccine-induced protection against rabies virus (RABV) infections in postexposure settings. In this report, we reinvestigated the potential for IgM to mediate protection in a mouse model of RABV vaccination. Immunocompetent mice immunized with an experimental live replication-deficient RABV-based vaccine produced virus neutralizing antibodies (VNAs) within 3 days of vaccination. However, mice unable to produce soluble IgM (sIgM(-/-)) did not produce VNAs until 7 days postimmunization. Furthermore, sIgM(-/-) mice were not protected against RABV infection when challenged 3 days postimmunization, while all wild-type mice survived challenge. Consistent with the lack of protection against pathogenic RABV challenge, approximately 50- to 100-fold higher viral loads of challenge virus were detected in the muscle, spinal cord, and brain of immunized sIgM(-/-) mice compared to control mice. In addition, IgG antibody titers in vaccinated wild-type and sIgM(-/-) mice were similar at all time points postimmunization, suggesting that protection against RABV challenge is due to the direct effects of IgM and not the influence of IgM on the development of effective IgG antibody titers. In all, early vaccine-induced IgM can limit dissemination of pathogenic RABV to the central nervous system and mediate protection against pathogenic RABV challenge. Considering the importance for the rapid induction of VNAs to protect against RABV infections in postexposure prophylaxis settings, these findings may help guide the development of a single-dose human rabies vaccine.
Assuntos
Anticorpos Antivirais/imunologia , Imunoglobulina M/imunologia , Profilaxia Pós-Exposição/métodos , Vacina Antirrábica/imunologia , Vírus da Raiva/imunologia , Raiva/prevenção & controle , Vacinação/métodos , Estruturas Animais/virologia , Animais , Modelos Animais de Doenças , Feminino , Imunoglobulina M/deficiência , Camundongos , Raiva/imunologia , Vacina Antirrábica/administração & dosagem , Análise de Sobrevida , Carga ViralRESUMO
Replication-deficient rabies viruses (RABV) are promising rabies postexposure vaccines due to their prompt and potent stimulation of protective virus neutralizing antibody titers, which are produced in mice by both T-dependent and T-independent mechanisms. To promote such early and robust B cell stimulation, we hypothesized that live RABV-based vaccines directly infect B cells, thereby activating a large pool of antigen-presenting cells (APCs) capable of providing early priming and costimulation to CD4(+) T cells. In this report, we show that live RABV-based vaccine vectors efficiently infect naive primary murine and human B cells ex vivo. Infection of B cells resulted in the significant upregulation of early markers of B cell activation and antigen presentation, including CD69, major histocompatibility complex class II (MHC-II), and CD40 in murine B cells or HLA-DR and CD40 in human B cells compared to mock-infected cells or cells treated with an inactivated RABV-based vaccine. Furthermore, primary B cells infected with a live RABV expressing ovalbumin were able to prime and stimulate naive CD4(+) OT-II T cells to proliferate and to secrete interleukin-2 (IL-2), demonstrating a functional consequence of B cell infection and activation by live RABV-based vaccine vectors. We propose that this direct B cell stimulation by live RABV-based vaccines is a potential mechanism underlying their induction of early protective T cell-dependent B cell responses, and that designing live RABV-based vaccines to infect and activate B cells represents a promising strategy to develop a single-dose postexposure rabies vaccine where the generation of early protective antibody titers is critical.
