Development of vaccines for HIV-1: relevance of subtype-specific cellular immunity

Han pasado casi 30 años de la detección de los primeros casos de infección con HIV-1 y aún no se ha conseguido desarrollar una vacuna efectiva y segura. A pesar del impacto positivo sobre la pandemia que se ha conseguido gracias a los avances en la terapia antirretroviral (TARV), el HIV/sida sigue constituyendo un grave problema para la salud pública, especialmente en los países en desarrollo, donde es difícil el acceso al tratamiento. En el mundo, 33 millones de personas viven con el virus del sida, mientras que en la Argentina se calcula que habría unos 120 000 infectados. Uno de los desafíos para lograr una vacuna contra el HIV es la variabilidad viral. El grupo M, responsable de la pandemia, se encuentra dividido en 10 subtipos y varios sub-subtipos, además de las 48 formas recombinantes circulantes y más de cien formas recombinantes únicas. La epidemia de HIV en nuestro país es tan compleja como en el resto del mundo, con la co-circulación principalmente de virus pertenecientes al subtipo B y recombinantes BF (CRF12_BF y derivadas). A pesar de la cantidad de trabajos dedicados a la caracterización de la respuesta inmune y al desarrollo de vacunas, no queda claro cuál es el impacto de la variabilidad en la elección del antígeno. Trabajos realizados en nuestro laboratorio demuestran el papel que juega la inmunidad celular con respecto a las variantes recombinantes BF, tanto en humanos como en modelos animales. Estos resultados son de importancia en el desarrollo de futuras vacunas para nuestra región.It has been almost 30 years since the detection of the first HIV-1 cases and yet an effective and safe vaccine has not been developed. Although, advances in antiretroviral therapy (HAART) have produced a major impact on the pandemic, and even though HIV/aids remains a major concern for developing countries, where access to therapy is limited. The last report from UNAIDS notified 33 million people living with HIV/aids, worldwide, while in Argentina it is estimated that 120 000 persons have been infected. One of the challenges to address and ultimately overcome when developing a vaccine is the high variability of HIV-1. The M group, responsible for the pandemic, is divided into 10 subtypes and several sub-subtypes, in addition to the 48 circulating recombinant forms (CRF) and over one hundred unique recombinant forms (URF). The HIV epidemic in Argentina is as complex as in the rest of the world, characterized by the high prevalence of infections caused by subtype B and BF variants. Despite the wide range of publications focused on the immune response against HIV as well as to vaccine development, how to overcome variability on vaccine antigen selection is still unclear. Studies performed in our laboratory showed the impact of the immunogenicity of BF recombinant variants, both in humans and in animal models. These results are of great concern in vaccine development for our region.Fil: Rodríguez, Ana María. Universidad de Buenos Aires. Facultad de Medicina. Departamento de Microbiología. Centro Nacional de Referencia del Sida; ArgentinaFil: Turk, Gabriela Julia Ana. Universidad de Buenos Aires. Facultad de Medicina. Departamento de Microbiología. Centro Nacional de Referencia del Sida; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Pascutti, María Fernanda. Universidad de Buenos Aires. Facultad de Medicina. Departamento de Microbiología. Centro Nacional de Referencia del Sida; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Falivene, Juliana. Universidad de Buenos Aires. Facultad de Medicina. Departamento de Microbiología. Centro Nacional de Referencia del Sida; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Gherardi, Maria Magdalena. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Departamento de Microbiología. Centro Nacional de Referencia del Sida; Argentin

Age-related increase in resistance to acute Trypanosoma cruzi infection in rats is associated with an appropriate antibody response

Inoculation at weaning with Trypanosoma cruzi in inbred 'l' rats resulted in a self-resolving acute infection characterized by marked parasitaemias, whereas challenge to adult rats revealed a mild disease with extremely low parasitaemias. To explore the mechanisms underlying such age-associated differences in disease outcome, we analysed the in vitro replication of T. cruzi, nitric oxide and tumour necrosis factor-α (TNF-α) production in peritoneal macrophages (PMs), the serum concentrations of the specific immunoglobulins (Igs) IgM and IgG, antibodies exhibiting lytic activity against bloodstream forms of T. cruzi and circulating levels of nitrate, TNF-α and interferon-γ (IFN-γ). Macrophages from young rats were as effective as their adult counterparts for restraining intracellular parasite replication. When stimulated with IFN-γ, culture supernatants from young PMs contained higher amounts of nitrite and TNF-α. Serum samples from 4 and 7 days post infection revealed easily detectable amounts of nitrate, with values being further augmented by day 7 post infection and significantly higher in the young group. TNF-α levels were only detected in the young group by day 7 post infection. Both groups had increased amounts of IFN-γ in their sera, although in adult rats, this trend was followed by a significant drop at day 7, with young rats showing values still higher by the same time point evaluation. In contrast, young rats presented significantly lower levels of IgM and IgG antibodies during the first week of infection. Increased resistance in adult rats seems to be the result of a more appropriate antibody production.Fil: Pascutti, María Fernanda. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Inmunología Clinica y Experimental de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Médicas. Instituto de Inmunología Clinica y Experimental de Rosario; ArgentinaFil: Bottasso, Oscar Adelmo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Inmunología Clinica y Experimental de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Médicas. Instituto de Inmunología Clinica y Experimental de Rosario; ArgentinaFil: Hourquescos, M. C.. Universidad Nacional de Rosario; ArgentinaFil: Wietzerbin, Jeanne. Institute Curie; FranciaFil: Revelli, Silvia Susana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Inmunología Clinica y Experimental de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Médicas. Instituto de Inmunología Clinica y Experimental de Rosario; Argentin

