J Virol. 2015; 89 (2): 970-988.

Perdiguero B, Gómez CE, Cepeda V, Sánchez-Sampedro L, García-Arriaza J, Mejías-Pérez E, Jiménez V, Sánchez C, Sorzano CÓ, Oliveros JC, Delaloye J, Roger T, Calandra T, Asbach B, Wagner R, Kibler KV, Jacobs BL, Pantaleo G, Esteban M.

The generation of vaccines against HIV/AIDS able to induce long-lasting protective immunity remains a major goal in the HIV field. The modest efficacy (31.2%) against HIV infection observed in the RV144 phase III clinical trial highlighted the need for further improvement of HIV vaccine candidates, formulation, and vaccine regimen.

n this study, we have generated two novel NYVAC vectors, expressing HIV-1 clade C gp140(ZM96) (NYVAC-gp140) or Gag(ZM96)-Pol-Nef(CN54) (NYVAC-Gag-Pol-Nef), and defined their virological and immunological characteristics in cultured cells and in mice. The insertion of HIV genes does not affect the replication capacity of NYVAC recombinants in primary chicken embryo fibroblast cells, HIV sequences remain stable after multiple passages, and HIV antigens are correctly expressed and released from cells, with Env as a trimer (NYVAC-gp140), while in NYVAC-Gag-Pol-Nef-infected cells Gag-induced virus-like particles (VLPs) are abundant. Electron microscopy revealed that VLPs accumulated with time at the cell surface, with no interference with NYVAC morphogenesis. Both vectors trigger specific innate responses in human cells and show an attenuation profile in immunocompromised adult BALB/c and newborn CD1 mice after intracranial inoculation. Analysis of the immune responses elicited in mice after homologous NYVAC prime/NYVAC boost immunization shows that recombinant viruses induced polyfunctional Env-specific CD4 or Gag-specific CD8 T cell responses. Antibody responses against gp140 and p17/p24 were elicited.

Our findings showed important insights into virus-host cell interactions of NYVAC vectors expressing HIV antigens, with the activation of specific immune parameters which will help to unravel potential correlates of protection against HIV in human clinical trials with these vectors.

J Virol. 2014; pii: JVI.02223-14.

Knudsen ML, Ljungberg K, Kakoulidou M, Kostic L, Hallengärd D, García-Arriaza J, Merits A, Esteban M, Liljeström P.

Alphavirus replicons are potent inducers of CD8⁺ T cell responses and thus constitute an attractive vaccine vector platform for developing novel vaccines. However, the kinetics and memory phenotype of CD8⁺ T cell responses induced by alphavirus replicons are not well characterized. Furthermore, little is known how priming with alphavirus replicons affects booster immune responses induced by other vaccine modalities. We demonstrate that a single immunization with an alphavirus replicon, administered as viral particles or naked DNA, induced an antigen-specific CD8⁺ T cell response that had a sharp peak, followed by a rapid contraction. Administering a homologous boost before contraction had occurred did not further increase the response. In contrast, boosting after contraction when CD8⁺ T cells had obtained a memory phenotype (based on CD127/CD62L expression), resulted in maintenance of CD8⁺ T cells with a high recall capacity (based on CD27/CD43 expression). Increasing the dose of replicon particles promoted T effector memory (Tem) and inhibited T central memory (Tcm) development. Moreover, infection with a replicating alphavirus induced a similar distribution of CD8⁺ T cells as the replicon vector. Lastly, the distribution of T cell subpopulations induced by a DNA-launched alphavirus replicon could be altered by heterologous boosts. For instance, boosting with a poxvirus vector (MVA) favored expansion of the Tem compartment. In summary, we have characterized the antigen-specific CD8⁺ T cell response induced by alphavirus replicon vectors and demonstrated how it can be altered by homologous and heterologous boost immunizations.

Importance Alphavirus replicons are promising vaccine candidates against a number of diseases and are by themselves developed as vaccines against for example chikungunya virus infection. Replicons are also considered to be used for priming followed by booster immunization using different vaccine modalities. In order to rationally design prime-boost immunization schedules with these vectors, characterization of the magnitude and phenotype of CD8⁺ T cell responses induced by alphavirus replicons is needed. Here, we demonstrate how factors such as timing and dose affect the phenotype of the memory T cell populations induced by immunization with alphavirus replicons. These findings are important for designing future clinical trials with alphaviruses, as they can be used to tailor vaccination regimens in order to induce a CD8⁺ T cell response that is optimal for control and/or clearance of a specific pathogen.