Assuntos
Linfócitos B/imunologia , Linfócitos B/virologia , Ativação Linfocitária , Vacina Antirrábica/imunologia , Vírus da Raiva/imunologia , Adulto , Animais , Linfócitos T CD4-Positivos/imunologia , Proliferação de Células , Células Cultivadas , Feminino , Humanos , Interleucina-2/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Vacinas Atenuadas/imunologiaRESUMO
A major goal in rabies virus (RV) research is to develop a single-dose postexposure prophylaxis (PEP) that would simplify vaccination protocols, reduce costs associated with rabies prevention in humans, and save lives. Live replication-deficient RV-based vaccines are emerging as promising single-dose vaccines to replace currently licensed inactivated RV-based vaccines. Nonetheless, little is known about how effective B cells develop in response to live RV-based vaccination. Understanding this fundamental property of rabies immunology may help in developing a single-dose RV vaccine. Typically, vaccines induce B cells secreting high-affinity, class-switched antibodies during germinal center (GC) reactions; however, there is a lag time between vaccination and the generation of GC B cells. In this report, we show that RV-specific antibodies are detected in mice immunized with live but not inactivated RV-based vaccines before B cells displaying a GC B cell phenotype (B220(+)GL7(hi)CD95(hi)) are formed, indicating a potential role for T cell-independent and early extrafollicular T cell-dependent antibody responses in the protection against RV infection. Using two mouse models of CD4(+) T cell deficiency, we show that B cells secreting virus-neutralizing antibodies (VNAs) are induced via T cell-independent mechanisms within 4 days postimmunization with a replication-deficient RV-based vaccine. Importantly, mice that are completely devoid of T cells (B6.129P2-Tcrß(tm1Mom) Tcrδ(tm1Mom)/J) show protection against pathogenic challenge shortly after immunization with a live replication-deficient RV-based vaccine. We show that vaccines that can exploit early pathways of B cell activation and development may hold the key for the development of a single-dose RV vaccine wherein the rapid induction of VNA is critical.
Assuntos
Linfócitos B/imunologia , Vacina Antirrábica/imunologia , Vírus da Raiva/imunologia , Animais , Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Linfócitos T CD4-Positivos/imunologia , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Vacina Antirrábica/administração & dosagem , Vacinas Atenuadas/administração & dosagem , Vacinas Atenuadas/imunologiaRESUMO
Recombinant rabies virus (RV)-based vectors have demonstrated their efficacy in generating long-term, antigen-specific immune responses in murine and monkey models. However, replication-competent viral vectors pose significant safety concerns due to vector pathogenicity. RV pathogenicity is largely attributed to its glycoprotein (RV-G), which facilitates the attachment and entry of RV into host cells. We have developed a live, single-cycle RV by deletion of the G gene from an RV vaccine vector expressing HIV-1 Gag (SPBN-DeltaG-Gag). Passage of SPBN-DeltaG-Gag on cells stably expressing RV-G allowed efficient propagation of the G-deleted RV. The in vivo immunogenicity data comparing single-cycle RV to a replication-competent control (BNSP-Gag) showed lower RV-specific antibodies; however, the overall isotype profiles (IgG2a/IgG1) were similar for the two vaccine vectors. Despite this difference, mice immunized with SPBN-DeltaG-Gag and BNSP-Gag mounted similar levels of Gag-specific CD8(+) T-cell responses as measured by major histocompatibility complex class I Gag-tetramer staining, gamma interferon-enzyme-linked immunospot assay, and cytotoxic T-cell assay. Moreover, these cellular responses were maintained equally at immunization titers as low as 10(3) focus-forming units for both RV vaccine vectors. CD8(+) T-cell responses were significantly enhanced by a boost with a single-cycle RV complemented with a heterologous vesicular stomatitis virus glycoprotein. These findings demonstrate that single-cycle RV is an effective alternative to replication-competent RV vectors for future development of vaccines for HIV-1 and other infectious diseases.
Assuntos
Vetores Genéticos , Vacina Antirrábica , Vírus da Raiva/imunologia , Raiva/prevenção & controle , Animais , Anticorpos Antivirais/genética , Anticorpos Antivirais/imunologia , Linfócitos T CD8-Positivos/imunologia , Feminino , Produtos do Gene gag/genética , Produtos do Gene gag/metabolismo , Vetores Genéticos/genética , Vetores Genéticos/imunologia , Humanos , Interferon gama/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Raiva/imunologia , Vacina Antirrábica/genética , Vacina Antirrábica/imunologia , Vírus da Raiva/patogenicidade , Linfócitos T Citotóxicos/imunologia , Internalização do Vírus , Replicação Viral/fisiologiaRESUMO
Although current postexposure prophylaxis rabies virus (RV) vaccines are effective, approximately 40,000-70,000 rabies-related deaths are reported annually worldwide. The development of effective formulations requiring only 1-2 applications would significantly reduce mortality. We assessed in mice and nonhuman primates the efficacy of replication-deficient RV vaccine vectors that lack either the matrix (M) or phosphoprotein (P) gene. A single dose of M gene-deficient RV induced a more rapid and efficient anti-RV response than did P gene-deficient RV immunization. Furthermore, the M gene-deleted RV vaccine induced 4-fold higher virus-neutralizing antibody (VNA) levels in rhesus macaques than did a commercial vaccine within 10 days after inoculation, and at 180 days after immunization rhesus macaques remained healthy and had higher-avidity antibodies, higher VNA titers, and a more potent antibody response typical of a type 1 T helper response than did animals immunized with a commercial vaccine. The data presented in this article suggest that the M gene-deleted RV vaccine is safe and effective and holds the potential of replacing current pre- and postexposure RV vaccines.