IL-12 and GM-CSF in DNA/MVA Immunizations against HIV-1 CRF12_BF Nef Induced T-Cell Responses With an Enhanced Magnitude, Breadth and Quality

In Argentina, the HIV epidemic is characterized by the co-circulation of subtype B and BF recombinant viral variants. Nef is an HIV protein highly variable among subtypes, making it a good tool to study the impact of HIV variability in the vaccine design setting. We have previously reported a specific cellular response against NefBF with low cross-reactivity to NefB in mice. The aim of this work was to analyze whether the co-administration of IL-12 and GM-CSF, using DNA and MVA vaccine vectors, could improve the final cellular response induced. Mice received three DNA priming doses of a plasmid that express NefBF plus DNAs expressing IL-12 and/or GM-CSF. Afterwards, all the groups were boosted with a MVAnefBF dose. The highest increase in the magnitude of the NefBF response, compared to that induced in the control was found in the IL-12 group. Importantly, a response with higher breadth was detected in groups which received IL-12 or GM-CSF, evidenced as an increased frequency of recognition of homologous (BF) and heterologous (B) Nef peptides, as well as a higher number of other Nef peptide pools representing different viral subtypes. However, these improvements were lost when both DNA cytokines were simultaneously administered, as the response was focused against the immunodominant peptide with a detrimental response towards subdominant epitopes. The pattern of cytokines secreted and the specific-T-cell proliferative capacity were improved in IL-12 and IL-12+GM-CSF groups. Importantly IL-12 generated a significant higher T-cell avidity against a B heterologous peptide

Adrenal steroids modulate the phenotype and function of M. tuberculosis-stimulated human dendritic cells

Cell-mediated immunity, cytokines induced during the specific immune response and T-cell populations are crucial factors for containing Mycobacterium tuberculosis infection. Recent reports suggest a cross-regulation between adrenal steroids (glucocorticoids and dehydroepiandrosterone, DHEA) and the function of antigen-presenting cells (APCs). Therefore, we investigated the role of adrenal hormones on the functional capacity of M. tuberculosis-induced dendritic cells (DCs). Cortisol significantly inhibited the functions of M. tuberculosis-induced DCs. Interestingly, the presence of DHEA enhanced the M. tuberculosis-induced expression of MHC I, MHC II and CD86 and also increased ERK1/2 phosphorylation. Moreover, DHEA improved the production of IL-12 in response to M. tuberculosis stimulation, diminished IL-10 secretion and could not modify TNF-α synthesis. Importantly, we observed that DHEA enhanced the antigen-specific T-cell proliferation and IFN-γ production induced by M. tuberculosis-stimulated DC. These data show for the first time the relevance of the adrenal axis (especially of DHEA) in the modulation of DC function in the context of tuberculosis, a disease where the induction of a Th1 environment by APCs is crucial for the development of an effective immune response to the mycobacteria.Fil: Angerami, Matías. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas en Retrovirus y Sida; Argentina; Universidad de Buenos Aires. Facultad de Medicina. Departamento de Microbiología. Centro Nacional de Referencia del Sida; Argentina;Fil: Suárez, Guadalupe Verónica. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas en Retrovirus y Sida; Argentina;Fil: Pascutti, María Fernanda. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas en Retrovirus y Sida; Argentina;Fil: Salomon, Horacio Eduardo. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas en Retrovirus y Sida; Argentina; Universidad de Buenos Aires. Facultad de Medicina. Departamento de Microbiología; Argentina;Fil: Bottasso, Oscar Adelmo. Universidad Nacional de Rosario. Facultad de Ciencias Médicas. Instituto de Inmunología; Argentina;Fil: Quiroga, Maria Florencia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas en Retrovirus y Sida; Argentina; Universidad de Buenos Aires. Facultad de Medicina. Departamento de Microbiología. Centro Nacional de Referencia del Sida; Argentina