Hum Vaccin Immunother. 2014; 10(8).

García-Arriaza J, Esteban M.

Attenuated recombinant poxvirus vectors expressing heterologous antigens from pathogens are currently at various stages in clinical trials with the aim to establish their efficacy. This is because these vectors have shown excellent safety profiles, significant immunogenicity against foreign expressed antigens and are able to induce protective immune responses.

In view of the limited efficacy triggered by some poxvirus strains used in clinical trials (i.e, ALVAC in the RV144 phase III clinical trial for HIV), and of the restrictive replication capacity of the highly attenuated vectors like MVA and NYVAC, there is a consensus that further improvements of these vectors should be pursuit. In this review we considered several strategies that are currently being implemented, as well as new approaches, to improve the immunogenicity of the poxvirus vectors. This includes heterologous prime/boost protocols, use of co-stimulatory molecules, deletion of viral immunomodulatory genes still present in the poxvirus genome, enhancing virus promoter strength, enhancing vector replication capacity, optimizing expression of foreign heterologous sequences, and the combined use of adjuvants. An optimized poxvirus vector triggering long-lasting immunity with a high protective efficacy against a selective disease should be sought.

J Virol. 2014; 88(6): 3527-3547.

García-Arriaza J1, Cepeda V, Hallengärd D, Sorzano CÓ, Kümmerer BM, Liljeström P, Esteban M.

There is a need to develop a single and highly effective vaccine against the emerging chikungunya virus (CHIKV), which causes a severe disease in humans. Here, we have generated and characterized the immunogenicity profile and the efficacy of a novel CHIKV vaccine candidate based on the highly attenuated poxvirus vector modified vaccinia virus Ankara (MVA) expressing the CHIKV C, E3, E2, 6K, and E1 structural genes (termed MVA-CHIKV). MVA-CHIKV was stable in cell culture, expressed the CHIKV structural proteins, and triggered the cytoplasmic accumulation of Golgi apparatus-derived membranes in infected human cells. Furthermore, MVA-CHIKV elicited robust innate immune responses in human macrophages and monocyte-derived dendritic cells, with production of beta interferon (IFN-β), proinflammatory cytokines, and chemokines. After immunization of C57BL/6 mice with a homologous protocol (MVA-CHIKV/MVA-CHIKV), strong, broad, polyfunctional, and durable CHIKV-specific CD8⁺ T cell responses were elicited.

The CHIKV-specific CD8⁺ T cells were preferentially directed against E1 and E2 proteins and, to a lesser extent, against C protein. CHIKV-specific CD8⁺ memory T cells of a mainly effector memory phenotype were also induced. The humoral arm of the immune system was significantly induced, as MVA-CHIKV elicited high titers of neutralizing antibodies against CHIKV. Remarkably, a single dose of MVA-CHIKV protected all mice after a high-dose challenge with CHIKV.

In summary, MVA-CHIKV is an effective vaccine against chikungunya virus infection that induced strong, broad, highly polyfunctional, and long-lasting CHIKV-specific CD8⁺ T cell responses, together with neutralizing antibodies against CHIKV. These results support the consideration of MVA-CHIKV as a potential vaccine candidate against CHIKV.

J Virol. 2014; 88(6): 3392-3410.

García-Arriaza J, Gómez CE, Sorzano CÓ, Esteban M.

A modified vaccinia virus Ankara poxvirus vector expressing the HIV-1 Env, Gag, Pol, and Nef antigens from clade B (MVA-B) is currently being tested in clinical trials. To improve its immunogenicity, we have generated and characterized the immune profile of MVA-B containing a deletion of the vaccinia viral gene N2L, which codes for an inhibitor of IRF3 (MVA-B ΔN2L).

Deletion of N2L had no effect on virus growth kinetics or on the expression of HIV-1 antigens; hence, the N2 protein is not essential for MVA replication. The innate immune responses triggered by MVA-B ΔN2L revealed an increase in beta interferon, proinflammatory cytokines, and chemokines. Mouse prime-boost protocols showed that MVA-B ΔN2L improves the magnitude and polyfunctionality of HIV-1-specific CD4⁺ and CD8⁺ T cell adaptive and memory immune responses, with most of the HIV-1 responses mediated by CD8⁺ T cells.