Assuntos
Anticorpos Antivirais/sangue , Vacina Antirrábica/imunologia , Vírus da Raiva/fisiologia , Raiva/prevenção & controle , Vacinas Atenuadas/imunologia , Animais , Afinidade de Anticorpos , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Feminino , Deleção de Genes , Macaca mulatta , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Raiva/imunologia , Vacina Antirrábica/efeitos adversos , Vacinas Atenuadas/efeitos adversos , Replicação ViralRESUMO
B cell activating factor (BAFF) is a member of the tumor necrosis factor (TNF) superfamily of cytokines that links innate with adaptive immunity. BAFF signals through receptors on B cells, making it an attractive molecule to potentiate vaccine-induced B cell responses. We hypothesized that a rabies virus (RABV)-based vaccine displaying both antigen and BAFF on the surface of the same virus particle would target antigen-specific B cells for activation and improve RABV-specific antibody responses. To test this hypothesis, we constructed a recombinant RABV-based vector expressing virus membrane-anchored murine BAFF (RABV-ED51-mBAFF). BAFF was incorporated into the RABV particle and determined to be biologically functional, as demonstrated by increased B cell survival of primary murine B cells treated ex-vivo with RABV-ED51-mBAFF. B cell survival was inhibited by pre-treating RABV-ED51-mBAFF with an antibody that blocks BAFF functions. RABV-ED51-mBAFF also activated primary murine B cells ex-vivo more effectively than RABV as shown by significant upregulation of CD69, CD40, and MHCII on the surface of infected B cells. In-vivo, RABV-ED51-mBAFF induced significantly faster and higher virus neutralizing antibody (VNA) titers than RABV while not adversely affecting the longevity of the vaccine-induced antibody response. Since BAFF was incorporated into the virus particle and genome replication was not required for BAFF expression in-vivo, we hypothesized that RABV-ED51-mBAFF would be effective as an inactivated vaccine. Mice immunized with 250 ng/mouse of ß-propriolactone-inactivated RABV-ED51-mBAFF showed faster and higher anti-RABV VNA titers compared to mice immunized with inactivated RABV. Together, this model stands as a potential foundation for exploring other virus membrane-anchored molecular adjuvants to make safer, more effective inactivated RABV-based vaccines.
Assuntos
Formação de Anticorpos/imunologia , Fator Ativador de Células B/imunologia , Linfócitos B/imunologia , Vacina Antirrábica/imunologia , Raiva/imunologia , Raiva/prevenção & controle , Vírion/imunologia , Adjuvantes Imunológicos , Animais , Anticorpos Antivirais/sangue , Citocinas/metabolismo , Feminino , Imunização , Cinética , Camundongos , Camundongos Endogâmicos C57BL , Vacina Antirrábica/efeitos adversos , Vírus da Raiva/genética , Vírus da Raiva/crescimento & desenvolvimento , Vírus da Raiva/imunologia , Vacinação , Vacinas Atenuadas/imunologia , Vacinas de Produtos Inativados/efeitos adversos , Vacinas de Produtos Inativados/imunologia , Vacinas SintéticasRESUMO
There is significant need to develop a single-dose rabies vaccine to replace the current multi-dose rabies vaccine regimen and eliminate the requirement for rabies immune globulin in post-exposure settings. To accomplish this goal, rabies virus (RABV)-based vaccines must rapidly activate B cells to secrete antibodies which neutralize pathogenic RABV before it enters the CNS. Increased understanding of how B cells effectively respond to RABV-based vaccines may improve efforts to simplify post-exposure prophylaxis (PEP) regimens. Several studies have successfully employed the TNF family cytokine a proliferation-inducing ligand (APRIL) as a vaccine adjuvant. APRIL binds to the receptors TACI and B cell maturation antigen (BCMA)-expressed by B cells in various stages of maturation-with high affinity. We discovered that RABV-infected primary murine B cells upregulate APRIL ex vivo. Cytokines present at the time of antigen exposure affect the outcome of vaccination by influencing T and B cell activation and GC