Deletion of A44L, A46R and C12L Vaccinia Virus Genes from the MVA Genome Improved the Vector Immunogenicity by Modifying the Innate Immune Response Generating Enhanced and Optimized Specific T-Cell Responses

MVA is an attenuated vector that still retains immunomodulatory genes. We have previously reported its optimization after deleting the C12L gene, coding for the IL-18 binding-protein. Here, we analyzed the immunogenicity of MVA vectors harboring the simultaneous deletion of A44L, related to steroid synthesis and A46R, a TLR-signaling inhibitor (MVAΔA44L-A46R); or also including a deletion of C12L (MVAΔC12L/ΔA44L-A46R). The absence of biological activities of the deleted genes in the MVA vectors was demonstrated. Adaptive T-cell responses against VACV epitopes, evaluated in spleen and draining lymph-nodes of C57Bl/6 mice at acute/memory phases, were of higher magnitude in those animals that received deleted MVAs compared to MVAwt. MVAΔC12L/ΔA44L-A46R generated cellular specific memory responses of higher quality characterized by bifunctionality (CD107a/b+/IFN-γ+) and proliferation capacity. Deletion of selected genes from MVA generated innate immune responses with higher levels of determining cytokines related to T-cell response generation, such as IL-12, IFN-γ, as well as IL-1β and IFN-β. This study describes for the first time that simultaneous deletion of the A44L, A46R and C12L genes from MVA improved its immunogenicity by enhancing the host adaptive and innate immune responses, suggesting that this approach comprises an appropriate strategy to increase the MVA vaccine potential

IFN-γ, TNF and IL-2 secretion pattern after re-stimulation with homologous or heterologous peptides.

<p>Spleen cells from mice immunized with 3xDNA/MVAnefBF plus IL-12 or/and GM-CSF (as depicted in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037801#pone-0037801-g001" target="_blank">Figure 1</a>) were re-stimulated during 72 h with the indicated peptides. Levels of IFN-γ, TNF and IL-2 secreted in supernatants were quantified by ELISA. Bars represent the average levels of cytokines secreted in cell supernatants from pooled samples of triplicate cultures. Negative controls (RPMIc) values were subtracted (<130 pg/ml for IFN-γ, <30 pg/ml for TNF and <14 pg/ml for IL-2). Cut-off levels (RPMI values+2xSD) are shown by horizontal grey lines. Significant differences between responses found in control vs the other three groups are indicated with ** (p<0.01) and * (p<0.05).</p

IL-12 and GM-CSF incremented the magnitude of the specific immune response.

<p><b>A</b>) Immunization schedule: Groups of 4 Balb/c mice received the different immunization regimes depicted. Immunization doses (both priming and boost doses) were spaced by 14 days. <b>B</b>) Nine days after the MVAnefBF boost dose, cellular immune responses against a pool of peptides representing <b>i-</b> NefBF or <b>ii-</b> NefB were evaluated in the spleen, by quantifying specific IFN-γ secreting cells by ELISPOT; <b>i-</b> Magnitude of the NefBF (homologous) response; n-fold increase represents the increments in the responses generated in relation to those found in the control group. <b>ii-</b> Magnitude of the cross-reactivity responses detected against NefB. Data represent the median values found in 4 to 5 independent experiments. Background values found in negative control wells were subtracted (5 to 50 spots/10⁶ cells). Mann Whitney test was applied, comparing NefBF responses between groups (* p<0.05). For the box and whisker plots, the horizontal line represents the median, the boxes represent the interquartile range and the whiskers represent the minimum and maximum values.</p

IL-12 and GM-CSF improved the quality of the cellular immune response.

<p><b>A</b>) Functional avidity of the BF33- and B84-specific T-cells: Groups of 4 mice received the immunization schemes depicted in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037801#pone-0037801-g001" target="_blank">Figure 1</a> for the group 1 (NefBF alone) and group 2 (plus IL-12 during the priming phase). Nine days after the MVA boost, T- cell avidity against BF33 and B84 peptides was evaluated using serial dilutions of the peptides. Peptide concentrations used ranged from 20 to 0.002 µg/ml. SD₅₀: sensitizing dose of peptides required to yield 50% maximal T-cell triggering of IFN-γ production. <b>B</b>) Spleen cells from the indicated group were evaluated for their proliferative capacity against the BF33 peptide, and the identification of the specific T-cell subset was performed. For this, cells were dyed with CFSE and stimulated with BF33 peptide during 5 days and then staining with surface antibodies was performed (CD3, CD4 and CD8). <b>i-</b> Representative dot plots. Numbers inside the graphs represent the average values for duplicated samples. <b>ii-</b> Bars represent the average of duplicated samples plus SD. Background values obtained in cell-cultures stimulated with RPMIc, were subtracted in each case. T test was applied, *p<0.05, * * p<0.005.</p