In the memory phase, HIV-1-specific CD8⁺ T cells with an effector phenotype were predominant and in a higher percentage with MVA-B ΔN2L than with MVA-B. In both immunization groups, CD4⁺ and CD8⁺ T cell responses were directed mainly against Env. Furthermore, MVA-B ΔN2L in the memory phase enhanced levels of antibody against Env. For the vector immune responses, MVA-B ΔN2L induced a greater magnitude and polyfunctionality of VACV-specific CD8⁺ T memory cells than MVA-B, with an effector phenotype.

These results revealed the immunomodulatory role of N2L, whose deletion enhanced the innate immunity and improved the magnitude and quality of HIV-1-specific T cell adaptive and memory immune responses. These findings are relevant for the optimization of poxvirus vectors as vaccines.

J Gen Virol. 2013 Sep 28

Di Pilato M, Mejías-Pérez E, Gómez CE, Perdiguero B, Sánchez-Sorzano CO, Esteban M.

Here we describe the design and strength of a new synthetic Late-Early Optimized (LEO) vaccinia virus (VACV) promoter used as transcriptional regulator of GFP expression during MVA infection.

In contrast to the described synthetic VACV promoter (pS), LEO induces in vitro significantly higher levels of GFP expression within the first hour after infection, which correlates with an enhancement in the GFP-specific CD8 T cell response detected in vivo demonstrating its potential use in future vaccines.

El CSIC ha renovado el panel de integrantes de su Comité Científico Asesor. La nueva lista de 23 miembros se ha cerrado con siete nuevas incorporaciones. Entre otras la de investigador del Centro Nacional de Biotecnología Mariano Esteban.

El órgano, cuya presidencia ostenta el presidente del CSIC, Emilio Lora-Tamayo, se compone de científicos y tecnólogos de las distintas áreas de conocimiento en las que está distribuida la actividad científica del CSIC. Según el Estatuto del organismo, tiene la función de informar y asesorar en aspectos científico-tecnológicos a la Presidencia del CSIC y al Consejo Rector.

Los siete vocales que se incorporan al comité son: Juan Albadalejo Montoro (profesor de Investigación del CSIC adscrito al Centro de Edafología y Biología Aplicada del Segura), Mariano Esteban Rodríguez (profesor de Investigación del CSIC adscrito al Centro Nacional de Biotecnología), Ángel Messeguer Peypoch (profesor de Investigación del CSIC adscrito al Instituto de Química Avanzada de Cataluña) y Juan Moreno Klemming (profesor de Investigación del CSIC adscrito al Museo Nacional de Ciencias Naturales)

Esteban es pionero en el campo de las vacunas, donde destaca en la lucha contra el sida empleando procedimientos de inmunización combinada de vectores. Sus trabajos, que están siendo financiados por distintos organismos nacionales e internacionales como la Fundación Bill y Melinda Gates, tienen aplicación en enfermedades como la hepatitis C, la gripe o el cáncer de próstata.

El investigador del Centro Nacional de Biotecnología del CSIC (CNB) Mariano Esteban sustituye a María Teresa Miras Portugal en la presidencia de la Real Academia Nacional de Farmacia. Y lo hace con la “ilusión de impulsar desde la Academia todo lo relacionado con el medicamento y la salud”, especialmente a través de conferencias, mesas redondas y simposios dirigidos a entender los mecanismos moleculares de la acción de los fármacos sobre el organismo.

Durante la Sesión Inaugural del curso del pasado jueves 17 de enero, Esteban tomó posesión como nuevo presidente de la institución. Desde su nuevo cargo anima a que en una época en la que la biología avanza a pasos agigantados, se apliquen esos conocimientos “para un mayor bienestar social”.

Pionero en el campo de las vacunas, caben destacar sus investigaciones en la lucha contra el sida empleando procedimientos de inmunización combinada de vectores. Sus trabajos, que están siendo financiados por distintos organismos nacionales e internacionales como la Fundación Bill y Melinda Gates, tienen aplicación en enfermedades como la hepatitis C, la gripe o el cáncer de próstata.

Para el que fuera director del CNB de 1992 a 2003, la Academia debe “fomentar el estudio de las llamadas enfermedades olvidadas”. Algo a lo que el mismo ha dedicado gran parte de su vida científica. No en vano, el propio Esteban cuenta con más de 270 artículos en revistas internacionales relacionados no solo con la producción de vacunas contra el VIH sino también contra enfermedades como la leishmaniasis o la malaria.

Para este licenciado en Farmacia (1967) y en Ciencias Biológicas (1972), la Academia “debe ser un foro donde brillen las ideas” y recalca la importancia de seguir colaborando con el sector farmacéutico ya que ambos se necesitan mutuamente.