formation. Therefore, we hypothesized that the presence of APRIL at the time of RABV-based vaccine antigen exposure would support the generation of protective antibodies against RABV glycoprotein (G). In an effort to improve the response to RABV vaccination, we constructed and characterized a live recombinant RABV-based vaccine vector which expresses murine APRIL (rRABV-APRIL). Immunogenicity testing in mice demonstrated that expressing APRIL from the RABV genome does not impact the primary antibody response against RABV G compared to RABV alone. In order to evaluate the necessity of APRIL for the response to rabies vaccination, we compared the responses of APRIL-deficient and wild-type mice to immunization with rRABV. APRIL deficiency does not affect the primary antibody response to vaccination. Furthermore, APRIL expression by the vaccine did not improve the generation of long-lived antibody-secreting plasma cells (PCs) as serum antibody levels were equivalent in response to rRABV-APRIL and the vector eight weeks after immunization. Moreover, APRIL is dispensable for the long-lived antibody-secreting PC response to rRABV vaccination as anti-RABV G IgG levels were similar in APRIL-deficient and wild-type mice six months after vaccination. Mice lacking the APRIL receptor TACI demonstrated primary anti-RABV G antibody responses similar to wild-type mice following immunization with the vaccine vector indicating that this response is independent of TACI-mediated signals. Collectively, our findings demonstrate that APRIL and associated TACI signaling is dispensable for the immune response to RABV-based vaccination.
Assuntos
Adjuvantes Imunológicos/metabolismo , Vacina Antirrábica/imunologia , Proteína Transmembrana Ativadora e Interagente do CAML/metabolismo , Membro 13 da Superfamília de Ligantes de Fatores de Necrose Tumoral/metabolismo , Animais , Anticorpos Antivirais/sangue , Imunoglobulina G/sangue , Camundongos , Camundongos Knockout , Membro 13 da Superfamília de Ligantes de Fatores de Necrose Tumoral/deficiência , Vacinas Atenuadas/imunologiaRESUMO
We previously showed that a matrix (M) gene-deleted rabies virus (RABV)-based vaccine (RABV-ΔM) is highly immunogenic and induces potent B cell responses in the context of RABV infection. We speculated that RABV-ΔM expressing HIV proteins would also induce potent B cell responses against HIV antigens. As a prerequisite to future studies in nonhuman primates, we completed immunogenicity studies in mice to confirm the ability of RABV-ΔM to induce polyfunctional B cell responses in the context of HIV. To that end, the envelope protein from the mac239 strain of SIV (SIVmac239Env) was cloned into RABV-ΔM, resulting in RABV-ΔM-Env. Infectious virus was recovered following standard methods and propagated on baby hamster kidney cells stably expressing RABV M [>10(7) focus forming units (ffu)/ml]. Western blot analysis of cell lysates or of purified virions confirmed Env expression on the surface of infected cells and within virus particles, respectively. Positive neutralization activity against a neutralization-sensitive SIV strain and to a lesser extent against a neutralization-resistant SIV strain was detected in mice after a single intramuscular inoculation with RABV-ΔM-Env. The quality, but not quantity, of the antibody response was enhanced via boosting with recombinant gp130 or RABV-ΔM-Env as measured by an increase in antibody avidity and a skewing toward a Th1-type antibody response. We also show that an intradermal inoculation induces higher antibodies than an intramuscular or intranasal inoculation. An intradermal inoculation of RABV-ΔM-Env followed by a boost inoculation with recombinant gp130 produced anti-SIV antibodies with neutralizing and nonneutralizing antibody (nNAb) effector functions. Together, RABV-ΔM-Env induces B cells to secrete antibodies against SIV with the potential to clear both "free" and cell-associated virus. Strategies capable of eliciting both NAbs as well as nNAbs might help to improve the efficacy of HIV-1 vaccines.
Assuntos
Anticorpos Antivirais/sangue , Formação de Anticorpos , Vacina Antirrábica/imunologia , Vírus da Raiva/imunologia , Vacinas contra a SAIDS/imunologia , Vírus da Imunodeficiência Símia/imunologia , Animais , Linfócitos B/imunologia , Feminino , Injeções Intradérmicas , Injeções Intramusculares , Camundongos Endogâmicos C57BL , Vacina Antirrábica/administração & dosagem , Vacina Antirrábica/genética , Vírus da Raiva/genética , Vacinas contra a SAIDS/administração & dosagem , Vacinas contra a SAIDS/genética , Vírus da Imunodeficiência Símia/genéticaRESUMO
The failure to develop vaccines to protect against important infectious diseases such as human immunodeficiency virus type I (HIV-1) or Hepatitis C virus (HCV) has increased the interest in new vaccine strategies. One of these methods is immunization with an attenuated recombinant viral vector expressing a foreign antigen, which could protect individuals from later exposure to the respective pathogen. A new method to recover a non-segmented negative-stranded RNA virus (NNSV) from cDNA was described for the first time for rabies virus (RV), a member of the rhabdovirus family. The same approach was successfully used for another rhabdovirus, vesicular stomatitis virus (VSV), and opened the possibility to use rhabdoviruses as vaccine vehicles and biomedical tools. Further research showed that the genomes of rhabdoviruses are highly flexible, easy to manipulate, and able to express large and even multiple foreign genes, and therefore are excellent vaccine candidates. In addition, it has been shown for both RV and VSV that their single surface glycoprotein G, which is responsible for attachment and fusion to the host cell, can functionally be replaced by other viral or cellular glycoproteins. This review gives an overview of the use of RV and VSV as promising new candidates in the fight against HIV-1 and other human diseases.
Assuntos
Vacinas contra a AIDS/imunologia , Infecções por HIV/imunologia , HIV-1 , Rhabdoviridae/imunologia , Vacinas contra a AIDS/genética , Animais , Vetores Genéticos/imunologia , Infecções por HIV/terapia , Humanos , Macaca mulatta , Camundongos , Vírus Reordenados/genética , Vírus Reordenados/imunologia , Rhabdoviridae/genéticaRESUMO
This article gives an overview about the development of an HIV-1 vaccine. Tremendous numbers of papers have been published on this topic during the last 10 years, and this article can only touch on the different directions taken toward the development of an HIV-1 vaccine, and not give a complete overview of the entire field.
Assuntos
Vacinas contra a AIDS , Anticorpos Anti-HIV/metabolismo , HIV-1/imunologia , HIV-1/metabolismo , Humanos , Imunização , Vacinas Sintéticas/imunologiaRESUMO
Dogs account for the majority of human exposures and deaths due to rabies virus (RABV) worldwide. In this report, we show that a replication-deficient RABV-based vaccine in which the matrix gene is deleted (RABV-ΔM) is safe and induces rapid and potent VNA titers after a single inoculation in dogs. Average VNA titers peaked at 3.02 or 5.11 international units (IU/ml) by 14 days post-immunization with a single dose of 10(6) or 10(7) focus forming units (ffu), respectively, of RABV-ΔM. By day 70 post immunization, all dogs immunized with either dose of vaccine showed VNA titers >0.5IU/ml, the level indicative of a satisfactory immunization. Importantly, no systemic or local reactions were noted in any dog immunized with RABV-ΔM. The elimination of dog rabies through mass vaccination is hindered by limited resources, requirement for repeat vaccinations often for the life of a dog, and in some parts of the world, inferior vaccine quality. Our preliminary safety and immunogenicity data in dogs suggest that RABV-ΔM might complement currently used inactivated RABV-based vaccines in vaccination campaigns by helping to obtain 100% response in vaccinated dogs, thereby increasing overall vaccination coverage.
Assuntos
Anticorpos Antivirais/sangue , Doenças do Cão/prevenção & controle , Vacina Antirrábica/imunologia , Raiva/veterinária , Animais , Anticorpos Neutralizantes/sangue , Doenças do Cão/virologia , Cães , Feminino , Deleção de Genes , Masculino , Raiva/prevenção & controle , Vacina Antirrábica/genética , Vírus da Raiva/genética , Proteínas da Matriz Viral/genéticaRESUMO
We have previously shown that live-attenuated rabies virus (RABV)-based vaccines infect and directly activate murine and human primary B cells in-vitro, which we propose can be exploited to help develop a single-dose RABV-based vaccine. Here we report on a novel approach to utilize the binding of Intracellular Adhesion Molecule-1 (ICAM-1) to its binding partner, Lymphocyte Function-associated Antigen-1 (LFA-1), on B cells to enhance B cell activation and RABV-specific antibody responses. We used a reverse genetics approach to clone, recover, and characterize a live-attenuated recombinant RABV-based vaccine expressing the murine Icam1 gene (rRABV-mICAM-1). We show that the murine ICAM-1 gene product is incorporated into virus particles, potentially exposing ICAM-1 to extracellular binding partners. While rRABV-mICAM-1 showed 10-100-fold decrease in viral titers on baby hamster kidney cells compared to the parental virus (rRABV), rRABV-mICAM-1 infected and activated primary murine B cells in-vitro more efficiently than rRABV, as indicated by significant upregulation of CD69, CD40, and MHCII on the surface of infected B cells. ICAM-1 expression on the virus surface was responsible for enhanced B cell infection since pre-treating rRABV-mICAM-1 with a neutralizing anti-ICAM-1 antibody reduced B cell infection to levels observed with rRABV alone. Furthermore, 100-fold less rRABV-mICAM-1 was needed to induce antibody titers in immunized mice equivalent to antibody titers observed in rRABV-immunized mice. Of note, only 10(3) focus forming units (ffu)/mouse of rRABV-mICAM-1 was needed to induce significant anti-RABV antibody titers as early as five days post-immunization. As both speed and potency of antibody responses are important in controlling human RABV infection in a post-exposure setting, these data show that expression of Icam1 from the RABV genome, which is then incorporated into the virus particle, is a promising strategy for the development of a single-dose RABV vaccine that requires only a minimum of virus.
Assuntos
Anticorpos Antivirais/biossíntese , Genoma Viral , Molécula 1 de Adesão Intercelular/imunologia , Vacina Antirrábica/imunologia , Vírus da Raiva/imunologia , Raiva/prevenção & controle , Proteínas Virais/imunologia , Animais , Linfócitos B/imunologia , Linfócitos B/virologia , Linhagem Celular , Cricetinae , Feminino , Expressão Gênica , Humanos , Imunidade Humoral/efeitos dos fármacos , Molécula 1 de Adesão Intercelular/genética , Antígeno-1 Associado à Função Linfocitária/genética , Antígeno-1 Associado à Função Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Raiva/imunologia , Raiva/virologia , Vacina Antirrábica/administração & dosagem , Vacina Antirrábica/genética , Vírus da Raiva/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Vacinas Atenuadas , Vacinas Sintéticas , Proteínas Virais/genética , Vírion/genética , Vírion/imunologiaRESUMO
Over two-thirds of the world's population lives in regions where rabies is endemic, resulting in over 15 million people receiving multi-dose post-exposure prophylaxis (PEP) and over 55,000 deaths per year globally. A major goal in rabies virus (RABV) research is to develop a single-dose PEP that would simplify vaccination protocols, reduce costs associated with RABV prevention, and save lives. Protection against RABV infections requires virus neutralizing antibodies; however, factors influencing the development of protective RABV-specific B cell responses remain to be elucidated. Here we used a mouse model of IL-21 receptor-deficiency (IL-21R-/-) to characterize the role for IL-21 in RABV vaccine-induced immunity. IL-21R-/- mice immunized with a low dose of a live recombinant RABV-based vaccine (rRABV) produced only low levels of primary or secondary anti-RABV antibody response while wild-type mice developed potent anti-RABV antibodies. Furthermore, IL-21R-/- mice immunized with low-dose rRABV were only minimally protected against pathogenic RABV challenge, while all wild-type mice survived challenge, indicating that IL-21R signaling is required for antibody production in response to low-dose RABV-based vaccination. IL-21R-/- mice immunized with a higher dose of vaccine produced suboptimal anti-RABV primary antibody responses, but showed potent secondary antibodies and protection similar to wild-type mice upon challenge with pathogenic RABV, indicating that IL-21 is dispensable for secondary antibody responses to live RABV-based vaccines when a primary response develops. Furthermore, we show that IL-21 is dispensable for the generation of Tfh cells and memory B cells in the draining lymph nodes of immunized mice but is required for the detection of optimal GC B cells or plasma cells in the lymph node or bone marrow, respectively, in a vaccine dose-dependent manner. Collectively, our preliminary data show that IL-21 is critical for the development of optimal vaccine-induced primary but not secondary antibody responses against RABV infections.
Assuntos
Interleucinas/imunologia , Vacina Antirrábica/imunologia , Vírus da Raiva/imunologia , Raiva/imunologia , Raiva/prevenção & controle , Animais , Anticorpos Antivirais/sangue , Modelos Animais de Doenças , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores de Interleucina-21/deficiênciaRESUMO
This study analyzed a heterologous prime-boost vaccine approach against HIV-1 using three different antigenically unrelated negative-stranded viruses (NSV) expressing HIV-1 Gag as vaccine vectors: rabies virus (RABV), vesicular stomatitis virus (VSV) and Newcastle disease virus (NDV). We hypothesized that this approach would result in more robust cellular immune responses than those achieved with the use of any of the vaccines alone in a homologous prime-boost regimen. To this end, we primed BALB/c mice with each of the NSV-based vectors. Primed mice were rested for thirty-five days after which we administered a second immunization with the same or heterologous NSV-Gag viruses. The magnitude and quality of the Gag-specific CD8(+) T cells in response to these vectors post boost were measured. In addition, we performed challenge experiments using vaccinia virus expressing HIV-1 Gag (VV-Gag) thirty-three days after the boost inoculation. Our results showed that the choice of the vaccine used for priming was important for the detected Gag-specific CD8(+) T cell recall responses post boost and that NDV-Gag appeared to result in a more robust recall of CD8(+) T cell responses independent of the prime vaccine used. However, the different prime-boost strategies were not distinct for the parameters studied in the challenge experiments using VV-Gag but did indicate some benefits compared to single immunizations. Taken together, our data show that NSV vectors can individually stimulate HIV-Gag specific CD8(+) T cells that are effectively recalled by other NSV vectors in a heterologous prime-boost approach. These results provide evidence that RABV, VSV and NDV can be used in combination to develop vaccines needing prime-boost regimens to stimulate effective immune responses.
Assuntos
HIV-1/imunologia , Imunização Secundária/métodos , Vírus de RNA/genética , Produtos do Gene gag do Vírus da Imunodeficiência Humana/genética , Produtos do Gene gag do Vírus da Imunodeficiência Humana/imunologia , Animais , Linfócitos T CD8-Positivos/imunologia , DNA Recombinante/genética , Feminino , Vetores Genéticos/genética , Camundongos , Camundongos Endogâmicos BALB CRESUMO
Rabies remains a global public health threat that kills more than 55,000 people per year. Rabies disproportionately affects children and, therefore, is ranked the seventh most important infectious disease due to years lost. Prevention of human rabies is accomplished by controlling rabies in domestic and wild animals, including the use of vaccination programs. The usefulness of human rabies vaccines is hampered by high cost, complicated vaccination regimens and lack of compliance, especially in areas of Africa and Asia where human rabies infections are endemic. A single-dose vaccine would greatly benefit efforts to combat this global health threat. However, a single-dose vaccine based on current inactivated vaccines does not appear feasible and other approaches are needed. Technology has advanced since modern human rabies vaccines were developed over 40 years ago. In addition, our understanding of immunological principles that influence the outcome of vaccination has increased. This article describes the current status of inactivated rabies virus vaccines and recent developments arising from the use of reverse genetics technologies designed to develop replication-deficient or single-cycle live rabies virus-based vectors for use as a single-dose rabies vaccine for humans.
Assuntos
Vacina Antirrábica , Vírus da Raiva/imunologia , Raiva/prevenção & controle , Vacinas de Produtos Inativados , Animais , Animais Selvagens , Vetores Genéticos , Humanos , Esquemas de Imunização , Vacina Antirrábica/administração & dosagem , Vacina Antirrábica/efeitos adversos , Vacina Antirrábica/genética , Vacina Antirrábica/imunologia , Vírus da Raiva/genética , Vacinação/tendências , Vacinas de Produtos Inativados/administração & dosagem , Vacinas de Produtos Inativados/efeitos adversos , Vacinas de Produtos Inativados/genética , Vacinas de Produtos Inativados/imunologia , Replicação ViralRESUMO
Rabies virus, the prototypical neurotropic virus, causes one of the most lethal zoonotic diseases. According to official estimates, over 55,000 people die of the disease annually, but this is probably a severe underestimation. A combination of virulence factors enables the virus to enter neurons at peripheral sites and travel through the spinal cord to the brain of the infected host, where it often induces aggression that facilitates the transfer of the virus to a new host. This Review summarizes the current knowledge of the replication cycle of rabies virus and virus- host cell interactions, both of which are fundamental elements in our quest to understand the life cycle of rabies virus and the pathogenesis of rabies.
Assuntos
Encéfalo/virologia , Sistema Nervoso Periférico/virologia , Vírus da Raiva/fisiologia , Medula Espinal/virologia , Animais , Humanos , Modelos Biológicos , Vírus da Raiva/patogenicidade , Proteínas Virais/fisiologia , Fatores de Virulência/fisiologiaRESUMO
Highly attenuated rabies virus (RV) vaccine vectors were evaluated for their ability to protect against highly pathogenic SIV(mac251) challenge. Mamu-A*01 negative rhesus macaques were immunized in groups of four with either: RV expressing SIV(mac239)-GagPol, a combination of RV expressing SIV(mac239)-Env and RV expressing SIV(mac239)-GagPol, or with empty RV vectors. Eight weeks later animals received a booster immunization with a heterologous RV expressing the same antigens. At 12 weeks post-boost, all animals were challenged intravenously with 100 TCID(50) of pathogenic SIV(mac251-CX). Immunized macaques in both vaccine groups had 1.3-1.6-log-fold decrease in viral set point compared to control animals. The GagPol/Env immunized animals also had a significantly lower peak viral load. When compared to control animals following challenge, vaccinated macaques had a more rapid induction of SIV(mac251) neutralizing antibodies and of CD8(+) T cell responses to various SIV epitopes. Moreover, vaccinated macaques better maintained peripheral memory CD4(+) T cells and were able to mount a poly-functional CD8(+) T cell response in the mucosa. These findings indicate promise for RV-based vectors and have important implications for the development of an efficacious HIV vaccine.
Assuntos
Anticorpos Neutralizantes/imunologia , Linfócitos T CD8-Positivos/imunologia , Macaca mulatta/imunologia , Macaca mulatta/virologia , Vírus da Raiva/imunologia , Vacinas contra a SAIDS/imunologia , Síndrome de Imunodeficiência Adquirida dos Símios/imunologia , Animais , Imunidade Celular/imunologia , Imunidade Humoral/imunologia , Vírus da Imunodeficiência Símia/imunologiaRESUMO
The type of immune response induced by a vaccine is a critical factor that determines its effectiveness in preventing infection or disease. Inactivated and live rabies virus (RV) vaccine strains elicit an IgG1-biased and IgG1/IgG2a-balanced antibody response, respectively. However, IgG2a antibodies are potent inducers of anti-viral effector functions, and therefore, a viral vaccine vector that can elicit an IgG2a-biased antibody response may be more effective against RV infection. Here we describe the humoral immune response of a live replication-deficient phosphoprotein (P)-deleted RV vector (SPBN-DeltaP), or a recombinant P-deleted virus that expresses two copies of the RV glycoprotein (G) gene (SPBN-DeltaP-RVG), and compare it to a UV-inactivated RV. Mice inoculated with UV-inactivated RV induced predominantly an IgG1-specific antibody response, while live recombinant SPBN-DeltaP exhibited a mixed IgG1/IgG2a antibody response, which is consistent with the isotype profiles from the replication-competent parental viruses. Survivorship in mice after pathogenic RV challenge indicates a 10-fold higher efficiency of live SPBN-DeltaP compared to UV-inactivated SPBN-DeltaP. In addition, SPBN-DeltaP-RVG induced a more rapid and robust IgG2a response that protected mice more effectively than SPBN-DeltaP. Of note, 10(3)ffu of SPBN-DeltaP-RVG-induced anti-RV antibodies that were 100% protective in mice against pathogenic RV challenge. The increased immune response was directed not only against RV G but also against the ribonucleoprotein (RNP), indicating that the expression of two RV G genes from SPBN-DeltaP-RVG enhances the immune response to other RV antigens as well. In addition, Rag2 mice inoculated intramuscularly with 10(5)ffu/mouse of SPBN-DeltaP showed no clinical signs of rabies, and no viral RNA was detected in the spinal cord or brain of inoculated mice. Therefore, the safety of the P-deleted vectors along with the onset and magnitude of the IgG2a-induced immune response by SPBN-DeltaP-RVG indicate that this vector holds great promise as either a therapeutic or preventative vaccine against RV or other infectious diseases.